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https://openalex.org/W4306175389	https://nutritionj.biomedcentral.com/counter/pdf/10.1186/s12937-022-00815-8	English	null	Validity and reliability of a semi-quantitative food frequency questionnaire in groups at high risk for cardiovascular diseases	Nutrition journal	2,022	cc-by	8,424	© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Weighted k statistics and Bland-Altman plots showed acceptable agreement between FFQs and 24HDRs. Conclusion: The FFQ developed for the population at high risk of cardiovascular and cerebrovascular diseases in areas of Ningxia can be used to measure the dietary intake of nutrients and food groups reliably and validly. Keywords: Food frequency questionnaire, Reliability, Validity, CVD, Food groups ischemic heart disease are the top two causes of death in China. Accounting for stroke and ischemic heart disease of deaths annually is 149 and 124 per 100,000 population [3]. Dietary risk factors have been found to influence the occurrence, progression and mortality of CVD [2, 4, 5]. Reasonable dietary intakes including Mediterranean Diet supplemented with extra-virgin olive oil and Mediterra- nean Diet supplemented with nuts can reduce the occur- rence of CVD by 50% [6]. Many epidemiological studies have also shown that appropriate dietary habits play a very important role in the protection and prevention of CVD and adapting appropriate dietary patterns such Yan et al. Nutrition Journal (2022) 21:63 https://doi.org/10.1186/s12937-022-00815-8 Open Access Abstract Background: Diet is a modifiable risk factor for cardiovascular diseases (CVD), but there is still a lack of tools to assess dietary intakes of this high-risk population in Ningxia, China. Background: Diet is a modifiable risk factor for cardiovascular diseases (CVD), but there is still a lack of tools to assess dietary intakes of this high-risk population in Ningxia, China. Objective: We aim to evaluate the validity and reliability of the semi-quantitative food frequency questionnaire (SFFQ) in the groups in Ningxia using a 24-hour dietary recall method. Objective: We aim to evaluate the validity and reliability of the semi-quantitative food frequency questionnaire (SFFQ) in the groups in Ningxia using a 24-hour dietary recall method. Method: Two hundred five participants were included in the analysis. The two FFQs were 6 months apart, and during this time two 24-hour dietary recalls (24HDRs) were completed. Statistical methods were compared using inter-class correlation coefficient, unadjusted, energy-adjusted, de-attenuated correlation coefficient, quartile classification, weighted K values, and 95% limits of agreement (LOA). Method: Two hundred five participants were included in the analysis. The two FFQs were 6 months apart, and during this time two 24-hour dietary recalls (24HDRs) were completed. Statistical methods were compared using inter-class correlation coefficient, unadjusted, energy-adjusted, de-attenuated correlation coefficient, quartile classification, weighted K values, and 95% limits of agreement (LOA). Method: Two hundred five participants were included in the analysis. The two FFQs were 6 months apart, and during this time two 24-hour dietary recalls (24HDRs) were completed. Statistical methods were compared using inter-class correlation coefficient, unadjusted, energy-adjusted, de-attenuated correlation coefficient, quartile classification, weighted K values, and 95% limits of agreement (LOA). Results: The inter-class correlation coefficients between FFQ1 and FFQ2 ranged from 0.25 to 0.73. The number of subjects classified as identical or adjacent was 72.2 to 85.9%. The crude correlation coefficient between FFQs and 24HDRs was 0.30 ~ 0.81, the energy-adjusted correlation coefficient was 0.16 ~ 0.83, and the de-attenuated correla- tion coefficient was 0.19 ~ 0.98. Weighted k statistics and Bland-Altman plots showed acceptable agreement between FFQs and 24HDRs. Results: The inter-class correlation coefficients between FFQ1 and FFQ2 ranged from 0.25 to 0.73. The number of subjects classified as identical or adjacent was 72.2 to 85.9%. The crude correlation coefficient between FFQs and 24HDRs was 0.30 ~ 0.81, the energy-adjusted correlation coefficient was 0.16 ~ 0.83, and the de-attenuated correla- tion coefficient was 0.19 ~ 0.98. Introduction Cardiovascular disease (CVD) is one of the lead- ing causes of death and disability globally. In the past 10 years, the global mortality rate of CVD has increased by 12.5% [1]. CVD is characterized by high morbidity, disability, mortality and recurrence rates [2]. Accord- ing to the Global Burden of Diseases 2017, stroke and Correspondence: zhaoyi751114@hotmail.com Correspondence: zhaoyi751114@hotmail.com *Correspondence: zhaoyi751114@hotmail.com 2 Key Laboratory of Environmental Factors and Chronic Disease Control, No.1160, Shengli Street, Xingqing District, Yinchuan, Ningxia, China Full list of author information is available at the end of the article 2 Key Laboratory of Environmental Factors and Chronic Disease Control, No.1160, Shengli Street, Xingqing District, Yinchuan, Ningxia, China Full list of author information is available at the end of the article 2 Key Laboratory of Environmental Factors and Chronic Disease Control, No.1160, Shengli Street, Xingqing District, Yinchuan, Ningxia, China Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Yan et al. Nutrition Journal (2022) 21:63 Yan et al. Nutrition Journal (2022) 21:63 Page 2 of 10 Therefore, the aim of this study was to assess the validity and reliability of an SFFQ used to assess dietary intakes in groups at high risk for CVD in Ningxia. as diets high in monounsaturated and polyunsaturated acids that favor metabolic markers may prevent CVD [7]. On the contrary, malnutrition may lead to an increase in morbidity and mortality. Considering that nutrition is an important modifiable risk factor for people at high risk of CVD, it is important to understand the current dietary intake [8]. Therefore need to have appropriate assessment tools for dietary patterns [9]. as diets high in monounsaturated and polyunsaturated acids that favor metabolic markers may prevent CVD [7]. On the contrary, malnutrition may lead to an increase in morbidity and mortality. Considering that nutrition is an important modifiable risk factor for people at high risk of CVD, it is important to understand the current dietary intake [8]. Therefore need to have appropriate assessment tools for dietary patterns [9]. Methodsh This study collected two FFQs over6 months. The first FFQ (FFQ1) was collected in December 2019 and the second FFQ (FFQ2) was collected in July 2020. Two dis- continuous 24-hour dietary recalls were collected, at the same time as FFQs. All the dietary interviewers were con- ducted on a random day because pre-trial tests showed there was no significant difference between weekdays and weekends in the diets of residents in the areas. In nutritional epidemiology, there have been numer- ous tools to assess dietary intake and each method has its own advantages and limitations [10–12]. The most commonly used assessment tools are the 24-hour dietary recall and dietary records containing weighing foods are also used to measure daily dietary intake, but these meth- ods are expensive, time-consuming, and not suitable for most large-scale studies [13]. Besides, short-term recalls and diet records are not representative of usual dietary intake. Therefore, it is not sufficient to assess dietary intakes over time. Food Frequency Questionnaire (FFQ) is considered to be a low-cost diet assessment method suitable for a large sample and is often used to research the relationship between dietary factors and diseases in various epidemiological studies [14, 15]. Due to its long reference period and pre-specified food list, the col- lected dietary intake information has limitations to accu- rately measure the dietary intakes [16]. In other words, the nutritional content of FFQ is mainly affected by sys- tematic errors and requires a careful evaluation when assessing diet-disease relationships [17, 18]. Also, the dif- ferences in demographic, socio-economic, cultural, and other points also influence the food intake of each group [19, 20]. Therefore, it is necessary to verify the validity and reliability of the FFQ in each group to evaluate the accuracy and precision of dietary intake.f Study populations h b Participants have been recruited from six villages from Qingtong Xia County of Ningxia province in China and they were same as the participants of project [23]. Based on the study of Willett [10], at least 110 participants were required to examine validity and reliability of dietary sur- veys. The study recruited 210 participants at high risk of CVD, defined as having a history of CVD. In each village, A history of CVD or a high risk of CVD, defined on the basis of a prior hospitalisation or male aged> 60 years, or female aged> 65 years, and with at least two of the follow- ing risk factors: a. Type 2 diabetes requiring treatment with at least two oral anti-hyperglycaemic agents and/ or insulin b. Systolic blood pressure > 140 mmHg while on one or more antihypertensive agents c. Current daily smoking. Participants with an intellectual disability, a cognitive disability or with any chronic medical condition which required dietary restriction were excluded. The doctors will inform those participants to prepare relevant supporting documents (medical records and physical evi- dence of cigarettes and drugs) prior to participating in screening interviews.hi A recent study evaluated the effectiveness of an SFFQ in a group with CVD [18]. The results showed that due to the short time span of SFFQ, there were large seasonal differences in nutrient estimates. Thus, there are some limitations in the research. In addition, the result can- not be transferred to other groups in different dietary regions, and each region should develop localized SFFQ based on its specific dietary habits and traditions rather than using a uniform SFFQ. The mortality rate of CVD in Ningxia residents showed an increasing trend from 2012 to 2016 and was mainly over 60 years old [21]. At the same time, the unreasonable dietary patterns of adult residents in Ningxia was manifested by insufficient intake of vegetables and fruits and excessive intake of salt and edible oil, which can increase the risk of related diseases [22]. We were interested in the consumption of polyun- saturated fatty acids in this population. Because the pro- gram was a long-term dietary intervention, FFQ was a relatively good dietary survey method. The study was reviewed and confirmed by the Eth- ics Committee of Ningxia Medical University (No. 2020–066). The SFFQh The SFFQ was developed from a pre-trial of 20 people, conducted by household interviews. In the pre-trial, food pictures were used to help recall food types. There were two standardized trained staff on site. The SFFQ was formulated by combining the types of foods that were based on the 3 days 24-hour dietary recalls survey in local household. The questionnaire concluded 173 types of typical foods from areas of Ningxia, including foods unique to northwest China and accounting for about 95% of the most commonly used foods in Ningxia. Par- ticipants had the option to type name of foods if not in the SFFQ. These food items were allocated to 15 food Yan et al. Nutrition Journal (2022) 21:63 Page 3 of 10 groups such as: cereals, beans and soy products, pota- toes, milk and dairy products, meat and meat products, eggs, aquatic products, vegetables, fruit, the fungus mushrooms, oils and fats, alcohol, beverages, snacks and condiments. For each food item, four categories were provided for frequencies (daily, weekly, monthly, annually or never) and the amount of consumption over the past 6 months was reported using the common weight unit in China (1 liang =50 g) [24]. The monthly consumption of the whole family was used for estimation of oil and con- diments consumption. The SFFQ was designed to survey the dietary intakes of participants over the past 6 months and was administered by two staff with standard training due to the low educational level of the participants. misclassification (quartiles method) analyses also were used to assess the reliability between FFQ1 and FFQ2. Among them, k values above 0.80 indicate very good agreement, 0.61 to 0.80 indicate substantial (good) agree- ment, 0.41 to 0.60 indicate moderate agreement, 0.21 to 0.40 indicate good agreement, and 0 to 0.20 indicate slight (poor) agreement [25].h The overall raw data were natural-log(ln) transformed to improve the normality of food groups and nutrients. Spearman’s rank correlation coefficient (r) of natural- log(ln) transformed values were calculated to evaluate the validity of FFQ and 24-hour dietary recalls. The FFQ and 24-hour dietary recall data were the mean of the two times. Energy-adjusted nutrients intakes estimates were obtained by the residual method [10]. All valid- ity coefficients were attenuated due to random errors in the 24-hour dietary recalls. Results Among the 210 selected participants, 205 agreed to take part in the study and completed the survey (response rate = 97.6%). The main reasons for not participating in the study included refusal, absence during the inves- tigation period, poor health and death. Table 1 lists the sociodemographic and anthropometric characteristics of the study population. The mean age of the subjects was 65.3 years old (male 46.3%), the mean height was 160.4 cm, the mean weight was 68.1 kg, and the mean body mass index was 26.4 kg /m2.h The median intakes of total energy, nutrients, and food groups derived from the two FFQ and the percentage of differences are presented in Table 2. The median intakes for all nutrients and food groups (except fruit) evalu- ated with FFQ2 were higher than or equal to the median intakes with FFQ1, with differences between 0 and 52.4%. The largest differences between the median intakes of the two FFQ were 36.0% for nutrients (cholesterol) and 52.4% for the food group (eggs). The 24‑hour dietary recall All recall interviews were conducted in the subjects’ homes to estimate commonly used home measurements more accurately and to limit the number of missing sub- jects. Participants were asked to recall all the foods or beverages they had eaten in the previous 24 hours and estimate the portion size. Common household meas- urements (the size of bowls and plates) are used to help estimate portions. Other dietary information includes recipe ingredients, the measures of cooking and the time and place of they had eaten (e.g. home or outside). Mixed foods in the 24-h dietary recall are converted to their ingredients to measure. IBM SPSS Statistics Version23.0 was used for all data analysis. All p values were double-tailed, and the p values less than 0.05 were considered evidence of a statistically significant correlation. The SFFQh This formula from Wil- let was used to calculate the de-attenuated correlation to eliminate within-person variability in 24-hour recalls [10]: rt = r0 √1 + r/n , where r0 is the observed correla- tion between FFQs and 24-hour recalls, and r is the rate of variation within- and between-person measured dur- ing two 24-hour recalls, and n is the number of days of dietary recall (n = 2). Bland-Altman plots were used to test the consistency of the two dietary assessment methods between different intakes. As suggested by Bland&Altman [26], the natural-log(ln) transformed was performed to narrow the 95% limits of agreement (LOA). Calculation of nutrient intakeh The data obtained from the FFQ was imported with Epi- Data software by two trained staff. The data from the 24-hour dietary recalls were inputted into the Nutrition Calculator (v2.7.5(k), Institute for Nutrition and Food Security, Chinese Center for Disease Control and Preven- tion). The daily nutrient intake of participants was calcu- lated using the China Food Composition Database. The nutritional values of each food were calculated by match- ing the food names in the database. If there is one food not listed in the database, the nutritional values were calculated using foods that contain similar ingredients. The data of food intake and nutrient intake output by the dietary software were imported into Microsoft Excel for statistical analysis. Discussionh This study aimed to examine the validity and reliability of SFFQ used to evaluate various food groups and nutri- ents in dietary intakes assessment of high-risk popula- tions of cardiovascular disease in Ningxia. In this study, two discontinuous 24-hour dietary recalls were collected to evaluate the reliability and validity of the SFFQ at 6-month intervals. We assessed the performance of SFFQ by comparing the food groups and nutrients intakes reported using the instrument with intakes obtained using the 24-hour dietary recalls. Overall, the SFFQ has shown acceptable reliability and validity in evaluating food groups and nutrient intakes in people at high risk for CVD. The ICCs of food groups ranged from 0.34 to 0.73 and nutrients ranged from 0.25 to 0.72 (Table 3). The energy- adjusted ICCs of food groups ranged from 0.26 to 0.73 and nutrients ranged from 0.26 to 0.71. When the food groups and nutrient intakes were divided into quartiles, the agreement rates of FFQ1 and FFQ2 in the same and adjacent quartile were 72.2 to 85.9%. Except for vitamin A(10.2%), the misclassification for all nutrients and food groups as extreme quartile was rare (< 10%). Most the weighted k statistics were moderate conformity, ranging from 0.40 to 0.54. Weighted k statistics for the five food groups and seven nutrients were general conformity, ranging from 0.29 to 0.39. Only vegetables showed poor consistency, with a low weighted k statistic (0.19). In previous studies [27, 28], the intervals between FFQs varied. Reliability is based on the results of diet between two questionnaires, ideally the shorter the interval, the better the reliability [10]. In this case, however, the sub- jects were more likely to remember and repeat their previous answers. In this study, there was a six-month interval between the two FFQs, which could relatively reduce the above bias. Similarly, for validation studies, it is crucial to select the appropriate reference method to evaluate FFQ. Previous studies have shown that FFQ and 24-hour dietary recalls have been validated in 75% of studies. Although FFQ and 24-hour dietary recall are both retrospective questionnaires, they are prone to the same recall bias, which may lead to overestimation of the correlation coefficient. However, 24-hour dietary recalls may be more appropriate for subjects with lower educa- tional levels [20, 29]. Since participants in this study were high-risk populations in areas with limited literacy, the 24-hour dietary recall was selected for assessment. Statistical analysis To measure the consistency of the first and second FFQ received by the same subject at different times, Spear- man’s rank correlation coefficient (r) was calculated to compare the energy, nutrients and food intakes between two FFQ administrations. For reliability analysis, we used the inter-class correlation coefficient (ICC) and 95% CI for each point. Weighted kappa (k) statistic, and Yan et al. Nutrition Journal (2022) 21:63 Page 4 of 10 Table 1 Characteristics and anthropometric measurements of the participants included (N = 205) a Continuous normally distributed variables are expressed as mean and SD b BMI body mass index, WHR Waist to hip ratio Characteristicsa Male(n = 110) Female(n = 95) All(n = 205) Age at recruitment (years) 65.0 ± 7.7 65.6 ± 6.8 65.3 ± 7.3 Age range (years) 34–80 42–77 34–80 Degree of education (number (%)) Primary school or below 71 (64.5) 79 (83.2) 150 (73.2) Middle school 29 (26.4) 8 (8.4) 37 (18.0) High school and above 10 (9.1) 8 (8.4) 18 (8.8) Height (cm) 165.8 ± 5.8 154.0 ± 4.7 160.4 ± 7.9 Weight (kg) 72.6 ± 10.5 62.8 ± 9.2 68.1 ± 11.0 BMI (kg/m2)b 26.3 ± 3.2 26.5 ± 3.6 26.4 ± 3.4 Waist (cm) 92.8 ± 9.8 89.4 ± 9.1 91.3 ± 9.6 Hip (cm) 98.1 ± 5.2 95.2 ± 5.4 96.8 ± 5.5 WHRb 0.94 ± 0.06 0.94 ± 0.06 0.94 ± 0.06 Table 1 Characteristics and anthropometric measurements of the participants included (N = 205) nutrients increased and ranged from 0.07 for folic acid to 0.98 for vitamin E. When nutrients intakes were divided into quartiles, the agreement rates between FFQs and 24-hour dietary recalls of the same and adjacent quartiles were 71.2 to 94.1%. The misclassification for all nutrients as extreme quartile was rare (< 10%), except for vitamin D (12.7%) and iodine (10.2%). The results of the Bland-Altman plots showed in Fig. 1 including energy, protein, fat, and carbohydrates (Other nutrients were listed in Annex). According to Fig. 1, most of the points fell within the 95% limits of agreement (LOAS), closer to the middle horizontal line and there was no linear trend between the differences and means for two FFQs and 24-hour dietary recalls. Discussionh y g Overall, the median daily intakes of nutrients, assessed by two FFQs average, were substantially higher than the average of the two 24-hour dietary recalls, except for protein, carbohydrates, and vitamin B1 (Table 4). The unadjusted Spearman correlation coefficients for nutri- ents ranged from 0.18 for vitamin A to 0.81 for vitamin E. After energy adjustment, Spearman correlation coef- ficients of all nutrients decreased except vitamin E, which increased slightly. Compared with the unadjusted values, niacin (0.59 ~ 0.27) and iron (0.45 ~ 0.14) had the most significant changes. Except for vitamin A, vitamin D, folic acid, and iodine, the energy-adjusted coefficients of the other nutrients were statistically significant (p < 0.01 or p < 0.05). The average of correlation coefficients improved from 0.32 to 0.39 after energy adjustment, and correction for random within-person variation and between-per- son variation. The de-attenuated correlations for all the In the reliability studies, results showed that the median intakes of all nutrient and food groups (except fruit) assessed with FFQ2 were higher or equal to the median intakes of all nutrients and food groups assessed with FFQ1. It could be that participants were more likely Yan et al. es: Vegetables including fresh vegetables and pickled vegetables; Aquatic products including fish, shrimp and seaweeds; SFA saturated fa yunsaturated fatty acids, MUFA monounsaturated fatty acids a and b mean P value for test of difference < 0.001 and < 0.05 respectively a and b mean P value for test of difference < 0.001 and < 0.05 respectively a and b mean P value for test of difference < 0.001 and < 0.05 respectively Notes: Vegetables including fresh vegetables and pickled vegetables; Aquatic products including fish shrimp and seaweeds; SFA saturated fatty acid PUFA Discussionh Nutrition Journal (2022) 21:63 Page 5 of 10 Table 2 Comparison of nutrients intakes between two FFQs for 205 participants (median and 25th - 75th percentiles) a and b mean P value for test of difference < 0.001 and < 0.05 respectively Notes: Vegetables including fresh vegetables and pickled vegetables; Aquatic products including fish, shrimp and seaweeds; SFA saturated fatty acid, PUFA polyunsaturated fatty acids MUFA monounsaturated fatty acids Food groups or nutrients FFQ1 FFQ2 Percentag of median difference Median 25–75th percentile Median 25–75th percentile Food groups(g/d) Tuber crops 47.0 21.6–94.0 47.0 25.9–70.5 0.0b Meats 29.5 14.7–63.7 37.5 19.1–69.1 21.2b Eggs 8.8 1.3–18.5 18.5 3.5–37.0 52.4a Grains 416.5 307.3–547.0 443.5 328.9–540.4 6.1 Oil 33.3 25.0–44.4 33.3 25.0–44.4 0.0 Vegetables 387.0 230.3–522.0 510.0 383.3–637.0 24.1a Fruits 4.2 0.4–14.0 4.0 0.1–11.2 −3.5b Aquatic products 42.3 20.0–100.0 64.2 40–101.4 34.1 Legumes and products 42.9 20.0–65.8 45.5 23.2–71.7 5.7 Dairy and products 16.8 0.0–84.0 40.0 0.0–87.6 58.0 Salty agent 10.0 7.4–13.1 10.0 7.4–13.3 0.0 Spicy agent 16.7 8.3–20.8 16.7 8.3–25.0 0.0 Sour agent 1.0 0.3–2.5 1.0 0.3–2.1 0.0 Nutrients Energy(kcal/d) 1633.1 1329.9–2064.2 1912.0 1483.4–2287.8 14.6a Carbohydrate(g/d) 61.0 44.9–79.0 70.4 57.6–88.8 13.4a Protein(g/d) 49.2 39.0–65.2 60.2 44.1–72.7 18.2a Dietary fiber(g/d) 11.8 8.0–15.1 14.9 11.6–17.9 21.2a Cholesterol(mg/d) 104.9 57.2–178.0 163.9 84.5–257.1 36.0a Fat(g/d) 222.4 171.2–289.7 248.1 187.3–310.9 10.4a SFA(g/d) 9.0 6.2–12.2 10.5 7.7–13.9 14.6a MUFA(g/d) 21.3 16.0–29.8 25.8 20.8–35.1 17.4b PUFA(g/d) 19.7 14.0–25.8 21.1 16.1–27.6 6.9a Vitamin A(μgRE/d) 263.4 174.5–380.7 350.6 270.0–447.7 24.9 Vitamin D(μg/d) 0.8 0.4–1.7 1.1 0.5–1.9 23.3 Vitamin E(mg/d) 82.6 22.3–146.7 88.2 30.2–151.2 6.3 Thiamin(mg/d) 0.8 0.6–1.0 0.9 0.7–1.1 9.8a Riboflavin(mg/d) 0.8 0.6–1.0 0.9 0.7–1.1 14.1a Pyridoxine(mg/d) 0.3 0.2–0.5 0.4 0.3–0.5 9.2a Vitamin C(mg/d) 100.3 60.5–140.4 133.7 98.9–167.5 25.0a Folate(μg/d) 159.1 103.8–220.6 208.7 160.3–267.8 23.8a Niacin(mg/d) 14.5 11.4–18.5 16.5 12.5–20.4 12.0a Calcium(mg/d) 383.7 277.8–499.6 492.0 378.2–605.6 22.0a Phosphorus(mg/d) 843.5 668.6–1080.0 998.2 788.1–1188.0 15.5a Potassium(mg/d) 1783.2 1331.8–2364.8 2251.3 1731.6–2621.4 20.8a Sodium(mg/d) 3804.4 3015.3–4748.1 3843.8 2968.8–4828.0 1.0 Magnesium(mg/d) 306.9 232.3–371.5 361.0 286.0–425.2 15.0a Iron(mg/d) 21.2 16.2–26.1 24.7 18.7–29.5 14.3a Zinc(mg/d) 9.0 7.1–11.4 10.4 8.0–12.6 14.2a Selenium(μg/d) 27.8 22.1–39.1 34.3 25.5–43.3 19.0a Copper(mg/d) 1.5 1.1–1.9 1.7 1.3–2.1 12.2a Iodine(μg/d) 31.0 20.8–58.5 42.1 28.0–68.4 26.4 Table 2 Comparison of nutrients intakes between two FFQs for 205 participants (median and 25th - 75th percentiles) Yan et al. Notes: FFQ1 first FFQ, FFQ2 FFQ after six months, ICC intra-group correlation coefficient, Weighted K weighted Kappa value, SFA saturated fatty acid, PUFA polyunsaturated fatty acids, MUFA monounsaturated fatty acids; Vegetables including fresh vegetables and pickled vegetables; Aquatic products including fish, shrimp and seaweeds; Residual method was adopted for energy adjustment p y FFQ2 FFQ after six months, ICC intra-group correlation coefficient, Weighted K weighted Kappa value, SFA saturated fatty acid, PUFA i Discussionh Nutrition Journal (2022) 21:63 Page 6 of 10 Table 3 Reliability of food groups and nutrients intakes between FFQ1 and FFQ2 a and b mean P value for ICC < 0.001 and < 0.05 respectively Notes: FFQ1 first FFQ, FFQ2 FFQ after six months, ICC intra-group correlation coefficient, Weighted K weighted Kappa value, SFA saturated fatty acid, PUFA polyunsaturated fatty acids, MUFA monounsaturated fatty acids; Vegetables including fresh vegetables and pickled vegetables; Aquatic products including fish, shrimp and seaweeds; Residual method was adopted for energy adjustment Food groups or nutrients ICC(95%CI) Percent of agreement(%) Weighted k Crude Energy adjusted Same quartile Adjacent quartile One quartile apart Opposite quartile Food groups(g/d) Tuber crops 0.65(0.54,0.74)a 0.66(0.55,0.74)a 43.9 42.0 12.2 1.9 0.52 Meats 0.58(0.45,0.68)a 0.53(0.38,0.64)a 36.6 41.5 17.6 4.3 0.39 Eggs 0.55(0.40,0.66)a 0.61(0.49,0.71)a 42.4 37.1 15.1 5.4 0.47 Grains 0.66(0.55,0.74)a 0.59(0.46,0.69)a 38.5 39.5 19.5 2.5 0.44 Oil 0.73(0.65,0.80)a 0.73(0.64,0.79)a 39.5 32.7 22.0 5.8 0.38 Vegetables 0.34(0.13,0.50)b 0.26(0.03,0.44)b 30.2 42.0 18.5 9.3 0.19 Fruits 0.39(0.19,0.53)a 0.47(0.30,0.59)a 44.9 35.6 15.6 3.9 0.46 Aquatic products 0.59(0.47,0.69)a 0.54(0.40,0.65)a 46.8 36.1 13.2 3.9 0.50 Legumes and products 0.68(0.58,0.76)a 0.59(0.46,0.69)a 45.9 38.0 13.7 2.4 0.54 Salty agent 0.57(0.43,0.67)a 0.49(0.33,0.61)a 34.2 39.5 21.0 5.3 0.32 Spicy agent 0.58(0.45,0.68)a 0.60(0.47,0.70)a 36.6 39.5 21.5 2.4 0.38 Sour agent 0.64(0.53,0.73)a 0.59(0.46,0.69)a 44.4 34.6 19.0 2.0 0.36 Nutrients Energy(kcal/d) 0.68(0.57,0.75)a 0.68(0.57,0.75)a 38.1 45.4 13.7 2.8 0.49 Carbohydrate(g/d) 0.65(0.53,0.73)a 0.56(0.42,0.66)a 38.1 44.9 13.7 3.3 0.48 Protein(g/d) 0.68(0.57,0.75)a 0.66(0.56,0.74)a 46.3 34.6 15.1 4.0 0.48 Dietary fiber(g/d) 0.58(0.45,0.68)a 0.60(0.47,0.69)a 35.6 43.9 15.6 4.9 0.4 Cholesterol(mg/d) 0.62(0.50,0.71)a 0.67(0.57,0.75)a 42.0 40.0 15.6 2.4 0.5 Fat(g/d) 0.65(0.54,0.73)a 0.62(0.50,0.71)a 36.1 41.0 18.1 4.8 0.37 SFA(g/d) 0.60(0.48,0.70)a 0.62(0.50,0.71)a 37.6 43.4 15.1 3.9 0.44 MUFA(g/d) 0.62(0.50,0.71)a 0.64(0.53,0.73)a 40.0 37.6 18.5 3.9 0.41 PUFA(g/d) 0.67(0.57,0.75)a 0.61(0.48,0.70)a 32.7 43.9 18.5 4.9 0.35 Vitamin A(μgRE/d) 0.27(0.04,0.44)b 0.26(0.02,0.44)b 37.6 35.1 16.6 10.7 0.33 Vitamin D(μg/d) 0.65(0.54,0.73)a 0.62(0.50,0.71)a 43.4 39.5 12.7 4.4 0.48 Vitamin E(mg/d) 0.72(0.63,0.79)a 0.69(0.60,0.77)a 46.3 37.1 13.7 2.9 0.53 Thiamin(mg/d) 0.63(0.51,0.72)a 0.61(0.48,0.70)a 41.5 38.1 15.6 4.8 0.42 Riboflavin(mg/d) 0.65(0.53,0.73)a 0.70(0.60,0.77)a 39.0 43.9 12.7 4.4 0.43 Pyridoxine(mg/d) 0.54(0.39,0.65)a 0.54(0.40,0.65)a 41.5 37.6 16.1 4.8 0.41 Vitamin C(mg/d) 0.25(0.02,0.43)b 0.28(0.05,0.45)b 36.1 36.1 22.0 5.8 0.29 Folate(μg/d) 0.53(0.38,0.64)a 0.50(0.35,0.62)a 35.1 42.9 16.1 5.9 0.36 Niacin(mg/d) 0.66(0.55,0.74)a 0.56(0.41,0.66)a 40.5 42.4 13.2 3.9 0.48 Calcium(mg/d) 0.62(0.50,0.71)a 0.64(0.52,0.72)a 40.5 37.6 15.6 6.3 0.37 Phosphorus(mg/d) 0.65(0.54,0.74)a 0.65(0.54,0.73)a 45.4 36.1 14.6 3.9 0.48 Potassium(mg/d) 0.65(0.54,0.73)a 0.58(0.44,0.68)a 45.4 36.1 13.2 5.3 0.45 Sodium(mg/d) 0.58(0.45,0.68)a 0.49(0.32,0.61)a 37.1 38.1 18.5 6.3 0.32 Magnesium(mg/d) 0.61(0.49,0.70)a 0.56(0.42,0.66)a 42.9 37.1 15.6 4.4 0.44 Iron(mg/d) 0.68(0.58,0.76)a 0.71(0.61,0.78)a 42.9 38.1 15.6 3.4 0.47 Zinc(mg/d) 0.65(0.53,0.73)a 0.62(0.50,0.71)a 41.0 42.4 11.2 5.4 0.46 Selenium(μg/d) 0.63(0.51,0.72)a 0.57(0.44,0.67)a 43.4 34.6 17.1 4.9 0.41 Copper(mg/d) 0.58(0.45,0.68)a 0.54(0.40,0.65)a 44.4 35.6 15.6 4.4 0.45 Iodine(μg/d) 0.65(0.53,0.73)a 0.68(0.58,0.76)a 42.0 42.9 9.8 5.3 0.48 Table 3 Reliability of food groups and nutrients intakes between FFQ1 and FFQ2 a and b mean P value for ICC < 0.001 and < 0.05 respectively Page 7 of 10 Yan et al. Discussionh Nutrition Journal (2022) 21:63 Page 8 of 10 Fig. 1 Bland-Altman plots for nutrients between FFQs and 24HDRs to assess the intake of: (A) protein, (B) energy, (C) carbohydrate, and (D) fat. The limits of agreement (dotted line) indicates the 95% confidence interval(mean ± 1.96SD) Fig. 1 Bland-Altman plots for nutrients between FFQs and 24HDRs to assess the intake of: (A) protein, (B) energy, (C) carbohydrate, and (D) fat. The limits of agreement (dotted line) indicates the 95% confidence interval(mean ± 1.96SD) Fig. 1 Bland-Altman plots for nutrients between FFQs and 24HDRs to assess the intake of: (A) protein, (B) energy, (C) carb limits of agreement (dotted line) indicates the 95% confidence interval(mean ± 1.96SD) reported in other studies [35–37]. In addition, our study collected FFQ data on household condiments commonly used, which made this dietary data more comprehen- sive than those reported in other studies [24]. In terms of results, FFQ showed acceptable reliability among participants.hfi to pay attention to their dietary intakes after the first FFQ, or it could be that over a longer span of time, the dietary of participants changed. The ICCs of most food groups and nutrients ranged from 0.53 to 0.73. After energy adjustment, most ICCs are 0.49 to 0.73. Other reliability studies have reported similar ICC [30–34]. When the food group and nutrient intake were divided into quartiles, the effective quartile coincidence rate between two FFQs was high (ranged from 72.2 to 85.9%). It is rare for all nutrient and food groups to be misplaced in the extreme quartile. Most of the weighted k values have a moderate degree of conformity, only vegetables showed poor consistency. The percentage of participants correctly classified into the same or adjacent categories was slightly higher than reported in other confirmatory studies, while the weighted K values were similar to those to pay attention to their dietary intakes after the first FFQ, or it could be that over a longer span of time, the dietary of participants changed. The ICCs of most food groups and nutrients ranged from 0.53 to 0.73. After energy adjustment, most ICCs are 0.49 to 0.73. Other reliability studies have reported similar ICC [30–34]. When the food group and nutrient intake were divided into quartiles, the effective quartile coincidence rate between two FFQs was high (ranged from 72.2 to 85.9%). Discussionh Nutrition Journal (2022) 21:63 Table 4 Validity of nutrients intakes between FFQs and 24HDRs a and b mean P value for test for difference and Spearman < 0.001 and < 0.05 respectively Notes: SFA saturated fatty acid, PUFA polyunsaturated fatty acids, MUFA monounsaturated fatty acids; Residual method was adopted for energy adjustment Nutrients FFQs 24HDRs Percentages of median difference Spearman (r) Percent of agreement(%) Median 25–75th percentiles Median 25–75th percentiles Crude Energy adjusted De-attenuated Same quartile Adjacent quartile One quartile apart Opposite quartile Energy(kcal/d) 1792.0 1475.3–2164.1 1787.0 1426.3–2146.0 0.3 0.61a – – 44.9 42.0 10.7 2.4 Carbohydrate(g/d) 235.4 189.4–299.1 272.6 202.1–328.0 −15.8a 0.63a 0.47a 0.55 48.8 37.1 12.2 1.9 Protein(g/d) 54.1 44.6–67.2 55.0 43.0–67.0 −1.7 0.58a 0.39a 0.45 38.5 45.9 13.7 1.9 Dietary fiber(g/d) 13.1 10.5–16.2 11.1 8.7–13.8 15.5a 0.46a 0.38a 0.47 41.5 38 17.6 2.9 Cholesterol(mg/d) 140.8 77.5–216.3 46.0 12.5–144.0 67.3a 0.43a 0.36a 0.44 35.6 41.5 18 4.9 Fat(g/d) 66.5 54.1–79.4 52.4 39.4–63.5 21.2a 0.55a 0.53a 0.63 43.9 37.6 15.6 2.9 SFA(g/d) 9.8 7.6–12.7 8.1 6.3–10.8 17.3a 0.35a 0.38a 0.46 39.5 42.0 14.1 4.4 MUFA(g/d) 20.9 16.5–26.0 15.8 12.5–21.1 24.4a 0.49a 0.56a 0.69 42.9 42.4 11.7 3.0 PUFA(g/d) 24.3 19.5–31.0 19.8 14.1–25.5 18.5 0.04a 0.11 0.13 36.6 49.8 10.7 2.9 Vitamin A(μgRE/d) 300.5 243.0–415.6 155.0 98.5–277.5 48.4a 0.18b 0.10 0.12 27.3 43.9 19.0 9.8 Vitamin D(μg/d) 1.0 0.6–1.9 0.0 0.0–0.6 100b 0.23b 0.08 0.10 31.7 35.1 20.5 12.7 Vitamin E(mg/d) 88.9 35.8–145.0 73.6 34.6–109.8 17.2a 0.81a 0.83a 0.98 62.4 31.7 5.4 0.5 Thiamin(mg/d) 0.8 0.7–1.1 1.1 0.8–1.4 −30.4a 0.55a 0.32a 0.37 46.8 38.0 11.2 4.0 Riboflavin(mg/d) 0.9 0.7–1.0 0.5 0.4–0.6 46.7a 0.55a 0.37a 0.45 37.6 44.4 16.6 1.4 Pyridoxine(mg/d) 0.4 0.3–0.5 0.3 0.2–0.4 21.0a 0.35a 0.22b 0.26 37.1 39.0 15.6 8.3 Vitamin C(mg/d) 115.1 90.3–148.8 81.4 54.1–114.7 29.3a 0.30a 0.16b 0.19 34.6 39.5 18.5 7.4 Folate(μg/d) 186.2 147.0–231.6 133.3 105.3–173.2 28.4a 0.31a 0.06 0.07 32.7 43.9 16.6 6.8 Niacin(mg/d) 16.0 12.4–19.4 10.4 8.3–13.1 34.6a 0.59a 0.27a 0.32 39.5 45.9 13.2 1.4 Calcium(mg/d) 449.0 359.4–535.3 263.0 199.0–326.8 41.4a 0.41a 0.33a 0.39 35.6 36.6 23.4 4.4 Phosphorus(mg/d) 917.1 762.6–1123.0 737.7 585.3–890.8 19.6a 0.59a 0.40a 0.47 46.3 37.6 15.1 1.0 Potassium(mg/d) 2000.4 1634.4–2452.6 1487.6 1181.9–1799.9 25.6a 0.48a 0.38a 0.45 43.4 36.1 18.0 2.5 Sodium(mg/d) 3961.4 3200.3–4623.2 2910.8 2431.4–3556.3 26.5a 0.53a 0.48a 0.59 42.0 38.5 16.6 2.9 Magnesium(mg/d) 320.3 277.5–398.5 258.0 202.0–311.5 19.4a 0.57a 0.40a 0.48 46.8 35.6 15.6 2.0 Iron(mg/d) 22.9 18.6–26.9 9.7 7.8–12.1 57.9a 0.45a 0.14b 0.17 37.6 40.5 19.5 2.4 Zinc(mg/d) 9.7 7.8–11.7 6.1 4.8–7.5 36.7a 0.54a 0.26a 0.31 48.3 34.6 15.6 1.5 Selenium(μg/d) 32.3 25.1–39.0 26.7 20.0–34.6 17.4a 0.59a 0.41a 0.49 42.4 42.9 12.7 2.0 Copper(mg/d) 1.6 1.3–2.0 1.2 0.9–1.5 22.3a 0.48a 0.33a 0.41 35.1 44.4 19.0 1.5 Iodine(μg/d) 40.6 27.2–62.1 21.3 15.7–28.7 47.6a 0.22b 0.10 0.12 29.8 42.4 17.6 10.2 Yan et al. Abbreviations d CVD: Cardiovascular and cerebrovascular diseases; 24HDR: 24-hour dietary recall; FFQ: Food frequency questionnaires; SFFQ: Semi-quantitative food frequency questionnaire; BMI: Body mass index; ICC: Inter-class correlation coefficient; SD: Standard deviation; WHR: Waist-to-hip ratio; LOA: Limits of Agreement. 5. Meier T, Gräfe K, Senn F, Sur P, Stangl GI, Dawczyns Ki C, et al. Cardiovascu- lar mortality attributable to dietary risk factors in 51 countries in the WHO European region from 1990 to 2016: a systematic analysis of the global burden of disease study. Eur J Epidemiol. 2019;34(1):37-55. 5. Meier T, Gräfe K, Senn F, Sur P, Stangl GI, Dawczyns Ki C, et al. Cardiovascu- lar mortality attributable to dietary risk factors in 51 countries in the WHO European region from 1990 to 2016: a systematic analysis of the global burden of disease study. Eur J Epidemiol. 2019;34(1):37-55. 6. Korakas E, Dimitriadis G, Raptis A, Lambadiari V. 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Nutrition and cardiovascular disease—an update. Curr Atheroscler Rep. 2018;20:8. 8. Brandhorst S, Longo VD. Dietary restrictions and nutrition in the preven- tion and treatment of cardiovascular disease. Circ Res. 2019;124:952–65. cardiovascular disease—an update. Curr Atheroscler Rep. 2018;20:8. 8. Brandhorst S, Longo VD. Dietary restrictions and nutrition in the preven- tion and treatment of cardiovascular disease. Circ Res. 2019;124:952–65. 8. Brandhorst S, Longo VD. Dietary restrictions and nutrition in the preven- tion and treatment of cardiovascular disease. Circ Res. 2019;124:952–65. Additional file 1. Annex. 9. He Y, Li Y, Lai J, Wang D, Zhang J, Fu P, et al. Dietary patterns as compared with physical activity in relation to metabolic syndrome among Chinese adults. Nutr Metab Cardiovasc Dis. 2013;23:920–8. 9. He Y, Li Y, Lai J, Wang D, Zhang J, Fu P, et al. Dietary patterns as compared with physical activity in relation to metabolic syndrome among Chinese adults. Nutr Metab Cardiovasc Dis. 2013;23:920–8. Author details 1 1 School of Public Health and Management, Ningxia Medical University, Yin- chuan, China. 2 Key Laboratory of Environmental Factors and Chronic Disease Control, No.1160, Shengli Street, Xingqing District, Yinchuan, Ningxia, China. Received: 7 June 2021 Accepted: 28 September 2022 Consent for publication Not applicable. Consent for publication Not applicable. Consent for publication Not applicable. Competing interests h h d l h The authors declare that they have no competing interests. Authors’ contributions Ni Yan wrote the original draft. Nan Li, Juan Li and Jiangwei Qiu conducted an investigated. Yuhong Zhang, Xiuying Liu, Pengju Zhang, Xiaoxia Li and Can Liu supervise and managed the project. The manuscript was revised by Yi Zhao. The author(s) read and approved the final manuscript. 12. Lee RD, Nieman DC. Nutritional assessment. 2nd ed; 1996. 13. Grandjean AC. Dietary intake data collection: challenges and limitations. Nutr Rev. 2012;70:S101–4. 14. Mokhtary N, Mousavi SN, Sotoudeh G, Qorbani M, Koohdani F. Associa- tion between dietary inflammatory indices (DII, EDII) and obesity with consideration of insertion/deletion Apo B polymorphism in type 2 diabetic patients. Obes Med. 2020;19:100241. 14. Mokhtary N, Mousavi SN, Sotoudeh G, Qorbani M, Koohdani F. Associa- tion between dietary inflammatory indices (DII, EDII) and obesity with consideration of insertion/deletion Apo B polymorphism in type 2 diabetic patients. Obes Med. 2020;19:100241. Discussionh It is rare for all nutrient and food groups to be misplaced in the extreme quartile. Most of the weighted k values have a moderate degree of conformity, only vegetables showed poor consistency. The percentage of participants correctly classified into the same or adjacent categories was slightly higher than reported in other confirmatory studies, while the weighted K values were similar to those The correlation coefficient of validity was less than that of reliability. It is possible that some foods were not consumed regularly and were not recorded in the 24-hour dietary recalls. The median intakes for most of the FFQ were higher than the 24-hour dietary recalls. This finding is consistent with other studies [38–40] that showed FFQ was overestimated. The study also found that the energy adjustment reduced the correla- tion coefficients of most nutrients, possibly because the nutrients intakes and types of foods varied from person Yan et al. Nutrition Journal (2022) 21:63 Page 9 of 10 Ethics approval and consent to participate This study was conducted according to the guidelines laid down in the Dec- laration of Helsinki and all procedures involving research study participants were approved by the Ethics Committee of Ningxia Medical University (No. 2020–066). Written informed consent was obtained from all subjects. It had to be admitted that this study has some limi- tations. As with all dietary assessment measures, FFQ and 24-hour dietary recall rely on self-reported data, which may be subject to some reporting bias [37]. On the other hand, only two 24-hour dietary recalls were conducted on a random day as there was no significant difference between weekdays and weekends in the diets of residents in areas in this study, and if the number is increased, the consistency between the dietary recall and FFQ will be more authentic and reliable. In addi- tion, this study only used the 24-hour dietary recall for validity study, and the results would be more convinc- ing if biomarkers were used. 13. Grandjean AC. Dietary intake data collection: challenges and limitations. Nutr Rev. 2012;70:S101–4. Funding This study was supported by the Ningxia Natural Science Foundation (No. 2020AAC03188). Yan et al. Nutrition Journal (2022) 21:63 Yan et al. Nutrition Journal (2022) 21:63 Page 10 of 10 Page 10 of 10 15. El Kinany K, Garcia-Larsen V, Khalis M, Deoula MMS, Benslimane A, Ibra- him A, et al. Adaptation and validation of a food frequency questionnaire (FFQ) to assess dietary intake in Moroccan adults. Nutr J. 2018;17:61. 37. Hollis JL, Craig LCA, Whybrow C, Kyle JAM, McNeill G. Assessing the rela- tive validity of the Scottish collaborative group FFQ for measuring dietary intake in adults. Public Health Nutr. 2017;20(3):449–55. 38. Dehghan M, Cerro SD, Zhang X, Cuneo JM, Linetzky B, Diaz R, et al. Valida- tion of a semi-quantitative food frequency questionnaire for Argentinean adults. PLoS One. 2012;7(5):e37958. 16. Naska A, Lagiou A, Lagiou P. Dietary assessment methods in epidemio- logical research: current state of the art and future prospects. F1000Res. 2017;6:926. 39. Molina MDCB, Benseor IM, Cardoso LO, Velasquez-Melendez G, Sichieri R. Reproducibility and relative validity of the food frequency questionnaire used in the ELSA-Brasil. Cad Saude Publica. 2013;29:379. 17. Leon Guerrero RT, Chong M, Novotny R, Wilkens LR, Badowski G, Blas- Laguana M, et al. Relative validity and reliability of a quantitative food fre- quency questionnaire for adults in Guam. Food Nutr Res. 2015;59:26276. 40. Dehghan M, Martinez S, Zhang X, Seron P, Lanas F. Relative validity of an FFQ to estimate daily food and nutrient intakes for Chilean adults. Public Health Nutr. 2013;16(10):1782–8. 18. Mumu SJ, Merom D, Ali L, Fahey PP, Hossain I, Rahman A, et al. Validation of a food frequency questionnaire as a tool for assessing dietary intake in cardiovascular disease research and surveillance in Bangladesh. Nutr J. 2020;19:42. 41. Zanolla AF, Olinto MTA, Henn RL, Wahrlich V, Anjos LAD. Assessment of reproducibility and validity of a food frequency questionnaire in a sample of adults living in Porto Alegre, Rio Grande do Sul state, Brazil. Cad Saude Publica. 2009;25:840. 19. Thompson FE, Subar AF. Chapter 1 - Dietary Assessment Methodology. In: Coulston AM, Boushey CJ, Ferruzzi MG, Delahanty LM, editors. Nutrition in the Prevention and Treatment of Disease. 4th ed. Cambridge: Academic Press; 2017. p. 5-48. 42. Block G, Wakimoto P, Jensen C, Mandel S, Green RR. Validation of a food frequency questionnaire for Hispanics. Prev Chronic Dis. 2006;3:A77. 20. Cade J, Thompson R, Burley V, Warm D. Publisher’s Note Reproduc- ibility and relative validity of a food frequency questionnaire developed for adults in Taizhou, China. PLoS One. 2012;7:e48341. 32. Ibiebele TI, Parekh S, Mallitt KA, Hughes MC, O’Rourke PK, Webb PM. Reproducibility of food and nutrient intake estimates using a semi-quan- titative FFQ in Australian adults. Public Health Nutr. 2009;12:2359. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? 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Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: 33. Pirjo P, Hartman AM, Eliina H, LEENA R, Jaason H, Juni P, et al. Reproduc- ibility and validity of dietary assessment instruments. I. a self-adminis- tered food use questionnaire with a portion size picture booklet. Am J Epidemiol. 1988;128(3):655-66. 34. Marques-Vidal P, Ross A, Wynn E, Rezzi S, Decarli B. Reproducibility and relative validity of a food-frequency questionnaire for French-speaking Swiss adults. Food Nutr Res. 2011;55. 35. Jackson MD, Motswagole BS, Kwape LD, Kobue-Lekalake RI, Rakgant- swana TB. Validation and reproducibility of an FFQ for use among adults in Botswana. Publisher’s Note 22. Fang M, Yin-e Z, Ya-nan JIN, Yuan Y, Yi Y, Jian-hua Z. Investigation and analysis of dietary intake status of adult residents in Ningxia [J]. Ningxia Med J. 2017;39(02):186–8. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 23. Liu Y, Li N, Yan N, Pan XF, Wu JHY. Protocol for a randomized controlled trial to test the acceptability and adherence to 6-months of walnut supplementation in Chinese adults at high risk of cardiovascular disease. Nutr J. 2021;20(1):3. 24. Zang J, Luo B, Chang S, Jin S, Wu F. Validity and reliability of a food frequency questionnaire for assessing dietary intake among Shanghai residents. Nutr J. 2019;18(1):30. 25. Koch LGG. The measurement of observer agreement for categorical data. Biometrics. 1977;33:159–74. 26. Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Int J Nurs Stud. 2010;47:931–6. 26. Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Int J Nurs Stud. 2010;47:931–6. 27. Hongmin Z, Xiang Q, Chunrong Z, Kewei M, Xiao N. Reproducibility and relative validity of a semi-quantitative food frequency questionnaire for Chinese pregnant women. Nutr J. 2015;14:56. 27. Hongmin Z, Xiang Q, Chunrong Z, Kewei M, Xiao N. Reproducibility and relative validity of a semi-quantitative food frequency questionnaire for Chinese pregnant women. Nutr J. 2015;14:56. 28. Qing Y, Xin H, Zhiyong W, Huafeng Y, Xupeng C. Reproducibility and validity of an FFQ developed for adults in Nanjing, China. Br J Nutr. 2016;115(5):887–94. 28. Qing Y, Xin H, Zhiyong W, Huafeng Y, Xupeng C. Reproducibility and validity of an FFQ developed for adults in Nanjing, China. Br J Nutr. 2016;115(5):887–94. 29. Ferreira-Sae MCS, Gallani MCB, Nadruz W, Rodrigues RC, Franchini KG, Cabral PC, et al. Reliability and validity of a semi-quantitative FFQ for sodium intake in low-income and low-literacy Brazilian hypertensive subjects. Public Health Nutr. 2009;12:2168–73. 30. Villegas R, Yang G, Liu DK, Xiang YB, Shu XO. Validity and reproducibility of the food-frequency questionnaire used in the Shanghai Men’s health study. Br J Nutr. 2007;97:993–1000. 31. Maoqiang Z, Ziyu Y, Lanfang L, Bin H, Xiaofeng W, Yajun Y, et al. Funding Development, validation and utilisation of food-frequency questionnaires – a review. Public Health Nutr. 2002;5:567–87. y 43. Barrat E, Aubineau N, Maillot M, Derbord É, Barthes P, Lescuyer J-F, et al. Repeatability and relative validity of a quantitative food-frequency ques- tionnaire among French adults. Food Nutr Res. 2012;56. 43. Barrat E, Aubineau N, Maillot M, Derbord É, Barthes P, Lescuyer J-F, et al. Repeatability and relative validity of a quantitative food-frequency ques- tionnaire among French adults. Food Nutr Res. 2012;56. 21. 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https://openalex.org/W4296027781	https://opus.bibliothek.uni-augsburg.de/opus4/files/98547/kaufmann_2022_oi_220906_1662670442.50229.pdf	English	null	Association of War Zone–Related Stress With Alterations in Limbic Gray Matter Microstructure	JAMA network open	2,022	cc-by	11,019	Original Investigation \| Psychiatry Association of War Zone–Related Stress With Alterations in Limbic Gray Matter Microstructure Original Investigation \| Psychiatry Association of War Zone–Related Stress With Alterations in Limbic Gray Matter Microstructure Elisabeth Kaufmann, MD; Philine Rojczyk, MSc; Valerie J. Sydnor, BA; Jeffrey P. Guenette, MD; Yorghos Tripodis, PhD; David Kaufmann, MD; Lisa Umminger, MD; Johanna Seitz-Holland, MD; Nico Sollmann, MD, PhD; Yogesh Rathi, PhD; Sylvain Bouix, PhD; Catherine B. Fortier, PhD; David Salat, PhD; Ofer Pasternak, PhD; Sidney R. Hinds, MD; William P. Milberg, PhD; Regina E. McGlinchey, PhD; Martha E. Shenton, PhD; Inga K. Koerte, MD Key Points Question Is war zone–related stress associated with limbic gray matter (GM) microstructure? IMPORTANCE Military service members returning from theaters of war are at increased risk for mental illness, but despite high prevalence and substantial individual and societal burden, the underlying pathomechanisms remain largely unknown. Exposure to high levels of emotional stress in theaters of war and mild traumatic brain injury (mTBI) are presumed factors associated with risk for the development of mental disorders. Findings In this cohort study of US veterans, exposure to war zone–related stress was associated with alterations in limbic GM microstructure, OBJECTIVE To investigate (1) whether war zone–related stress is associated with microstructural alterations in limbic gray matter (GM) independent of mental disorders common in this population, (2) whether associations between war zone–related stress and limbic GM microstructure are modulated by a history of mTBI, and (3) whether alterations in limbic GM microstructure are associated with neuropsychological functioning. independent of the diagnosis of mental disorder or mild traumatic brain injury. Furthermore, GM microstructure was associated with cognitive functioning. Meaning These findings suggest that war zone–related stress may lead to limbic GM microstructure alterations, which may underlie the deleterious outcomes of war zone–related stress on brain health and that military service members may benefit from early therapeutic interventions following deployment. DESIGN, SETTING, AND PARTICIPANTS This cohort study was part of the TRACTS (Translational Research Center for TBI and Stress Disorders) study, which took place in 2010 to 2014 at the Veterans Affair Rehabilitation Research and Development TBI National Network Research Center. Participants included male veterans (aged 18-65 years) with available diffusion tensor imaging data enrolled in the TRACTS study. Data analysis was performed between December 2017 to September 2021. EXPOSURES The Deployment Risk and Resilience Inventory (DRRI) was used to measure exposure to war zone–related stress. The Boston Assessment of TBI-Lifetime was used to assess history of mTBI. Stroop Inhibition (Stroop-IN) and Inhibition/Switching (Stroop-IS) Total Error Scaled Scores were used to assess executive or attentional control functions. + Supplemental content Author affiliations and article information are listed at the end of this article. + Supplemental content Author affiliations and article information are listed at the end of this article. Abstract (continued) response inhibition (Stroop-IS left cingulate: P < .001, partial r = −0.440; Stroop-IS right cingulate: P < .001, partial r = −0.372; Stroop-IS left medial orbitofrontal cortex: P < .001, partial r = −0.304; Stroop-IS right medial orbitofrontal cortex: P < .001, partial r = −0.340; Stroop-IN left cingulate: P < .001, partial r = −0.421; Stroop-IN right cingulate: P < .001, partial r = −0.300; Stroop-IN left medial orbitofrontal cortex: P = .01, partial r = −0.223; Stroop-IN right medial orbitofrontal cortex: P < .001, partial r = −0.343), whereas higher FAT in the mesial temporal regions was associated with improved short-term memory and processing speed (left amygdala-hippocampus complex: P < .001, partial r = −0.574; right amygdala-hippocampus complex: P < .001, partial r = 0.645; short-term memory left amygdala-hippocampus complex: P < .001, partial r = 0.570; short-term memory right amygdala-hippocampus complex: P < .001, partial r = 0.633). A history of mTBI did not modulate the association between war zone–related stress and GM diffusion. CONCLUSIONS AND RELEVANCE This study revealed an association between war zone–related stress and alteration of limbic GM microstructure, which was associated with cognitive functioning. These results suggest that altered limbic GM microstructure may underlie the deleterious outcomes of war zone–related stress on brain health. Military service members may benefit from early therapeutic interventions after deployment to a war zone. JAMA Network Open. 2022;5(9):e2231891. doi:10.1001/jamanetworkopen.2022.31891 MAIN OUTCOMES AND MEASURES Diffusion characteristics (fractional anisotropy of tissue [FAT]) of 16 limbic and paralimbic GM regions and measures of functional outcome. MAIN OUTCOMES AND MEASURES Diffusion characteristics (fractional anisotropy of tissue [FAT]) of 16 limbic and paralimbic GM regions and measures of functional outcome. RESULTS Among 384 male veterans recruited, 168 (mean [SD] age, 31.4 [7.4] years) were analyzed. Greater war zone–related stress was associated with lower FAT in the cingulate (DRRI-combat left: P = .002, partial r = −0.289; DRRI-combat right: P = .02, partial r = −0.216; DRRI-aftermath left: P = .004, partial r = −0.281; DRRI-aftermath right: P = .02, partial r = −0.219), orbitofrontal (DRRI- combat left medial orbitofrontal cortex: P = .02, partial r = −0.222; DRRI-combat right medial orbitofrontal cortex: P = .005, partial r = −0.256; DRRI-aftermath left medial orbitofrontal cortex: P = .02, partial r = −0.214; DRRI-aftermath right medial orbitofrontal cortex: P = .005, partial r = −0.260; DRRI-aftermath right lateral orbitofrontal cortex: P = .03, partial r = −0.196), and parahippocampal (DRRI-aftermath right: P = .03, partial r = −0.191) gyrus, as well as with higher FAT in the amygdala-hippocampus complex (DRRI-combat: P = .005, partial r = 0.254; DRRI-aftermath: P = .02, partial r = 0.223). Lower FAT in the cingulate-orbitofrontal gyri was associated with impaired Open Access. This is an open access article distributed under the terms of the CC-BY License. September 16, 2022 1/17 JAMA Network Open. 2022;5(9):e2231891. doi:10.1001/jamanetworkopen.2022.31891 ( Downloaded From: https://jamanetwork.com/ by a Universitaet Augsburg User on 10/10/2022 Downloaded From: https://jamanetwork.com/ by a Universitaet Augsburg User on 10/10/2022 Association of War Zone–Related Stress With Alterations in Limbic Gray Matter Microstructure JAMA Network Open \| Psychiatry Introduction Military personnel serving in theaters of war are at increased risk for physical and mental health problems following deployment.1-3 Mental health–related disorders are pervasive; up to 30% of service members returning from Operation Enduring Freedom (OEF), Operation Iraqi Freedom (OIF), or Operation New Dawn (OND) receive a diagnosis of a mental illness, such as posttraumatic stress disorder (PTSD), anxiety, or depression.4-6 Known factors associated with postdeployment mental disorders include combat exposure and associated psychosocial stressors.7-9 Importantly, service members exhibit symptoms related to war zone stress and experience low quality of life even if they do not meet the diagnostic criteria for a mental disorder.10 Furthermore, despite the prevalence and adversity of war zone–related stress, the majority of previous studies have not specifically investigated the impact of war zone–related stress, and even fewer have used quantitative questionnaires such as the Deployment Risk and Resilience Inventory (DRRI) to quantify perceived war zone–related stress.11-14 Although mental health problems are highly prevalent in postdeployed military service members15 and war zone–related stress has been discussed as a risk factor, the underlying pathomechanisms remain poorly understood. Furthermore, approximately 12% to 35% of OEF, OIF, and OND veterans have sustained a mild traumatic brain injury (mTBI).16-19 Evidence suggests that mTBI is not only a highly prevalent comorbidity but is also considered a potential risk factor for the development of mental disorders. In fact, service members who have sustained mTBI have a significantly increased risk for developing PTSD1,16,20-22 and depression.1,23,24 Moreover, they exhibit poorer neurocognitive functioning, worse long-term recovery,25 and more severe neurological impairment26,27 compared with those who have not sustained mTBI. However, it is unknown whether comorbidity with mTBI modulates a possible association between war zone–related stress and alterations of brain structure and neuropsychological functioning. A better understanding of the outcomes of war zone–related stress on brain microstructure and function is critical for improving long-term health and quality of life of military service members returning from theaters of war. Magnetic resonance imaging (MRI) provides a noninvasive way to study brain alterations as it allows for the in vivo, 3-dimensional investigation of brain macrostructure and microstructure.28 Neuroimaging studies have linked neuropsychiatric disorders, including PTSD and mTBI, to September 16, 2022 2/17 JAMA Network Open. 2022;5(9):e2231891. doi:10.1001/jamanetworkopen.2022.31891 Downloaded From: https://jamanetwork.com/ by a Universitaet Augsburg User on 10/10/2022 Methods This cohort study was approved by the institutional review board of human studies research at the Veterans Affair Boston Healthcare System and all participants provided written informed consent. The study follows the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline for observational studies. Downloaded From: https://jamanetwork.com/ by a Universitaet Augsburg User on 10/10/2022 Downloaded From: https://jamanetwork.com/ by a Universitaet Augsburg User on 10/10/2022 Association of War Zone–Related Stress With Alterations in Limbic Gray Matter Microstructure JAMA Network Open \| Psychiatry macrostructural brain alterations.29 However, although an association between diagnoses and abnormal brain structure has been established, research on the outcomes of war zone–related stress on brain structure is sparse. Combat exposure has been found to be associated with lower volume of limbic or limbic-associated gray matter (GM) regions, such as the amygdala,30 hippocampus,31,32 orbitofrontal gyrus,33 posterior insula,34 ventromedial prefrontal cortex, and dorsal anterior cingulate cortex.35 Of note, although lower limbic GM volumes have been associated with PTSD symptom severity and extent of alcohol use, other disorders commonly seen in this population have previously not been considered. Diffusion-weighted MRI (dMRI) has been shown to be sensitive to subtle microstructural brain alterations associated with neuropsychiatric disorders, such as PTSD and mTBI.29 Complementary to volumetric measures, dMRI has the potential to reveal alterations in tissue composition (eg, glial changes36-38 and atrophy39) and tissue morphologic changes (eg, alterations in dendritic arborization40-42), thereby providing insight into underlying pathomechanisms. Although most research to date has focused on the microstructure of connecting white matter (WM) fiber tracts,43-46 studies on the limbic GM microstructure are sparse. Importantly, to our knowledge, no study to date has investigated the association between combat exposure and limbic GM diffusion, although limbic GM constitutes an essential neuroanatomical correlate of mental and neuropsychological functioning as suggested previously by volumetric studies31,32,47 of limbic system structures in postdeployed veterans. The aim of this study is to investigate (1) whether war zone– related stress is associated with microstructural alterations in limbic GM independent of mental disorders, (2) whether associations between war zone–related stress and limbic GM microstructure are modulated by a history of mTBI, and (3) whether alterations in limbic system GM microstructure are associated with neuropsychological functioning. JAMA Network Open. 2022;5(9):e2231891. doi:10.1001/jamanetworkopen.2022.31891 Assessment of Psychiatric Disorders The nonpatient edition of the Structured Clinical Interview for DSM-IV Axis I Disorders (SCID-I/NP)49 was used to detect the presence of psychopathological disorders. The following modules were administered: module D, mood disorders; module E, substance use disorders; module F, anxiety disorders (except PTSD); module H, eating disorders; and module I, adjustment disorders. Presence and history of PTSD were determined according to the Clinician-Administered PTSD Scale (CAPS)50 using the Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition) (DSM-IV) standard scoring rule.51 Assessment of War Zone–Related Stress Stressors associated with deployment to war zones were assessed via selected scales from the DRRI.52 The combat experiences and aftermath of battle scales were used to assess perceived war zone–related stress. Both DRRI subscales (called hereafter DRRI-combat and DRRI-aftermath) consist of 16 questions concerning combat or war zone–related events. The DRRI-combat uses a 5-point Likert frequency scale (0 = never; 4 = daily or almost daily), yielding a maximum possible score of 64 points. The DRRI-aftermath scale uses a binary response (0 = no and 1 = yes), resulting in a maximum score of 16 points. Higher scores on both the DRRI-combat and DRRI-aftermath scale reflect greater exposure to deployment-related stressors. Assessment of mTBI The Boston Assessment of TBI-Lifetime (BAT-L)17 was conducted to diagnose lifetime history of TBI. Specifically, mTBI was defined by the following criteria: loss of consciousness for 30 minutes or less, posttraumatic amnesia for 24 hours or less, or altered mental status for 24 hours or less.17 Participants The Translational Research Center for TBI and Stress Disorders (TRACTS) study is a longitudinal prospective cohort study that aims to assess and track the potential outcomes of psychologically and physically traumatic experiences related to military deployment over time. Inclusion criteria for enrollment into the TRACTS study were (1) age 18 to 65 years, (2) male sex, and (3) service in OEF, OIF, or OND, or scheduled deployment.48 Exclusion criteria were (1) history of neurological illness other than TBI; (2) current diagnosis of schizophrenia spectrum or other psychotic disorders; (3) current diagnosis of bipolar or related disorders; (4) active suicidal and/or homicidal ideation, intent, or plan requiring crisis intervention; and (5) cognitive disorder due to general medical condition other than TBI. Parameters with potential impact on cerebral microstructure and resilience such as eduction, socioeconomic status, race and ethnicity were collected via interview. Of the first 384 consecutively recruited veterans, 273 consented to share their data with investigators outside of TRACTS. Of these 273 veterans, several had to be excluded from the present study for the following reasons: predeployment status (ie, military service members who had not yet been deployed to combat zones) (15 participants), postenrollment report of neurological disorders (ie, history of meningitis, or brain surgery; 4 participants), history of moderate or severe TBI (15 participants), and exposure to neurotoxic chemicals or anoxia (30 participants). Another 26 cases did not pass the rigorous quality control of the MRI data, and 15 cases had missing clinical variables required for this study. The selection process is summarized in Figure 1. JAMA Network Open. 2022;5(9):e2231891. doi:10.1001/jamanetworkopen.2022.31891 September 16, 2022 3/17 Downloaded From: https://jamanetwork.com/ by a Universitaet Augsburg User on 10/10/2022 Downloaded From: https://jamanetwork.com/ by a Universitaet Augsburg User on 10/10/2022 Association of War Zone–Related Stress With Alterations in Limbic Gray Matter Microstructure JAMA Network Open \| Psychiatry MRI indicates magnetic resonance imaging; TBI traumatic brain injury; TRACTS, Translational Research Center for TBI and Stress Disorders; VA, Veterans Affairs. Assessment of Functional Outcome The World Health Organization Disability Assessment Schedule II (WHODAS II)53 is a 36-item self- report questionnaire that was designed to measure disability associated with all physical and mental disorders including cognition, mobility, self-care, getting along, life activities, and participation. Functional impairments within the last 30 days are rated on a 5-point scale (0 = no disability; Figure 1. Flowchart of the Cohort Selection Process 384 Veterans enrolled in TRACTS 273 Veterans available for study 111 Excluded did not agree to share the data with investigators outside of VA 209 Clinically preselected cohort 15 Missing clinical variables required for the study analyses 194 Full data sets 168 Final study cohort 26 Excluded insufficient MRI quality 64 Excluded 15 Predeployment status 15 History of moderate or severe TBI 4 Postenrollment report of neurological disorders 30 Exposure to neurotoxic chemicals or anoxia MRI indicates magnetic resonance imaging; TBI traumatic brain injury; TRACTS, Translational Research Center for TBI and Stress Disorders; VA, Veterans Affairs. JAMA Network Open. 2022;5(9):e2231891. doi:10.1001/jamanetworkopen.2022.31891 (Reprinted) September 16, 2022 4/17 Figure 1. Flowchart of the Cohort Selection Process 384 Veterans enrolled in TRACTS 273 Veterans available for study 111 Excluded did not agree to share the data with investigators outside of VA 209 Clinically preselected cohort 15 Missing clinical variables required for the study analyses 194 Full data sets 168 Final study cohort 26 Excluded insufficient MRI quality 64 Excluded 15 Predeployment status 15 History of moderate or severe TBI 4 Postenrollment report of neurological disorders 30 Exposure to neurotoxic chemicals or anoxia JAMA Network Open. 2022;5(9):e2231891. doi:10.1001/jamanetworkopen.2022.31891 (Reprinted) Figure 1. Flowchart of the Cohort Selection Process 111 Excluded did not agree to share the data with investigators outside of VA MRI indicates magnetic resonance imaging; TBI traumatic brain injury; TRACTS, Translational Research Center for TBI and Stress Disorders; VA, Veterans Affairs. September 16, 2022 4/17 ork Open. 2022;5(9):e2231891. doi:10.1001/jamanetworkopen.2022.3 Downloaded From: https://jamanetwork.com/ by a Universitaet Augsburg User on 10/10/2022 Downloaded From: https://jamanetwork.com/ by a Universitaet Augsburg User on 10/10/2022 Association of War Zone–Related Stress With Alterations in Limbic Gray Matter Microstructure JAMA Network Open \| Psychiatry JAMA Network Open \| Psychiatry 4 = extreme disability/cannot do). A total disability score is calculated by summing the scores across all subscales. Higher scores reflect greater disability. Effort Testing Performance validity was assessed via the Verbal Multiple Symptom Validity Test (MSVT).63 The MSVT evaluates verbal learning, memory, and response consistency. It is composed of the subtests immediate recall, delayed recognition, consistency of responding across immediate recall, and delayed recognition, as well as paired associates and free recall. Study participants who failed the MSVT (8 participants) were excluded from the post hoc analyses as they were suspected of potential reduced effort or malingering. Assessment of Functional Outcome The Neurobehavioral Symptom Inventory (NSI) is a 22-item self-report questionnaire used to assess postconcussion symptoms following TBI.54 Tested symptoms include sensory, affective, vestibular, and cognitive symptoms, rated on a 5-point Likert scale (0 = none; 4 = very severe). Higher scores reflect more severe neurobehavioral symptoms. According to identified limbic regions with GM diffusion alterations, the Digit Span Total Score (DSTot) and the Coding Raw Scores55 were chosen from the comprehensive neuropsychological test battery,48 as they reflect functions of the frontal and temporal lobe (ie, verbal short-term memory performance and processing speed). In addition, Stroop Inhibition (Stroop-IN) and Inhibition/ Switching (Stroop-IS) Total Error Scaled Scores56 were selected to assess more specifically executive or attentional control functions associated with the prefrontal and cingulate cortex,57-62 whereby higher Total Error Scaled Scores reflect impaired response inhibition and vice versa. Assessment of Hypervigilance The CAPS criterion D was used to assess the frequency and intensity of symptoms of hypervigilance at postdeployment, including difficulty sleeping, irritability, difficulty concentrating, hypervigilance, and exaggerated startle response. Answers were rated on a 5-point Likert scales ranging from 0 to 4 and summarized in a total score, resulting in a maximum score of 40 points. MRI Acquisition and Data Processing Amygdala and hippocampus were combined into 1 region of interest to ensure higher parcellation accuracy.69 For each of these 8 bihemispheric regions of interest (16 in total), the mean of the diffusion measure (FAT) was calculated. paralimbic GM regions in each hemisphere were evaluated—that is, cingulate gyrus, amygdala- hippocampus complex, parahippocampal gyrus, entorhinal cortex, lateral and medial orbitofrontal cortex, insula, and temporal pole. Amygdala and hippocampus were combined into 1 region of interest to ensure higher parcellation accuracy.69 For each of these 8 bihemispheric regions of interest (16 in total), the mean of the diffusion measure (FAT) was calculated. Results The final study cohort encompassed 168 male veterans with a mean (SD) age of 31.4 (7.4) years. Sample demographic characteristics are summarized in Table 1. The vast majority of participants were White (130 participants [77%]), followed by 24 Hispanic participants (14%) and 11 Black participants (6%) (Table 1). Although the level of education was balanced across the cohort (mean [SD] 13.9 [1.9] school years), potentially relevant differences were observed for the family status as only 38% (64 participants) were married or cohabiting. Statistical Analysis Statistical analyses were performed using SAS statistical software version 9.4 (SAS Institute). Means and SDs are displayed for continuous parameters, while absolute and relative frequencies are provided for noncontinuous variables. Generalized linear models for repeated measures using the restricted maximum likelihood approach and an unstructured covariance matrix across brain regions were used to evaluate the association of war zone–related stress with regional diffusion measures. The following parameters were selected a priori as covariates: age, diagnosis of current PTSD, mood, anxiety, substance use disorder, and weight-corrected lifetime drinking history (LDH). To test the outcomes of mTBI on the association between war zone–related stress and limbic GM diffusion, the number of lifetime mTBIs was added as fixed effect as well as modifier to the main effect. Post hoc analyses were conducted to test for associations between diffusion measures that were significantly associated with war zone stress and neurobehavioral symptoms (NSI), cognitive (DSTot, Coding Raw Score, and Stroop IN/IS Total error scaled score), and disability (WHODAS). Participants who failed error testing (MSVT) were excluded from the post hoc analyses. Age, diagnosis of current PTSD, mood, anxiety, and substance use disorder, and LDH were included as covariates. A false discovery rate70 was set at 5% to correct for multiple comparisons, using the Benjamini- Hochberg method. A corrected 2-tailed P < .05 was considered significant. Data were analyzed December 2017 to September 2021. Associations of War Zone–Related Stress With Limbic GM Diffusion In the cohort of 168 veterans, greater war zone–related stress as assessed by DRRI-combat and DRRI- aftermath was negatively associated with FAT in the bilateral cingulate gyri (DRRI-combat left: P = .002, partial r = −0.289, df = 167; DRRI-combat right: P = .02, partial r = −0.216, df = 167; DRRI- aftermath left: P = .004, partial r = −0.281, df = 167; DRRI-aftermath right: P = .02, partial r = −0.219, df = 167) and bilateral medial orbitofrontal gyri (DRRI-combat left medial orbitofrontal cortex: P = .02, partial r = −0.222, df = 167; DRRI-combat right medial orbitofrontal cortex: P = .005, partial r = −0.256, df = 167; DRRI-aftermath left medial orbitofrontal cortex: P = .02, partial r = −0.214, df = 167; DRRI-aftermath right medial orbitofrontal cortex: P = .005, partial r = −0.260, df = 167; DRRI-aftermath right lateral orbitofrontal cortex: P = .03, partial r = −0.196, df = 167). Notably, these associations were observed while controlling for age, PTSD diagnosis, mood disorder, anxiety disorder, and substance use disorder as well as LDH. Moreover, a negative association was observed between DRRI-aftermath and the right lateral orbitofrontal gyrus FAT and right parahippocampal gyrus FAT (P = .03, partial r = −0.191, df = 167). In contrast, a positive association was found for both measures of war zone–related stress and FAT in September 16, 2022 6/17 JAMA Network Open. 2022;5(9):e2231891. doi:10.1001/jamanetworkopen.2022.31891 Downloaded From: https://jamanetwork.com/ by a Universitaet Augsburg User on 10/10/2022 MRI Acquisition and Data Processing MRI of the brain was performed using a 3-Tesla TIM Trio scanner (Siemens Healthineers) located at the VA Medical Center in Boston, Massachusetts. T1-weighted (T1w) gradient-echo sequence parameters were field of view, 256 mm; 256 sections; inversion time, 1.000 ms; repetition time, 2.530 ms; echo time, 3.32 ms; flip angle, 7°; and isotropic resolution, 1 × 1 × 1 mm3. dMRI was acquired using a single-shot, echo-planar sequence with a twice-refocused spin-echo pulse and the following parameters: field of view, 256 mm; 64 axial sections with no intersection gap; 60 gradient directions with a b-value of 700 seconds/mm2; 10 b = 0 volumes; repetition time, 10 000 ms; echo time, 103 ms; and isotropic resolution, 2 × 2 × 2 mm3. dMRI data were corrected for motion and eddy current distortions via affine registration to the first b = 0 volume using FMRIB Software Library, version 5.1 (The Oxford Centre for Functional MRI of the Brain).64 Brain masks were created and manually edited in 3D Slicer, version 4.5 (Surgical Planning Laboratory, Brigham and Women´s Hospital).65 Automated segmentation of brain regions from the T1w data was performed using FreeSurfer66 (version 5.1.0).67 Free water (FW)–corrected diffusion tensor measures were derived from dMRI using in-house software.68 FW imaging separates the dMRI signal into 2 compartments: a FW and a tissue compartment. FW in the brain is expected where water molecules are free to diffuse, such as in cerebrospinal fluid, and large extracellular spaces. We calculated a fractional anisotropy of tissue (FAT) map from the FW-corrected diffusion tensor, which serves as a more accurate marker of anisotropy in brain tissue than the conventional FA measure. To obtain diffusion metrics for selected regions, FreeSurfer parcellation label maps were nonlinearly registered from the individual T1w space to the respective dMRI space to obtain diffusion metrics for selected regions. Eight limbic and JAMA Network Open. 2022;5(9):e2231891. doi:10.1001/jamanetworkopen.2022.31891 September 16, 2022 5/17 k Open. 2022;5(9):e2231891. doi:10.1001/jamanetworkopen.2022.318 Downloaded From: https://jamanetwork.com/ by a Universitaet Augsburg User on 10/10/2022 Downloaded From: https://jamanetwork.com/ by a Universitaet Augsburg User on 10/10/2022 JAMA Network Open \| Psychiatry Association of War Zone–Related Stress With Alterations in Limbic Gray Matter Microstructure paralimbic GM regions in each hemisphere were evaluated—that is, cingulate gyrus, amygdala- hippocampus complex, parahippocampal gyrus, entorhinal cortex, lateral and medial orbitofrontal cortex, insula, and temporal pole. Downloaded From: https://jamanetwork.com/ by a Universitaet Augsburg User on 10/10/2022 Downloaded From: https://jamanetwork.com/ by a Universitaet Augsburg User on 10/10/2022 Association of War Zone–Related Stress With Alterations in Limbic Gray Matter Microstructure JAMA Network Open \| Psychiatry Association of War Zone–Related Stress With Alterations in Limbic Gray Matter Microstructure the right amygdala-hippocampus complex (DRRI-combat: P= .005, partial r = 0.254, df = 167; DRRI- aftermath: P = .02, partial r = 0.223, df = 167). Results are summarized in Table 2. Outcomes of mTBI on the Association of War Zone–Related Stress and Limbic GM Diffusion The majority of veterans (109 of 168 [64.9%]) sustained at least 1 mTBI before or during deployment. They reported having experienced a mean (SD) of 1.38 (2.23) mTBIs throughout life with a maximum number of 18 mTBIs. Number of lifetime mTBIs was not associated with limbic GM diffusion and did not mediate the association between war zone–related stress and limbic GM FAT. Table 1. Demographics, Deployment-Related Factors, and Postdeployment Characteristics of Study Cohort Variable Participants, No. (%) (N = 168) Demographics Age, mean (SD), y 31.36 (7.43) Race and ethnicity Asian 2 (1.19) Black 11 (6.55) Hispanic 24 (14.29) Unknown 1 (0.60) White 130 (77.38) Education mean (SD), school years 13.86 (1.93) Married or cohabitating 64 (38.10) Deployment factors OEF, OIF, or OND deployments, mean (SD), No. 1.4 (0.7) Other stressful deployments, mean (SD), No. 0.41 (0.79) Duration of OEF, OIF, or OND deployments, mean (SD), mo 13.82 (8.45) Service in army branch 101 (60.12) DRRI total score, mean (SD) Combat experience (DRRI-combat) 17.31 (12.02) Aftermath exposure (DRRI-aftermath) 7.65 (4.7) Military mTBIs, mean (SD), No. 0.63 (1.53) Wounded or injured in combat 35 (20.83) Postdeployment characteristics Time since last deployment, mean (SD), mo 40.07 (29.98) Disorder Mood 35 (20.83) Anxiety 28 (16.67) PTSD diagnosis 112 (66.67) Clinician-Administered PTSD Scale, mean (SD)a 78.35 (22.9) Substance use disorder 25 (14.88) Lifetime drinking history, weight corrected, mean (SD) 1790.6 (2092.7) Lifetime TBIs, mean (SD) 1.38 (2.23) Abbreviations: DRRI, Deployment Risk and Resilience Inventory; mTBI, mild traumatic brain injury; OEF, Operation Enduring Freedom; OIF, Operation Iraqi Freedom; OND, Operation New Dawn; PTSD, posttraumatic stress disorder; TBI, traumatic brain injury. a Clinician-Administered PTSD Scale score was evaluated for 112 veterans who met diagnostic criteria for postdeployment PTSD. September 16, 2022 7/17 Abbreviations: DRRI, Deployment Risk and Resilience Inventory; FDR, false b Denotes significant results. Association of Limbic GM Diffusion and Functional Outcome Results of the post hoc analysis of diffusion and associated functioning are shown in Table 3. Decreased FAT in the cingulate gyri and the medial orbitofrontal cortex was associated with impaired response inhibition (Stroop-IS left cingulate: P < .001, partial r = −0.440, df = 151; Stroop-IS right cingulate: P < .001, partial r = −0.372, df = 151; Stroop-IS left medial orbitofrontal cortex: P < .001, partial r = −0.304, df = 151; Stroop-IS right medial orbitofrontal cortex: P < .001, partial r = −0.340, df = 151; Stroop-IN left cingulate: P < .001, partial r = −0.421, df = 151; Stroop-IN right cingulate: P < .001, partial r = −0.300, df = 151; Stroop-IN left medial orbitofrontal cortex: P = .01, partial r = −0.223; df = 151; Stroop-IN right medial orbitofrontal cortex: P < .001, partial r = −0.343, df = 151), but with better frontotemporal functions (DSTot left amygdala-hippocampus complex: P < .001, partial r = −0.574, df = 159; DSTot right amygdala-hippocampus complex: P < .001, partial r = 0.645, df = 159; short-term memory left amygdala-hippocampus complex: P < .001, partial r = 0.570, df = 156; short-term memory right amygdala-hippocampus complex: P < .001, partial r = 0.633, df = 156). In contrast, impaired response inhibition and improved verbal short-term memory Table 2. Association of War Zone–Related Stress and Limbic Gray Matter Diffusion Using Fractional Anisotropy of Tissue Region Combat exposure (DRRI-combat) Aftermath exposure (DRRI-aftermath) Left hemisphere Right hemisphere Left hemisphere Right hemisphere Partial ra FDR corrected P value Partial ra FDR corrected P value Partial ra FDR corrected P value Partial ra FDR corrected P value Amygdala-hippocampus complex 0.158 .09 0.254 .005b 0.136 .14 0.224 .02b Cingulate gyrus −0.289 .002b −0.216 .02b −0.281 .004b −0.219 .02b Entorhinal cortex 0.020 .80 0.121 .21 −0.023 .88 0.049 .65 Insular cortex −0.058 .52 −0.057 .52 −0.138 .14 −0.061 .57 Lateral orbitofrontal cortex −0.081 .43 −0.151 .10 −0.083 .41 −0.196 .03b Medial orbitofrontal cortex −0.222 .02b −0.256 .005b −0.214 .02b −0.260 .005b Parahippocampal gyrus −0.059 .52 −0.166 .08 −0.009 .97 −0.191 .03b Temporal pole −0.089 .40 0.053 .52 −0.224 .39 −0.003 .97 Table 2. Association of War Zone–Related Stress and Limbic Gray Matter Diffusion Using Fractional Anisotropy of Tissue a The higher the partial r, the stronger the linear association between 2 variables. Positive values represent positive correlations, and negative values represent negative or inverse correlations. Downloaded From: https://jamanetwork.com/ by a Universitaet Augsburg User on 10/10/2022 Downloaded From: https://jamanetwork.com/ by a Universitaet Augsburg User on 10/10/2022 Association of War Zone–Related Stress With Alterations in Limbic Gray Matter Microstructure JAMA Network Open \| Psychiatry b Denotes significant results. a The higher the partial r, the stronger the linear association between 2 variables. Abbreviations: DRRI, Deployment Risk and Resilience Inventory; FDR, false discovery rate. b Denotes significant results b Denotes significant results. Positive values represent positive, and negative values represent negative or inverse correlations a The higher the partial r, the stronger the linear association between 2 variables. Positive values represent positive, and negative values represent negative or inverse y Abbreviations: DRRI, Deployment Risk and Resilience Inventory; FDR, false discovery rate. b Denotes significant results. a The higher the partial r, the stronger the linear association between 2 variables. Positive values represent positive, and negative values represent negative or inverse correlations b Denotes significant results. JAMA Network Open. 2022;5(9):e2231891. doi:10.1001/jamanetworkopen.2022.31891 Association of Limbic GM Diffusion and Functional Outcome Table 3. Association of Limbic Gray Matter Diffusion Using Fractional Anisotropy of Tissue and Cognitive Functioning Table 3. Association of Limbic Gray Matter Diffusion Using Fractional Anisotropy of Tissue and Cognitive Functioning Region Digit Span Total Score Coding Raw Score Stroop inhibition Total error scaled score Switching total error scaled score Partial ra FDR corrected P value Partial ra FDR corrected P value Partial ra FDR corrected P value Partial ra FDR corrected P value Left amygdala-hippocampus comp 0.574 <.001b 0.570 <.001b 0.443 <.001b 0.483 <.001b Left cingulate gyrus −0.393 <.001b −0.330 <.001b −0.421 <.001b −0.440 <.001b Left lateral orbitofrontal cortex −0.058 .74 −0.006 .94 −0.036 .79 −0.044 .78 Left medial orbitofrontal cortex −0.202 .02b −0.193 .03b −0.223 .01b −0.304 <.001b Left parahippocampal gyrus 0.042 .80 0.007 .94 0.059 .79 0.013 .95 Right amygdala-hippocampus comp 0.645 <.001b 0.633 <.001b 0.500 <.001b 0.518 <.001b Right cingulate gyrus −0.290 <.001b −0.237 .007b −0.300 <.001b −0.372 <.001b Right lateral orbitofrontal cortex 0.041 .80 0.024 .76 −0.038 .79 0.005 .95 Right medial orbitofrontal cortex −0.263 .002b −0.262 .003b −0.343 <.001b −0.340 <.001b Right parahippocampal gyrus −0.001 .99 −0.021 .79 −0.032 .79 −0.103 .35 JAMA Network Open. 2022;5(9):e2231891. doi:10.1001/jamanetworkopen.2022.31891 September 16, 2022 8/17 Abbreviation: FDR false discovery rate. b Denotes significant results. e. the partial r the stronger the linear association between 2 variables Discussion This cohort study found an association between war zone–related stress and microstructure of limbic GM in veterans. Importantly, these findings were observed while accounting for common comorbidities, including PTSD, mood, anxiety, and substance use disorder. Furthermore, mTBI had no significant effect on the association between war zone–related stress and limbic GM microstructure. Finally, characteristics of limbic GM microstructure were associated with cognitive performance including verbal short-term memory, processing speed, and response inhibition, while no associations with overall disability and neurobehavioral symptoms were found. Downloaded From: https://jamanetwork.com/ by a Universitaet Augsburg User on 10/10/2022 JAMA Network Open \| Psychiatry Association of War Zone–Related Stress With Alterations in Limbic Gray Matter Microstructure performance and processing speed were associated with increased FAT in the amygdala- hippocampal region (Figure 2). No significant associations were revealed for limbic GM diffusion and (postconcussion) neurobehavioral symptoms or disability (eTable 1 in the Supplement). Association of Limbic GM Diffusion and Hypervigilance State Hypervigilance at postdeployment was positively associated with FAT in the amygdala-hippocampal region (left: P < .001, partial r = 0.325, df = 165; right: P <.001, partial r = 0.309; df = 165) and negatively associated with FAT in the cingulate gyri (left: P < .01, partial r = −0.253 df = 165; right: P < .01; partial r = −0.261 df = 165). The results are summarized in eTable 2 in the Supplement. Association Between Limbic GM Diffusion and Functional Outcome We observed improved frontotemporal brain functions (ie, short-term memory and processing speed) in association with increased FAT in the amygdala-hippocampal complex (Figure 2), which is in line with previous studies that report a link between processing speed and hippocampal FA.78-80 Our study results further suggest an association between improved frontotemporal brain functions with war zone–related stress.81 It has been hypothesized that hypervigilance and readiness to respond to combat-related challenges may be advantageous adaptations to the highly stressful environment. However, it may be challenging to transition back to normal states of alertness when returning from deployment. The chronic activated state may consequently lead to a functional overuse of frontotemporal brain functions. This overuse may induce neuroplastic changes as suggested by the increased FAT in the amygdala-hippocampal complex73 found in this study. This hypothesis is supported by our finding of a significant association between hypervigilance state at postdeployment and increased FAT in the amygdala-hippocampal complex. At the same time, we observed impaired prefrontal-cingulate functions (response inhibition) in association with lower FAT in prefrontal regions. This is thought to result from functional (emotional or stress) overuse of mesial temporal structures, as described previously, which may, in turn, lead to poorer performance in other cognitive tasks, a phenomenon called interference.82-84 Interference or shift of emotion and cognition has previously been described in patients with PTSD85 as well as in veterans. More specifically, impaired memory consolidation and reduced learning speed were observed in veterans returning from OEF, OIF, or OND.86,87 Of note, those functions are typically associated with the prefrontal-cingulate cortex,86-89 regions that have been found to have lower FAT in association with war zone stress in the current study. Taken together, we hypothesize that the outcomes of war zone–related stress outlast deployment, leading to attentional interference with increased functional use of mesial temporal structures and decreased use or impaired retrieval of prefrontal-cingulate functions. This hypothesis is further supported by functional MRI studies,90 which have reported a hypoconnectivity of mesial temporal and prefrontal brain regions under conditions of stress. The functional interference may, in turn, lead to microstructural adaptations, reflected by increased FAT in the amygdala-hippocampus complex and decreased FAT in the cingulate and orbitofrontal gyri (Figure 2). War Zone–Related Stress and Limbic GM Diffusion This study revealed a co-occurring decrease and increase in limbic GM FAT. More specifically, the greater the experienced war zone–related stress, the lower FAT was in the cingulate gyri, the medial orbitofrontal gyri, the right lateral orbitofrontal gyrus, and the right parahippocampal gyrus. Moreover, the greater the experienced war zone–related stress, the higher the FAT in the amygdala- hippocampus complex. Importantly, associations described previously were independent of diagnosis of mental disorders as well as mTBI. The interpretation of diffusion measures in GM is challenging as data linking diffusion to histologic profile is sparse.37,71-74 FA in GM likely reflects diffusion properties of the main GM components (ie, astroglia, neurons, and axons). For example, a study in mice reveals an association Figure 2. Model of Structural Brain Alterations and Associated Cognitive Function in the Context of War Zone–Related Stress Figure 2. Model of Structural Brain Alterations and Associated Cognitive Function in the Context of War Zone–Related Stress Figure 2. Model of Structural Brain Alterations and Associated Cognitive Function in the Context of War Zone–Related Stress Increase in FAT and processing speed and verbal short-term memory Amygdala-hippocampus complex Cingulate gyrus Decrease in FAT and response inhibition Orbitofrontal gyrus Decrease in FAT War zone–related JAMA Network Open. 2022;5(9):e2231891. doi:10.1001/jamanetworkopen.2022.31891 (Reprinted) Increase in FAT and processing speed and verbal short-term memory Amygdala-hippocampus complex Cingulate gyrus Decrease in FAT and response inhibition Orbitofrontal gyrus Decrease in FAT War zone–related War zone–related stress was associated with decreased fractional anisotropy of tissue (FAT) in cingulate and orbitofrontal cortex, as well as increased FAT in the amygdala-hippocampus complex. Limbic gray matter FAT measures were further associated with cognitive function (ie, impaired response inhibition as well as improved verbal short-term memory and processing speed). Taken together with the current literature on functional imaging in posttraumatic stress disorder, we propose that the observed diffusion alterations may result from a functional shift from frontolimbic toward mesial temporal structures (shift of functional demand from cingulate and orbitofrontal regions toward mesial temporal regions). September 16, 2022 9/17 JAMA Network Open. 2022;5(9):e2231891. War Zone–Related Stress and Limbic GM Diffusion doi:10.1001/jamanetworkopen.2022.31891 Downloaded From: https://jamanetwork.com/ by a Universitaet Augsburg User on 10/10/2022 Downloaded From: https://jamanetwork.com/ by a Universitaet Augsburg User on 10/10/2022 Association of War Zone–Related Stress With Alterations in Limbic Gray Matter Microstructure JAMA Network Open \| Psychiatry between decreased FA and decreased astrocyte density in the hippocampus.71 Astrocytes play a crucial role in complex brain functions, such as neurotransmitter homeostasis and blood-brain barrier maintenance.75 Moreover, a decrease in astrocytes predisposes the brain to inflammatory states.75,76 Another dMRI study77 in a murine model of Parkinson disease found an association between decreased FAT in the substantia nigra and neuronal loss. Taken together, the association between war zone–related stress and decreased FAT in the cingulate, orbitofrontal gyri, and right parahippocampal gyrus may potentially be due to a decrease in astrocytes and/or neurons. Interestingly, a positive association was found for greater war zone–related stress and higher FAT in the amygdala-hippocampus complex. Increased FAT in GM and WM has been associated with neuroplastic remodeling.72,73 In rodents, long-term learning and memory tasks induced an FA increase, particularly in limbic system structures such as the amygdala, the parahippocampal gyrus, and the cingulate cortex, which correlated with an increase in a myelin marker (myelin basic protein) in the histological analysis.72,73 The authors72,73 hypothesized that oligodendrocytes, which form the myelin sheaths in the central nervous system, produced more myelin basic protein postlearning to allow for the required flow of information. Taken together, findings of our study suggest regional differences in the association between war zone–related stress and alterations in GM microstructure that may be due to neurodegenerative and neuroplastic processes. JAMA Network Open. 2022;5(9):e2231891. doi:10.1001/jamanetworkopen.2022.31891 Limitations Our study has limitations. We investigated a representative subsample of OEF, OIF, or OND veterans48 and we accounted for common comorbidities in the statistical analysis. However, we used dichotomous variables based on the DSM-IV classifications to account for the presence of psychopathologic disorders. Future studies should consider using dimensional assessments of psychopathologic disorders, to further investigate the spectrum of psychopathologic disorders. Furthermore, we did not account for service branch, race, or socioeconomic status,91-98 which might be of importance for resilience, stress exposure, management, and rehabilitation and should be considered in future analyses. The vast majority of participants were White, followed by Hispanic and Black participants (Table 1). Although the level of education was balanced across the cohort, potentially relevant differences were observed for the family status as only 38% were married or cohabiting. A further limitation is that this study was limited to male participants only. The cross- sectional design of this study further limits the interpretation of our findings as well as the identification of additional factors associated with risk and causal relationship between war zone– related stress and alterations in limbic GM may not be drawn. Moreover, we did not differentiate between the amygdala and hippocampus as we aimed for the highest possible accuracy in the segmentation. Previous research of imaging data has demonstrated that the use of the combined amygdala-hippocampus complex represents a methodologically more rigorous and accurate approach of segmentation using FreeSurfer.69 Against the background of our study findings, future studies should strive to retest our hypothesis on manually segmented limbic GM. Additionally, although all interviews were conducted by doctoral level psychologists, their administration at long- term follow-up might have been inevitably biased by participant subjective memory and reporting. Of further note, multishell dMRI data would have improved the FW model fit but was not available in the study. In addition, the analysis of GM is highly sensitive to misalignment of the diffusion space and T1 space, which may have caused inflation in the FW measure. Despite the FW-correction, the FAT measures remain unspecific and can only serve as a gross estimation of the underlying microstructure. JAMA Network Open \| Psychiatry JAMA Network Open \| Psychiatry Association of War Zone–Related Stress With Alterations in Limbic Gray Matter Microstructure No significant associations were found between limbic GM diffusion and more general measures of functional outcome following mTBI (ie, the WHODAS and NSI). We thus speculate that abnormalities in the limbic system may need to be more severe to cause impairments in everyday functioning. Furthermore, the observed limbic alterations may represent a minor contributor to everyday functioning as assessed using WHODAS and NSI, whereas the individual comorbidities may be the main drivers of the functional impairment. Association Between Limbic GM Diffusion and Functional Outcome This biological adaptive response may potentially, in addition to preexisting biological predisposition for deployment, mean that service members with outstanding processing speed and verbal short-term memory might be more likely to join the military and to be deployed. JAMA Network Open. 2022;5(9):e2231891. doi:10.1001/jamanetworkopen.2022.31891 September 16, 2022 10/17 k Open. 2022;5(9):e2231891. doi:10.1001/jamanetworkopen.2022.318 Downloaded From: https://jamanetwork.com/ by a Universitaet Augsburg User on 10/10/2022 Downloaded From: https://jamanetwork.com/ by a Universitaet Augsburg User on 10/10/2022 ARTICLE INFORMATION September 16, 2022 11/17 Conclusions In this study, war zone–related stress was associated with alterations in limbic GM microstructure, which, in turn, were associated with cognitive function independent of the diagnosis of mental disorders and mTBI commonly observed in this population. Taken together, findings from this study suggest that alterations in limbic GM microstructure may underlie the deleterious outcomes of exposure to war zone–related stress. Thus, military service members exposed to war zone–related stress may benefit from early therapeutic intervention even in the absence of a diagnosed mental disorder. ARTICLE INFORMATION Accepted for Publication: August 1, 2022. Published: September 16, 2022. doi:10.1001/jamanetworkopen.2022.31891 JAMA Network Open \| Psychiatry Open Access: This is an open access article distributed under the terms of the CC-BY License. © 2022 Kaufmann E et al. JAMA Network Open. Corresponding Author: Inga K. Koerte, MD, Psychiatry Neuroimaging Laboratory, Department of Psychiatry, Brigham and Women’s Hospital, 1249 Boylston St, Boston, MA 02215 (ikoerte@bwh.harvard.edu). Author Affiliations: Psychiatry Neuroimaging Laboratory, Department of Psychiatry, Brigham and Women’s Hospital, Boston, Massachusetts (E. Kaufmann, Rojczyk, Sydnor, Guenette, D. Kaufmann, Umminger, Seitz- Holland, Sollmann, Rathi, Bouix, Pasternak, Shenton, Koerte); Department of Neurology, University Hospital, Ludwig-Maximilians-Universität, Munich, Germany (E. Kaufmann, Shenton); cBRAIN, Department of Child and Adolescent Psychiatry, Psychosomatics, and Psychotherapy, Ludwig-Maximilians-Universität, Munich, Germany (E. Kaufmann, Rojczyk, D. Kaufmann, Umminger, Sollmann, Koerte); Department of Radiology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts (Guenette); Department of Biostatistics, Boston University School of Public Health, Boston, Massachusetts (Tripodis); Boston University Alzheimer’s Disease and CTE Center, Boston University School of Medicine, Boston, Massachusetts (Tripodis); Department of Diagnostic and Interventional Radiology and Neuroradiology, Klinikum Augsburg, Germany (D. Kaufmann); Department of Diagnostic and Interventional Neuroradiology, School of Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany (Sollmann); TUM-Neuroimaging Center, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany (Sollmann); Department of Diagnostic and Interventional Radiology, University Hospital Ulm, Ulm, Germany (Sollmann); Translational Research Center for TBI and Stress Disorders and Geriatric Research, Education and Clinical Center, VA Boston Healthcare System, Boston, Massachusetts (Fortier, Salat, Milberg, McGlinchey); Department of Psychiatry, Harvard Medical School, Boston, Massachusetts (Fortier, Milberg, McGlinchey); Neuroimaging Research for Veterans Center, VA Boston Healthcare System, Boston, Massachusetts (Salat); Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Charlestown, Massachusetts (Salat); Department of Neurology, Uniformed Services University of the Health Science, Bethesda, Maryland (Hinds); Department of Psychiatry, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts (Shenton, Koerte). Author Contributions: Drs E. Kaufmann and Koerte had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Drs Shenton and Koerte contributed equally. Concept and design: E. Kaufmann, Rojczyk, Rathi, Sylvain, Brawn Fortier, Milberg, Shenton, Koerte. Acquisition, analysis, or interpretation of data: All authors. Drafting of the manuscript: E. Kaufmann, Rojczyk, Sollmann, Shenton, Koerte. Critical revision of the manuscript for important intellectual content: Rojczyk, Sydnor, Guenette, Tripodis, D. Kaufmann, Umminger, Seitz-Holland, Sollmann, Rathi, Sylvain, Brawn Fortier, Salat, Pasternak, Hinds, Milberg, McGlinchey, Shenton, Koerte. September 16, 2022 12/17 JAMA Network Open \| Psychiatry Statistical analysis: E. Kaufmann, Rojczyk, Tripodis, D. Kaufmann, Seitz-Holland, Sollmann, Koerte. Obtained funding: Brawn Fortier, Salat, Milberg, McGlinchey, Shenton. Administrative, technical, or material support: Sollmann, Rathi, Sylvain, Brawn Fortier, Hinds, Milberg, McGlinchey, Shenton, Koerte. Supervision: Rathi, Sylvain, Brawn Fortier, Shenton, Koerte. Conflict of Interest Disclosures: Dr E. Kaufmann reported receiving grants from the German Society of Clinical Neurophysiology and Functional Imaging (DGKN) during the conduct of the study. Dr Rojczyk reported receiving grants from Evangelisches Studienwerk Villigst outside the submitted work. Dr Tripodis reported receiving grants from National Institutes of Health during the conduct of the study. Dr Hinds reported being employed by Wounded Warrior Project June 2021 to June 2022 and being a consultant/owner for SCS Consulting LLC May 2020 to present outside the submitted work. Dr Koerte reported receiving personal fees from Siemens Thieme Publisher (royalties from book chapters) and grants from Abbott, National Institutes of Health, and European Research Council, outside the submitted work; and serving as Vice President of the European Neurotrauma Organization and as European Editor of the Journal of Neurotrauma. No other disclosures were reported. Funding/Support: This research was supported by a fellowship from the DGKN to Dr E. Kaufmann. Dr Shenton was supported by a Veterans Affairs (VA) Merit award to as well as the Translational Research Center for TBI and Stress Disorders (TRACTS), a VA Rehabilitation Research and Development (RR&D) Traumatic Brain Injury Center of Excellence (B6796-C). Dr Koerte is supported by National Institute of Neurological Disorders and Stroke (R01NS100952). Dr Pasternak was supported by National Institute of Biomedical Imaging and Bioengineering (P41EB015902). JAMA Network Open. 2022;5(9):e2231891. doi:10.1001/jamanetworkopen.2022.31891 September 16, 2022 12/17 Downloaded From: https://jamanetwork.com/ by a Universitaet Augsburg User on 10/10/2022 Downloaded From: https://jamanetwork.com/ by a Universitaet Augsburg User on 10/10/2022 Association of War Zone–Related Stress With Alterations in Limbic Gray Matter Microstructure JAMA Network Open \| Psychiatry REFERENCES 1. Lippa SM, Fonda JR, Fortier CB, et al. Deployment-related psychiatric and behavioral conditions and their association with functional disability in OEF/OIF/OND veterans. J Trauma Stress. 2015;28(1):25-33. doi:10.1002/ jts.21979 2. Elder GH Jr, Shanahan MJ, Clipp EC. Linking combat and physical health: the legacy of World War II in men’s lives. 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https://openalex.org/W616585381	https://hal.archives-ouvertes.fr/hal-03704553/file/3.pdf	English	null	Towards a critical realist-inspired economic methodology	The Journal of Philosophical Economics	2,010	cc-by	9,483	To cite this version: Bjørn-Ivar Davidsen. Towards a critical realist-inspired economic methodology. Journal of Philosoph- ical Economics, 2010, Volume III Issue 2 (2), pp.74-96. 10.46298/jpe.10595. hal-03704553 Towards a critical realist-inspired economic methodology Bjørn-Ivar Davidsen Abstract: This paper argues that critical realism conceived as a meta-theory for scientific activities offers a consistent set of helpful philosophical resources from which a potentially fruitful position of economic methodology may be developed. When fully developed, a critical realist-inspired economic methodology may in turn underlabour for more concrete scientific undertakings, economic theorising and applied analyses. Adopting such a strategy for further advancement of the critical realist project would prove a much needed supplement to, or perhaps even substitute for, the currently dominating strategy of grand scale philosophical underlabouring aimed at reorienting more or less the whole discipline of economics. The main trust of the argument made then, is that critical realism comes with a constructive and practical potential that goes beyond critiques of mainstream economics and philosophical underlabouring for already existing schools of thought within economics and that it is time for this potential to be actualised. Keywords: critical realism, meta-theory, ontology, epistemology, methodology HAL Id: hal-03704553 https://hal.science/hal-03704553v1 Submitted on 25 Jun 2022 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 - NonCommercial - ShareAlike 4.0 International License Distributed under a Creative Commons Attribution - NonCommercial - ShareAlike 4.0 International License Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 at the outset are sympathetic to the critical realist project. Moreover, arguments made for establishing critical realism as a unifying basis for various heterodox positions have been questioned on the grounds that they seem to overrate the credibility of critical realist ontological theories as well as the degree of ontological agreement among adherents of different economic schools of thought. [3] The critical realist project within the discipline of economics may thus be in danger of losing momentum and eventually of running into stagnation. Against this background it is my purpose here to explore a somewhat different strategy for developing critical realist approaches to economics; a strategy that takes a more practical turn. This turn is motivated by the view that in order to be convincing, the potential fruitfulness of critical realism needs to be demonstrated through analyses of relevant economic questions and issues. It has to be demonstrated that critical realist-informed approaches to economics actually make a difference. To contribute to a development in this direction I will, in what follows, inquire into the prospects for establishing a helpful critical realist-inspired economic methodology. More specifically I will argue the case for a critical realist meta-theory that offers a consistent set of philosophical resources, tenets and heuristic ideas that may underpin a self-contained and potentially fruitful position of economic methodology. When fully developed, such a methodology may in turn underlabour for more specific economic theorising and for concrete analyses of substantial economic issues and phenomena. Against what many advocates of critical realism within the discipline of economics seem to uphold then, I do indicate that there may be something like a critical realist-inspired approach to economic theorising and economic analyses. The approach suggested will also represent a contribution to the current debate over the development of economic methodological thinking more generally. Introduction [1] Thus far critical realism has first of all been noted for its sustained critique of mainstream economics. More recently, however, efforts have also been made in order to render critical realist ontological theories a common philosophical basis for various economic heterodox schools of thought. [2] These strategies for situating the critical realist project within economics have, however, been somewhat wanting. The critique launched against mainstream economics has failed to convince, not only mainstream economists, but also commentators who Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 philosophical sub-fields of ontology and epistemology and to possible interrelations between them. philosophical sub-fields of ontology and epistemology and to possible interrelations between them. Within the realm of ontology, addressing basic questions of being or existence, the critical realist position takes as its point of departure the view that the world, social and natural, exists independently of our investigations of it. Most realists would subscribe to such a claim, which separate them from philosophical idealists or anti-realists more generally. What makes critical realism stand out as a position of philosophy in its own right, then, is to a large extent due to its further elaborations of some substantial, yet abstract, claims about social and natural reality. In the critical realist view of things, the world is seen as structured, stratified, and differentiated. Encompassed in this account is also a particular understanding of causation and of open versus closed systems. The general argument of stratification implies a world view in which we move from a basic physical level to strata of chemistry, biology, psychology and society. At each stratum we find structured objects; that is, objects moulded through combinations of more basic entities. Typically, objects at a higher stratum possess properties that cannot be reduced to its parts. These properties are considered acquired through processes of emergence, and they contribute in a significant way to the causal powers and capabilities of the objects in question. Water has acquired causal powers through emergence that cannot be reduced to its component parts hydrogen and oxygen; human beings have powers and capabilities that cannot be fully explained in terms of their constituent parts, and so on. As a consequence, causal powers are seen to reside with structured objects in virtue of these objects being what they are. When triggered, causal powers turn into operating mechanisms. Causation then, on this account of reality, is nothing like event regularities. Rather, causation is viewed as powers and capabilities ascribed to structured objects at different strata of a hierarchically organised reality, by way of their nature. [5] The social realm has been paid special attention to in the critical realist account of existence or being. In his ‘transformational model of social activity’ Bhaskar (1989A, 1989B) argues that social reality must be understood as made up by intentional actors, as well as social structures with emergent powers which enable and facilitate, restrict and direct, individual action. Critical realism as a meta-theory The critical realist position discussed in what follows is outlined and developed in the seminal works of Roy Bhaskar dating back to the 1970s and 1980s, and later elaborated upon by contributors within various social sciences. [4] In bringing out the main tenets of this position, seen as a meta-theory for scientific activities, particular attention will be paid to claims made within the Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 for their existence. By employing social structures in planning and performing activities of various kinds, individual actors contribute to the reproduction or transformation of such structures. Bhaskar, consequently, outlines a transformational view of social reality in which both intentional individuals and social structures have real existence while they at the same time are interdependently related. Margaret Archer (1995 and 2003) elaborates this agent-structure model further by discriminating between cultural and structural emergent structures and introducing more nuanced concepts of agents and actors in the social realm. In a broader perspective then, the critical realist account depicts a stratified world view in which what happens at any one stratum should be viewed as the outcome of powers and mechanisms existing and acting at the stratum in question and potentially at all underlying strata. Events and states of affairs at the social stratum of reality, for example, will on this account be the combined results of powers and capabilities residing with structured objects at the social level as well as at underlying psychological and natural strata. According to this view, critical realists see the world as differentiated in the sense that an ontological distinction is posited between three domains of reality called the ‘empirical’, the ‘actual’ and the ‘deep’ domain. Events and states of affairs that are potentially observable comprise the domain of the actual. Phenomena that are actually observed make up the domain of the empirical. Causal powers and mechanisms, which in various ways govern and influence what goes on at the domain of the actual, are seen as existing and acting, at least partly, at the underlying deep domain of reality. As different mechanisms and powers in action may countervail or reinforce one another they cannot be readily identified through empirical observations at the actual level. On this view then, there tends to be a lack of spontaneous regularities between observable objects, which render the domains of the actual and the empirical ‘open’ systems in the critical realist terminology. [6] Effects of mechanisms and powers in operation, consequently, manifest themselves as tendencies rather than law-like empirical regularities. Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Social structures are conceived as pre-existing individual actions, and are thus irreducible to them. At the same time social structures are obviously dependent upon individual actions Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 experience. We do not, in other words, have access to all aspects of the world by way of observation or any other direct methods. Rather, knowledge is something we have to work for in various ways and with different means. Moreover, the ontological claims made by critical realists indicate that uncovering and understanding underlying structures, mechanisms and their emergent properties are the most rewarding goals of knowledge production. When it comes to the question of how to discriminate between competing claims to knowledge of phenomena of interest, and to the question of how to evaluate claims made to knowledge of such phenomena, critical realists entertain a position of judgemental rationality. All empirical observations and descriptions are considered theory-laden. They are, in complex ways, seen as conceptually mediated. More generally, the critical realist position maintains that there can be no neutral or value-free knowledge. All knowledge is considered a social product. Advocates of critical realism do steer clear of full-blown relativism, however, by invoking a claim for judgmental rationality. It is argued that rational judgements can be passed on claims to knowledge despite the fact that empirical ‘data’ will not do as a sole, or final, arbiter. [7] There is nothing like empirical aversion implied by this. Empirical identifications of underlying forces in operation in the natural sphere, for example, are highly acclaimed by critical realists when conducted under reliable experimental control. In the notoriously open system conditions characterising the social realm, however, the possibilities for controlled experiments are scant or almost non-existent. Consequently, investigators of social phenomena, according to the critical realist view, will to some extent have to rely on mental activities like abstractions and thought-experiments as vehicles of inquiry, having the outcomes checked against various forms of empirical material whenever possible. What seems to be entailed in the critical realist approach, then, is a non-foundational stance on questions epistemological in which evaluations of claims to knowledge have to be based upon a mix of empiricist and rationalist considerations. Even if admittedly overly difficult to achieve, the overall goal of critical realist inquiries is true statements of the world; and at this point a final arbiter is invoked: ‘claims are true or false in virtue of the way the world is.’ (Fleetwood 2006, p. 172). Following Outhwaite (1987, p. Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Turning to epistemology, addressing questions of knowledge production and evaluation of claims to knowledge, the critical realist position claims that knowledge of the depicted structured, stratified and differentiated world is in principle possible. It is argued, however, in accordance with the ontological views advanced, that knowledge of these phenomena is not readily given to us in Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 This statement seems to imply that ontology and epistemology are two distinct or separate departments of the critical realist meta-theory. It is clearly indicated that it is possible to be bold regarding questions of ontology while at the same time entertaining a cautious stand on questions epistemological. Or rather, as many protagonists of the critical realist project within economics seem to uphold, that a particular attention to questions of ontology can somehow help us get around tricky questions of epistemology. I seriously doubt the tenacity of such an allegation. In order to argue my case here, I find it expedient to invoke the distinction made in critical realist vocabulary between the ‘transitive’ and the ‘intransitive’ dimension of knowledge. Objects and phenomena of the world taken to exist independently of our investigations of them constitute, on this account, the intransitive dimension of knowledge. They are our objects of knowledge, so to speak. The knowledge we at any point in time claim of these objects and state in the form of observations, descriptions, hypotheses, theories and so on, belong to the transitive dimension. This material is considered a social product, fallible and liable to change and transformation as our insights progress and develop. Now, what is at stake here then, are the ontological claims made in the critical realist meta-theory as set out above. What status or credibility accrues to these claims? Are they in any way separate from epistemological arguments? The claim made by critical realists to the effect that the world exists independently of our investigations of it, I would characterise as a basic philosophical stance. You either take it onboard or you discard it. There is hardly any way to argue sensibly and convincingly pro or contra this stance. It is a matter of belief, or some would say informed belief, as it were. So, there is a least one element in the critical realist ontology distinct from matters epistemological. As soon as some substantial claims about reality are made, however abstract and general these claims may be, things change radically. When critical realists claim that not only does the world exist independently of our investigations of it, it is also structured, stratified and differentiated (in the critical realist understanding of these concepts), we enter the realm of theories and empirics. Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 34), critical realists often characterise their meta-theoretical position as ‘ontologically bold and epistemologically cautious’. Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 We are thoroughly within what critical realists call the transitive dimension of knowledge production. Critical realists’ claim to a structured, stratified and differentiated world is, in other words, an ontological theory. And a theory will have to be supported by arguments in its favour if it is to be accepted or approved. Such arguments are, of course, epistemological in nature. ct and general these claims may be, things change radically. Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 social constructivism on the other. Accordingly, the critical realist tenets as set out above qualify as a separate and distinguished meta-theoretical position in its own right. The one feature which most of all characterises this critical realist meta-theory is its explicit concern with questions of ontology. The epistemic status of critical realist ontological theories, however, needs to be paid attention to. They are to be seen as conjectures expressing carefully argued theories or hypotheses about how the social and natural world is constituted. Consequently, they are conditional and revisable in the event of convincing arguments offered to this effect. This is the main reason why critical realist ontological theories are not suitable as a basis, or point of departure, for critical assessments of practices based on a different methodology and ontology. Being interesting and potential fruitful hypotheses about the social and natural world, however, critical realist ontological theories, and the critical realist position of which they are a part, offers a rich and fertile basis for more practical scientific endeavours, including endeavours directed towards developing a viable economic methodology. Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 To take stock then, the claims made with respect to basic questions of ontology and epistemology obviously delineates critical realism from other meta-theories like variants of positivism on the one hand, and post-modernism and strong Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 To realise this, consider how the critical realist ontological theories are set forth. They are established and elaborated mainly by way of transcendental arguments. To claim absolute truth, or some close equivalent, for the conclusions drawn from such arguments would, of course, verge on dogmatism. Rather, the conclusions offered should be seen as contested and fallible theories dependent for their epistemic status upon evaluations made of the initial premises chosen as well as of the subsequent inferences made. Bhaskar, being keenly aware of these facts, and consequently also of the need for additional support for his transcendental elaborations, invokes the method of immanent critique of competing positions for this purpose. In the case of the natural realm, Bhaskar’s transcendental arguments in favour of ontological depth are bolstered by an immanent critique of the contrasting ‘flat’ ontology accruing to the empirical realist account of reality (Bhaskar 1979). And in the case of the social realm Bhaskar invokes immanent critiques of voluntarism, collectivism and the ‘dialectic’ approach of Berger and Luckmann in order to further substantiate the propositions advanced in his ‘transformational model of society’ (Bhaskar 1989A). Questions of ontology and questions of epistemology then, are not separate realms in the critical realist meta-theory. To be consistent, therefore, the critical realist position of epistemological prudence should carry over to a position of ontological prudence. If critical realist ontological theories are supported by cautious epistemological arguments, it should follow that the ontological theories in question are viewed likewise. I would suggest, therefore, that critical realist meta-theory should be characterised as ‘ontologically and epistemologically cautious with prominence given to questions ontological’, rather than ‘ontologically bold and epistemologically cautious’. It is, or should be, cautious in the sense of entertaining an anti-foundational stance on all knowledge production, including knowledge of basic properties of existence or being. Ontological theories should, on this account, be considered fallible and contingent and subject to revision and adjustments, or even possibly replacement, in the face of convincing arguments to this effect. This insight ought, moreover, to be taken seriously when critical realism is evaluated as a potential meta-theory for scientific undertakings. Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 such undertakings then, would be to uncover and describe causal powers and capabilities pertaining to objects of relevance for the issue or problem at hand, and their ways of working as mechanisms whenever triggered. Several aspects need to be addressed in this process: What powers and capabilities do the relevant objects possess, under what circumstances will these powers be triggered and turned into operating mechanisms, what will be the effect of an operating mechanism when taken in isolation and what will happen when several mechanisms operate in concert? In a particular research project then, having depicted objects of relevance for explaining the issues at hand, researchers will be advised to address the task of uncovering necessary and constitutive properties of these objects. According to the critical realist view, however, causal powers and capabilities may exist without being exercised, and when triggered the resulting mechanisms may or may not imply observable effects, depending, among other things, on the question whether other mechanisms in operation will act in countervailing or reinforcing ways. As a consequence, causal powers are rarely directly observable and may in many cases not even be indirectly identified through their effects. Investigations, therefore, will normally have to be undertaken by conceptual abstractions invoking structural analysis and the method of retroductive inference. Researchers will have to find answers to questions like: What is fundamentally constitutive for object X? What properties will have to be in place for X to be what X is? What causal powers and capabilities follow from these answers? (Danermark et al. 2003, p. 110). To be somewhat more concrete, within economics it may for example be relevant to ask what social and cultural structures and mechanisms must be in place for exchange of commodities to take place. Contexts will then have to be envisaged in which causal powers and capabilities intrinsic to the objects in question are triggered, turning them into operative mechanisms. Moreover the effects of such mechanisms in operation will have to be studied. In the natural sciences researchers will in many cases be able to study the effects of separate mechanisms in controlled experiments in which closed systems are artificially established. Under the notoriously open systems conditions of social sciences like economics, however, there are hardly any possibilities for creating experimental settings of this kind. From meta-theory to methodology Let us turn then to considerations of how the critical realist meta-theory may be turned into an operational position of economic methodology. As we have seen above, the critical realist meta-theory gives prominence to matters ontological. At the heart of this position we find a systematic account of some basic features of social and natural reality. Invoking the critical realist meta-theory as a philosophical underpinning for a position of economic methodology will then to a large extent turn on taking advantage of these ontological insights. Whether starting out from empirical observations of some new or surprising phenomena or addressing more traditional economic issues, researchers informed by the critical realist meta-theory would be advised to seek explanations of the phenomena in question in terms of underlying powers and mechanisms, being particularly aware of the fact that such powers and mechanisms reside with structured objects at different strata of reality. Adding to this general picture, researchers would also be urged to make sure they amply capture the interplay between economic agents and relevant underlying enabling and constraining social structures, paying due attention to processes of reproduction and transformation of structures and further consequences thereof. The main aim of Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 introduced work themselves out. By combining the workings of several mechanisms, processes of countervailing and reinforcing forces may be studied in more complex theoretical models. It must be underlined, however, that critical realist-inspired tasks of this kind would always pay heed to the need to work with proper abstractions of reality, as set forth in critical realist ontological arguments, rather than mere fictions. introduced work themselves out. By combining the workings of several mechanisms, processes of countervailing and reinforcing forces may be studied in more complex theoretical models. It must be underlined, however, that critical realist-inspired tasks of this kind would always pay heed to the need to work with proper abstractions of reality, as set forth in critical realist ontological arguments, rather than mere fictions. The notion of natural necessity, central to the critical realist understanding of causality, needs to be commented somewhat further upon here. Structured objects are seen to have emergent properties, like powers and capabilities necessary for the objects in question to exist and to be what they are. This, however, has nothing to do with unchangeable essence. The notion of necessity as applied here refers to properties which at a certain point in time determine what an object is. As time goes by, however, these properties may change turning the object in question into a different entity. Analyses according to a critical realist meta-theory would, in other words, be open to the possibility of qualitative change in its subject matter. [8] When R. B. Walters and D. Young (2001) more or less discard critical realism as a basis for a viable position of economic methodology, their concern is mainly with questions of epistemology. In their view the critical realist “ontological boldness has never been supported by any plausible epistemology” (Walters and Young 2001, p. 500). As argued in the previous section, I find it contentious to treat ontology and epistemology as though they were separate departments of the critical realist, or indeed any, meta-theory. Rather, ontology and epistemology should to a large extent be viewed as being intimately related while pertaining to different aspects of the meta-theory in question. Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Social scientists, therefore, will, to a large extent, have to take resort to abstract thought-experiments in which they may sort out how conceptualised mechanisms thought-experiments in which they may sort out how conceptualised mechanisms Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 [9] In considering the plausibility or credibility of economic analyses conducted according to a critical realist-inspired methodology, therefore, one has to address the whole package, so to speak: the ontological theories introduced as well as the more concrete scientific work undertaken. In this respect it should be noted that critical realist ontological theories are not introduced as dogmatic or metaphysical assertions about the social and natural world. Rather, they are developed by transcendental arguments from a set of premises. The premises are painstakingly chosen to encompass what is believed to be widely accepted features of the world in which we live; the success of experimental natural sciences and the notion of intentional actors in the social Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 In a positivist conception of science, still influencing the minds of many, case studies are invariably regarded as appropriate only for investigations of particular or unique instances with no claims to generalised knowledge. In a broader picture, moreover, case studies are often seen as useful preparations for large-scale data collection schemes and subsequent analyses. The demise of positivism has however, among other things, brought with it a new conception of the role of case studies in scientific undertakings. Case studies are generally acknowledged as a methodological approach consistent with depth ontologies. And when theoretically based and structured, case studies are now becoming increasingly recognised as supporting claims to generalised or theoretical knowledge of transfactual phenomena like underlying powers and mechanisms (R. K. Yin, 1989, G. Easton, 1992). There are, consequently, no scientific hazards involved in adopting case studies as an integral part of a critical realist-inspired approach to economics. Rather, this seems to be a potential road for empirical support of abstract reasoning which deserves closer attention in endeavours to develop a critical realist-inspired economic methodology. To sum up then, epistemic evaluations of critical realist-inspired analyses of economic phenomena or issues of interest will have to involve several elements; the arguments advanced to anchor the analyses in reality from the outset, the consistency and creativity exhibited in envisaging powers and mechanisms explaining the phenomena at hand, and the degree to which empirical support are found in case studies or by other means being the most central. Moreover, when competing accounts of explanatory forces and mechanisms are set forth, criteria like explanatory power may also be invoked to discriminate between the candidates. In any case, no single or simple algorithm for theory evaluation or theory choice will be available. Rather, rational judgements will have to be made taking into consideration material of different character and content. Now, for some, such a broad and encompassing approach to questions of epistemology, invoking among other things the need to make rational and argued judgements, may seem less than satisfactory or even unconvincing. Difficult problems of epistemology, however, seem to haunt all philosophical meta-theories and consequently all methodologies and practical academic approaches that build upon them. Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 sphere being the most central ones. By invoking transcendental reasoning, then, an account is offered of what the world must be like for these features to be possible or intelligible. The resulting ontological theories, being admittedly contingent and fallible and consequently open to critical assessment, nevertheless anchor critical realist-inspired analyses in a carefully argued account of what exists rather than in some lofty speculations or plainly fictional statements of what the world might be like. While abstract reasoning and theorising will be indispensable in critical realist-inspired economic analyses, empirical evaluations and support should be brought in whenever possible. In considering how this can be done, critical realists will insist upon paying due attention to the nature of the subject matter in question. In any critical realist-inspired approach to scientific work the methods applied should be adjusted to the basic characteristics and properties of the objects under investigation, rather than the other way around. A critical realist-inspired economic methodology will, in other words, reject evaluative procedures that invoke particular test methods or techniques in a compulsory or mandatory way. What methods and techniques are considered adequate or suitable for the purpose at hand will have to be decided upon and defended by practical researchers with due regard being paid to the nature of the objects into which they inquire. Due to the nature of critical realist-inspired analyses, in which the direction of investigations is from observations of issues of interest to underlying forces and mechanisms that may explain such phenomena, the empirical side of research undertaken may in many instances most productively take the form of case studies. By focusing upon a few carefully selected cases researchers will be able to carry out investigations in the necessary depth and detail. Contexts crucial for triggering mechanisms, and allowing them to work their effects out, may be studied more efficiently and closely on a case study basis. Following a case in point over a period of time, moreover, during which relevant contexts may change, will offer opportunities for empirical material to be collected which may support or question claims made to knowledge of how envisaged mechanisms actually work. The unavoidable openness of such studies and the socially mediated quality pertaining to any empirical data implies, however, that ‘tests’ of this kind will only be one element in an overall judgement of what credibility accrues to explanations offered. Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 considered a serious drawback or weakness accruing to critical realist-inspired approaches. [10] considered a serious drawback or weakness accruing to critical realist-inspired approaches. [10] considered a serious drawback or weakness accruing to critical realist-inspired approaches. [10] Turning finally to more specific questions of methods to be applied in investigations as set out above, a position of pluralism will typically be endorsed. This, however, is not to be understood as anything like methodological relativism or ‘anything goes’ at the level of method. Rather, it indicates that there are no a priori preferences for any particular method and no methods are banned or excluded at the outset. [11] The choice of the best methods is left to the discretion of researchers in each particular case within the restrictions set by the ontological considerations at the heart of a critical realist approach. In general, critical realist approaches according to the methodological reflections set out above seem to invite combinations of a range of various methods. Approaches of method triangulation or conscious mixes of qualitative and quantitative methods according to the insights and creativity of the researchers will be seen as potentially fruitful. [12] Regarding questions of methods to be applied then, the critical realist approach offers broad but helpful guidelines with final decisions to be made by the researchers. According to Dow (2003, p. 17) critical realists see their project “as being in support of an approach to economics rather than a specific methodology, suggesting that the approach can support a range of methodologies.” My above arguments and deliberations do support a somewhat different view. According to this view the critical realist meta-theory offers a consistent set of philosophical resources that will underpin a self-contained and potentially fruitful approach sufficiently distinguished and characteristic to warrant the label ‘a critical realist-inspired approach to economic methodology’. Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 The fact that critical realists entertain an anti-foundational position in the realm of epistemology, invoking recourse to rational judgements rather than some untenable simple test-criterion or reliance upon ‘self-evident’ axioms and pure logic, therefore, should not in any way be Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired econom The Journal of Philosophical Economics, III:2, 74-96 Critical realist-inspired economic methodology in perspective Traditionally economic methodology has been prescriptivist and empiricist focusing upon formulation and implementation of precepts for ‘good’ scientific practice within the discipline of economics. These precepts were invariably based upon, or taken directly from, different positions within the philosophy of natural science, like positivism, Popper’s falsificationism and Lakatos’ methodology of scientific research programmes. [13] Little or nothing was said Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 about problems related to the question of how theories might be established and developed. Emphasis was, in other words, upon issues belonging to the ‘context of justification’ to the almost total neglect of questions pertaining to the ‘context of discovery’. Wade Hands (1994) succinctly terms this the ‘shelf-of-scientific-philosophy’ approach to economic methodology. During the 1980s and the 1990s, however, things changed profoundly. Leading economic methodologists, with some remarkable exceptions, now turned away from prescriptive approaches and even critical analyses. In the ensuing ‘new economic methodology’ as Hands (2001) terms it, focus has been upon descriptive accounts of on-goings mainly within mainstream economics, including among other things rhetorical, social, and economic aspects of this activity. The underlying rationale for this descriptive turn seems to be predicated upon the view that economic methodologists can best contribute to the future development of economics by enhancing our understanding of what actually goes on within the discipline. [14] The critical realist-inspired position of economic methodology set forth above represents a distinct alternative to both the ‘old’ and the ‘new’ approaches to economic methodology. The suggested approach differs from the ‘new’ descriptive economic methodology by being more normative, indicating that methodology can, and should, have a constructive role aimed at influencing academic economic practice. Moreover, the critical realist-inspired methodology, as against most positions within the ‘new’ methodology, is explicitly developed from a meta-theory or a position within the philosophy of science. According to the critical realist-inspired view there still is, in other words, an important role for explicit philosophical reflection in matters methodological. The approach set forth above will also, in several respects, stand apart from ‘old’ or ‘traditional’ normative approaches to economic methodology. The normative content of the suggested approach is introduced as argued advice and constructive guidance rather than prescriptions. And even though the process delineated starts out from philosophical reflections, insights from scientific work may well feed back upon these reflections and possibly induce changes and adjustments in them, indicating a cautious approach in which philosophical deliberations and scientific practices are considered mutually interrelated activities. A critical realist-inspired economic methodology will thus not be a rules-based methodology. In fact, it will not be a top-down methodology at all. Every Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 In sum, then, a critical realist-inspired position of economic methodology stands out as viable and helpful in the areas of ‘discovery’ as well as ‘justification’. Moreover, it is flexible and open-ended in many respects, leaving much to the creativity and discretion of practising researchers without lapsing into an approach of ‘anything goes’, or what goes is decided by the ‘best practices’ of the discipline. Finally, a critical realist-inspired economic methodology would encourage analyses of economic issues and problems which draw upon insights from several other disciplines, quite different from the disciplinary separateness which, as Hausman rightly argues, has for a long period of time dominated and circumscribed much of mainstream economics (Hausman, 1992). Consequently, the suggested critical realist-inspired approach exemplifies a third way with respect to questions methodological within the discipline of economics, a constructive and potentially fruitful alternative to the traditional rules-based approaches on the one hand and the new ‘recovering practice’ approaches on the other hand. In sum, then, a critical realist-inspired position of economic methodology stands out as viable and helpful in the areas of ‘discovery’ as well as ‘justification’. Moreover, it is flexible and open-ended in many respects, leaving much to the creativity and discretion of practising researchers without lapsing into an approach of ‘anything goes’, or what goes is decided by the ‘best practices’ of the discipline. Finally, a critical realist-inspired economic methodology would encourage analyses of economic issues and problems which draw upon insights from several other disciplines, quite different from the disciplinary separateness which, as Hausman rightly argues, has for a long period of time dominated and circumscribed much of mainstream economics (Hausman, 1992). Consequently, the suggested critical realist-inspired approach exemplifies a third way with respect to questions methodological within the discipline of economics, a constructive and potentially fruitful alternative to the traditional rules-based approaches on the one hand and the new ‘recovering practice’ approaches on the other hand. Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 element in the suggested methodological scheme will at all times and in all respects be open to critical assessments and possible changes, even as a consequence of results and insights gained through scientific work itself. A critical realist-inspired economic methodology will, moreover, through its preoccupation with ontological reflection, offer helpful and constructive precepts and guidelines for activities directed towards establishing and developing economic theories and analyses. And it will come with consistent and accommodating epistemological considerations indicating how theories and analyses may be evaluated and supported by various kinds of argument. A critical realist-inspired economic methodology, thus, will be balanced in the sense that it encompasses a mix of elements belonging to the context of discovery as well as the context of justification, as opposed to the context of justification-bias that characterises the traditional ‘shelf-of-scientific-philosophy’ approach to economic methodology. Scientific practice in accordance with, or informed by, the main tenets of critical realist ontological theories may also imply a more multidisciplinary approach to economic analyses and economic theorising. According to Bhaskar’s transformational model of social activities, researchers will be advised to make sure that they amply capture the depicted interplay between economic agents and relevant enabling and restricting social structures. Processes of reproduction and transformation and the further consequences thereof, should be built into the analyses. This indicates the need for economists to draw upon insights developed within disciplines like sociology and political science. The critical realist vision of a structured and multi-layered world may, moreover, be applied also at the level of the individual. According to such a view, there will be more to human nature than instrumental rationality. Critical realist-inspired economic analyses would, consequently, induce increased openness towards knowledge produced within the discipline of psychology. Finally, it should be remembered that critical realist ontological theories of the social and natural spheres of reality are not separate or unrelated entities. Rather, as argued above, what happens at the social stratum should be seen as the result of mechanisms and forces existing and acting at this stratum of reality as well as all underlying strata, including the entire natural world. Critical realist ontology thus suggests that economic analyses would benefit from, and ought to be more integrated with, lines of thought developed within natural sciences like biology and ecology. Such integration would entail not only inter-disciplinary but even trans-disciplinary approaches to questions and issues under investigation. Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 non-foundational stand. Rather than invoking a single test procedure or some decisive evaluative criterion, the notion of judgemental rationalism is invoked. This notion points to the need for taking into consideration all material relevant for evaluating the case at hand, theoretical as well as empirical, and to pass an argued judgement made open to critical assessment. Attention has also been drawn to the fact that a critical realist-inspired economic methodology would induce a more interdisciplinary approach to analyses of economic issues and problems and stimulate increased openness to, and awareness of, insights from other disciplines within the natural as well as other social science disciplines. The argument of this paper, encouraging more concrete theoretical and practical work inspired by a critical realist meta-theory, may perhaps seem less glorious and prestigious than the currently dominating critical realist strategy of grand scale philosophical underlabouring with the overriding goal to reorient economic thinking more generally. The strategy suggested here may, however, turn out to be more viable with a real potential of making a difference. At the bottom line ‘action speaks louder than words’, and the future prospects of the critical realist project within economics will depend crucially upon the ability of its adherents to address substantial economic issues in ways that demonstrate the benefits from what should be the hallmark of any scientifically oriented critical realist project: Explicit ontological reflection as an integral part of scientific analyses of substantial issues and problems. At the present stage there is room for further improvements of, and amendments to, the suggested critical realist-inspired position of economic methodology. Even at this stage, however, the suggested approach stands out as a constructive, self-contained and potentially fruitful alternative to the traditional rules-based approaches to economic methodology, on the one hand, and the so-called ‘new’ descriptive or ‘recovering practice’ approaches on the other. Concluding remarks The arguments set forth in this paper have been predicated upon the view that the critical realist projects within economics need a new turn, or at the very least a third leg, directed more explicitly towards the development of economic theories and concrete analyses of substantial economic issues and problems. More specifically, the paper has inquired into the question whether, and how, a critical realist-inspired economic methodology may be developed and elaborated, which in turn would underlabour for more concrete critical realist-informed economic theorising and applied analyses. Starting out with critical realism conceived as a meta-theory for scientific activities it has been argued that this position comes with a consistent set of helpful philosophical resources addressing relevant questions of ontology and epistemology. Considerations of necessary interrelations between ontology and epistemology in the critical realist meta-theory led to the view that the ontological theories at the heart of critical realism have properties which render them potentially fruitful as underpinnings for a critical realist-inspired methodology. As a particularly attractive feature a position of economic methodology predicated upon the critical realist meta-theory would offer helpful guidelines for establishing and developing economic theories and analyses. When it comes to theory evaluation the critical realist meta-theory suggests a basically Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Endnotes [1] I would like to thank the journal’s referees for helpful and constructive comments on an earlier draft of this paper. The usual disclaimer, of course, applies. [2] The most central contributions to the process of establishing and situating the critical realist project within economics are T. Lawson (1997, 2003) and a Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 number of articles by the same author. Discussions of various aspects of these endeavours are found in S. Fleetwood (ed) (1999), P. Downward (ed) (2003) and P. Lewis (ed) (2004). [3] The critical realist critique of mainstream economics is discussed in D. W. Hands (1999), B. Waltres and D. Young (2001), J. Reiss (2004), G. Hodgson (2009) and B.-I. Davidsen (2005). The endeavours to establish critical realism as common ontological basis for various heterodox positions within economics are addressed in C. S. Dow (2004, 2006), J. Vromen (2009), B.-I. Davidsen (2009) and a number of contributions in Lewis (ed) (2003). [4] A non-exhaustive list of contributions would include R. Bhaskar (1979, 1989A, 1989B), A. Collier (1994), M. Archer (1995), M. Archer et al (1998), A. Sayer (1992, 2000), B. Danermark et al (2002), J. Cruickshank (2003), S. Ackroyd and S. Fleetwood (eds) (2000), S. Fleetwood and S. Ackroyd (eds) (2004) and publications by T. Lawson mentioned in note 1. [5] R. Groff (ed) (2008) offers a thorough exploration of the notion of causality in a realist perspective. In a special issue of the Journal of Critical Realism, vol. 8, no. 3, devoted to causal powers, R. Groff situates critical realist views of causation within the broader realist take on this issue; Groff (2009). [6] In fact, the critical realist definitions of open and closed systems are more elaborate than suggested here, turning on what Bhaskar (1978 ch. 2) terms requirements for internal and external closure (also attended to in Lawson 1997, pp. 77-79). In practice, however, the simpler definition turning on event regularities is often invoked. The critical realist critique of mainstream economics, for example, takes at its point of departure the view that “[s]ystems in which [event] regularities occur are said to be closed.” (Lawson 2003, p. 5; italics in original). This definition does not, however, follow from Bhaskar’s stated requirement for external and internal closure, which does not deny ontological depth as indicated by the ‘event-regularity’-definition of closed systems. Endnotes [7] C. Wight (2006, pp. 53-56) offers an insightful and constructive discussion of epistemological questions and judgements along a cautious and non-foundational line of reasoning. [8] The debate over essence has a long history in the literature of philosophy and even within the discipline of economics. Meikle (1985), O’Neill (2001) and Ellis Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 (2001) offer a relevant and balanced survey and discussion of some main questions here. (2001) offer a relevant and balanced survey and discussion of some main questions here. (2001) offer a relevant and balanced survey and discussion of some main questions here. (2001) offer a relevant and balanced survey and discussion of some main questions here. [9] Critical realism, for example, focus on questions of ontology. As argued above, however, the ontological theories of critical realism are necessarily supported by epistemological arguments (necessary if they are not to be stated as mere assertions or dogmatic beliefs). In other meta-theories, giving prominence to questions of epistemology, a particular view of questions ontological will follow, whether they are explicitly stated or not. [10] The problem of substantiating claims to knowledge of underlying powers and mechanisms and of discriminating between competing claims to such knowledge should not, however, be underestimated. This is a feature of a critical realist-inspired economic methodology in need of further elaboration. J. Runde (1998) offers some important reflections on these problems which may prove a fruitful instigation of such processes. [11] The question of legitimate methods has caused some controversy in the literature pertaining to the critical realist project. T. Lawson (1997) and S. Fleetwood (2001) represent in a way the ‘hardliners’ in this debate being strongly negative to almost any use of mathematical and econometric methods (at least in their traditional forms) in critical realist-inspired economic analyses. This view, however, has been contested by a number of commentators, many of them sympathetic to the critical realist project. The debate over methods seems to be closely related to the parallel debate over definitions and applications of closed and open systems in economic analyses. If the constructive and insightful arguments offered in S. Nash (2004) and V. Chick and S. Dow (2001, 2006) on these problems were taken onboard, the critical realist project might also get the debate over methods onto a more productive track. [12] P. Downward and A. Endnotes Mearman (2003, 2006) and W. Olsen (2003, 2004) are examples of recent work addressing the question of method triangulation in a critical realist perspective. [13] Economic methodology according this view is thoroughly exposed by M. Blaug (1980/92) and B. Caldwell (1982/94). [13] Economic methodology according this view is thoroughly exposed by M. Blaug (1980/92) and B. Caldwell (1982/94). [14] Dow (1997, 2002) offers excellent overviews over the developments of and tensions between ‘old’ and ‘new’ approaches to economic methodology. Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 References Ackroyd, S. and Fleetwood, S. (eds) (2000), Realist Perspectives on Management and Organisations, London: Routledge Archer, M. S. (1995), Realist Social Theory: the Morphogenetic Approach, Cambridge: Cambridge University Press Archer, M. S. (2003), Structure, Agency and the Internal Conversation; Cambridge University Press Archer, M. et al (1998), Critical Realism – Essential Readings, London: Routledge Archer, M. et al (1998), Critical Realism – Essential Readings, London: Routledge Bhaskar, R. (1978), A Realist Theory of Science, Hemel Hempstead: Harvester Wheatsheaf Bhaskar, R. (1978), A Realist Theory of Science, Hemel Hempstead: Harvester Wheatsheaf Bhaskar, R. 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(2003), ‘Triangulation, time and the social objects of econometrics’, in P Downward (ed), Applied Economics and the Critical Realist Critique, London: Routledge, 153-159 Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Davidsen, Bjørn-Ivar (2010) ‘Towards a critical realist-inspired economic methodology’, The Journal of Philosophical Economics, III:2, 74-96 Olsen, W. (2004), ‘Triangulation in social research: qualitative and quantitative methods can really be mixed’, in M. Bjørn-Ivar Davidsen is associate professor at Ostfold University College, Halden, Norway (bjorn.i.davidsen@hiof.no) References Holborn (ed), Developments in Sociology: An Annual Review, Ormskirk: Causeway Press Outhwaite, W. (1987), New Philosophies of the Social Sciences: Realism, Hermeneutics and Critical Theory, London: Routledge Reiss, J. (2004), ‘Critical realism and the mainstream, Roundtable: Tony Lawson’s Reorienting Economics’, Journal of Economic Methodology 11: 3, 321-327 Runde, J. (1998), ‘Assessing causal economic explanations’, Oxford Economic Papers, 50, 151-172 Sayer, A. (1992), Method in Social Science, 2nd ed., London: Routledge Sayer, A. (2000), Realism and Social Science, London: Sage Vromen, J. (2009), ‘Conjectural revisionary ontology’, in E. Fullbrook (ed): Ontology and Economics: Tony Lawson and his Critics; London: Routledge; pp. 325-334 Walters, B. & Young, D. (2001), ‘Critical realism as a basis for economic methodology: a critique’, Review of Political Economy, 13:2, 483-501 Yin, R. K. (1989), Case Study Research: Design and Methods, revised edition, London: Sage Bjørn-Ivar Davidsen is associate professor at Ostfold University College, Halden, Norway (bjorn.i.davidsen@hiof.no)
https://openalex.org/W4390583976	https://ijoerar.net/index.php/ijoerar/article/download/12/9	English	null	Analysis of Cambridge Curriculum Implementation in Character Education Aspects in Economic Learning (Case Study at Singapore Intercultural School Semarang)	International Journal of Emerging Research and Review	2,023	cc-by	8,604	ABSTRACT Objective: This study aims to analyze the implementation of Cambridge curriculum in the field of Economic and to find out the implications for developing student’s character. Method: This research is conducted at Singapore Intercultural School (SIS) Semarang and carried out by interview, observation and documentation. The validity technique used is triangulation. Results: The results showed that the implementation of Cambridge Curriculum at SIS Semarang is very high in terms of the planning, implementation, and evaluation stages of learning, and has integrated character education through aspects of school input, process, and output. Novelty: This study combines educational character variables in the Cambridge International Curriculum and the Indonesian National Curriculum with research objects in international schools. IJOERAR, Vol. 1, No. 2, June 2023 © 2023 IJOERAR : IJOERAR, Vol. 1, No. 2, June 2023 © 2023 IJOERAR : International Journal of Emerging Research and Review Homepage : https://ijoerar.net/index.php/ijoerar p-ISSN : 3024-8825; e-ISSN : 3021-8950 IJOERAR, Vol. 1, No. 2, June 2023 © 2023 IJOERAR : International Journal of Emerging Research and Review International Journal of Emerging Research and Review DOI: https://doi.org/10.56707/ijoerar.v1i2.12 Analysis of Cambridge Curriculum Implementation in Character Education Aspects in Economic Learning (Case Study at Singapore Intercultural School Semarang) Rafida Yosaria Anindya1, Heni Purwa Pamungkas1 1Universitas Negeri Surabaya, Surabaya, Indonesia INTRODUCTION 2, June 2023 © 2023 IJOERAR : International Journal of Emerging Research and Review IJOERAR, Vol. 1, No. 2, June 2023 © 2023 IJOERAR : J International Journal of Emerging Research and Review problem solving together with students (Cambridge Assessment International Education, 2017b). Thus it is important for every teacher to understand and prepare for learning starting from the process of planning, implementing and evaluating according to the curriculum guidelines used by the school (Islam & Fajaria, 2022). Despite the increasing number of international school establishments, globalization in education also followed by developments of technology used in learning, such as Google Classroom, Google Form, Google Drive, Microblog, and so on. Known as the impact of globalization, the negative use of technology information and communication has exceeded its usefulness and its main purpose and is considered to have contributed to weakening national character, reducing human quality, and damaging the degree of quality of life of the nation and state (Soesatyo et al., 2015). Advances in science and technology are like a double-edged sword, because even though these advances are able to provide more opportunities for global cooperation and progress, on the other hand they can create challenges to the character and morals of every human being (Bialik et al., 2015). ) The development of the world in the 21st century has led to many developments and advances, especially in terms of technology (Khumairoh, 2022). Facing the challenges of the 21st century requires efforts to grow the personality and abilities of students so they are able to fulfill social responsibilities (Bialik et al., 2015). In this case, the role of the school as an educational institution, besides evaluating goals, it is also necessary to develop a curriculum that clearly includes character education (Matsutono, 2020). This is because learning practices in schools include various pedagogical activities such as playing, investigating, debating, designing, sports and so on which can be integrated with character education (Bialik et al., 2015). Character education aims to improve the quality of implementation and teaching results in schools which lead to the formation of holistic, integrated and balanced character and noble character of students (Darmawati et al., 2013). Learners who apply a good attitude culture tend to also apply cognitive skills and socio-emotional skills, including personality qualities, such as self-resilience, self-motivation, and self- management to their learning process effectively (Cambridge Assessment International Education, 2021). INTRODUCTION Globalization in the field of education is carried out in order to fulfill the needs of a competitive workforce (Kholillah et al., 2022). The globalization of education can be seen in the establishment of international schools. In line with that, every schools must determine a curriculum that suits with the needs of their students. The position of the curriculum as a set of systems, plans and arrangements to achieve learning objectives. One of the international curricula that emphasizes the teaching and learning process is the Cambridge curriculum. The Cambridge curriculum is part of the University of Cambridge Local Examination Syndicate (UCLES) which has been used in more than 150 countries (Cambridge Assessment International Education, 2021). While focusing on building knowledge, this curriculum also shapes students' mindsets to be independent and prepares them for social life. The Cambridge learning direction is intended so that students are able to have knowledge, understanding and skills, are able to think critically and involve personality qualities (Christiana et al., 2022). In the Cambridge curriculum, there are five character developments or called as Cambridge Learner Attributes which consist of confident, reflective, responsible, engaged and innovative (Cambridge Assessment International Education, 2021). 000012 - 1 https://ijoerar.net/index.php/ijoerar One of the international school using Cambridge curriculum is Singapore Intercultural School (SIS) Semarang. In implementing the curriculum, teacher need to develop a lesson plan that refers to the curriculum syllabus used (Diocolano & Nafiah, 2019). Application of the curriculum in structured classroom learning requires control by the teacher through the concept of lifelong learning (Boyle & Charles, 2016). This is in line with the concept of the teacher's role in the Cambridge curriculum, where the teacher has a role more than being a facilitator, but also a leader in carrying out discussions and 000012 - 1 International Journal of Emerging Research and Review Homepage : https://ijoerar.net/index.php/ijoerar p-ISSN : 3024-8825; e-ISSN : 3021-8950 IJOERAR, Vol. 1, No. 2, June 2023 © 2023 IJOERAR : International Journal of Emerging Research and Review International Journal of Emerging Research and Review Homepage : https://ijoerar.net/index.php/ijoerar p-ISSN : 3024-8825; e-ISSN : 3021-8950 IJOERAR, Vol. 1, No. 2, June 2023 © 2023 IJOERAR : International Journal of Emerging Research and Review IJOERAR, Vol. 1, No. 2, June 2023 © 2023 IJOERAR : International Journal of Emerging Research and Review IJOERAR, Vol. 1, No. INTRODUCTION In line with these national education goals, the government of Indonesia also formulate a Pancasila Student Profile as the main reference that directs policies in building the character and competence of students (RI Ministry of Education and Culture, 2022). The profile of Pancasila Student Profile consists of six dimensions, namely: 1) faith, fear of God Almighty and have noble character, 2) independence, 3) mutual cooperation, 4) global diversity, 5) critical reasoning, and 6) creative. g y g Character education has a close relationship with its implementation in the social and economic life of society. Economics is a science that studies human behavior and actions to meet their needs throughvariousproduction, consumption and/or distribution activities (Darmawati et al., 2013). Economics learning based on character education can be carried out by instilling a wise, rational and responsible attitude with knowledge and 000012 - 2 000012 - 2 https://ijoerar.net/index.php/ijoerar International Journal of Emerging Research and Review Homepage : https://ijoerar.net/index.php/ijoerar p-ISSN : 3024-8825; e-ISSN : 3021-8950 IJOERAR, Vol. 1, No. 2, June 2023 © 2023 IJOERAR : International Journal of Emerging Research and Review International Journal of Emerging Research and Review Homepage : https://ijoerar.net/index.php/ijoerar p-ISSN : 3024-8825; e-ISSN : 3021-8950 International Journal of Emerging Research and Review Homepage : https://ijoerar.net/index.php/ijoerar p-ISSN : 3024-8825; e-ISSN : 3021-8950 IJOERAR, Vol. 1, No. 2, June 2023 © 2023 IJOERAR : International Journal of Emerging Research and Review IJOERAR, Vol. 1, No. 2, June 2023 © 2023 IJOERAR : International Journal of Emerging Research and Review skills that are useful for oneself, the household, the community and the country in the fields of economics, management and accounting, as well as responsible decisions about values. socio-economic values in a pluralistic society, at the national and international levels (Darmawati et al., 2013). Character education in economics learning is expected to be able to provide understanding and experience to actualize the rational, formal juridical, procedural, morality, politeness and appropriateness of every fulfillment of personal needs and dealing with phenomena that occur in society (Soesatyo et al., 2015). In connection with these conditions, questions arise in society regarding the establishment of international schools, namely concerns about the influence of culture and the character or personality of students (Arifa, 2021). Besides that, the role of educational institutions is needed in directing students to use technology in a positive direction. INTRODUCTION skills that are useful for oneself, the household, the community and the country in the fields of economics, management and accounting, as well as responsible decisions about values. socio-economic values in a pluralistic society, at the national and international levels (Darmawati et al., 2013). Character education in economics learning is expected to be able to provide understanding and experience to actualize the rational, formal juridical, procedural, morality, politeness and appropriateness of every fulfillment of personal needs and dealing with phenomena that occur in society (Soesatyo et al., 2015). In connection with these conditions, questions arise in society regarding the establishment of international schools, namely concerns about the influence of culture and the character or personality of students (Arifa, 2021). Besides that, the role of educational institutions is needed in directing students to use technology in a positive direction. https://ijoerar.net/index.php/ijoerar RESEARCH METHOD This research was conducted using a qualitative approach. Qualitative research are methods for exploring and understanding the meaning of problems through specific data collection procedures from participants, analyzing data inductively, and interpreting the meaning of data (Creswell, 2019). The research design used is a case study or case study. Case studies are research designs that are mostly devoted to the evaluation of a case, program, event, activity, or process that is limited in time and observed through observation and interview procedures (Creswell, 2019). Collecting data in this study using observation, interviews and documentation. Observation techniques are carried out by observing all processes or activities related to research topics regarding the implementation of the Cambridge curriculum in Economics learning including the teaching and learning process and the process of instilling character education through certain activities at SIS Semarang. In the interview technique, the informants in this study were school principals, coordinators of the Cambridge curriculum, economics teachers and representatives of Semarang SIS students. Several considerations for the qualifications of the informants included having competent school administration knowledge, knowing the direction of school development in accordance with the school's vision and mission, understanding Cambridge learning procedures, understanding Cambridge learning concepts and knowing the implementation of the Cambridge curriculum for character education. Documentation techniques are carried out through data collection including, SIS Semarang school profiles, syllabus, lesson plans, documentation of student activities and achievements, as well as Cambridge Assessment International Education documents, such as Implementing the Curriculum with Cambridge, Code of Practice, IGCSE syllabus, approach to textbooks, etc. The author uses a documentation study to strengthen the results of the analysis. The data analysis technique used is triangulation of methods and data sources. Triangulation (triangulate) is done by collecting information and then developing research topics in an integrated manner (Creswell, 2019). 000012 - 3 000012 - 3 000012 - 3 https://ijoerar.net/index.php/ijoerar Meta-Analysis of Problem Based Learning Models Impact towards of Problem Solving Skills in Physics Learning https://ijoerar.net/index.php/ijoerar 000012 - 4 https://ijoerar.net/index.php/ijoerar Meta-Analysis of Problem Based Learning Models Impact towards of Problem Solving Skills in Physics Learning g RESULTS AND DISCUSSION Results The following table is a summary of the research results obtained from several data collection techniques, both interviews, observation and documentation. Table 1. Results The following table is a summary of the research results obtained from several data collection techniques, both interviews, observation and documentation. Evidence Mapping Unit Plan using IGCSE and AS/A Level for Economics Syllabus Mapping Unit Plan using IGCSE and AS/A Level for Economics Syllabus Use English as the first language, provide regular training for teachers, use textbooks from the Cambridge curriculum, adequate facilities and infrastructure that support Cambridge learning, and provide remedial activities rovide pretest questions, completing teacher dministration before learning, applying several earning strategies, an environment that supports earning, using several learning media, having eference benchmarks for attitudes and learning motivation, and non-written test assessments Applying attitudes to Cambridge Learner Attributes, such as responsibility, confidence, reflective, innovative, and engagaed g attitudes to Cambridge Learner s, such as responsibility, confidence, , innovative, and engagaed Characters Building in National Curriculum (Profil Pelajar Pancasila) RESEARCH METHOD Summary of the Research Results Aspects Emersion Evidence Yes No Cambridge Curriculum Planning ✓ Mapping Unit Plan using IGCSE and AS/A Level for Economics Syllabus Cambridge Curriculum Implementation ✓ Use English as the first language, provide regular training for teachers, use textbooks from the Cambridge curriculum, adequate facilities and infrastructure that support Cambridge learning, and provide remedial activities Cambridge Curriculum Evaluation ✓ Provide pretest questions, completing teacher administration before learning, applying several learning strategies, an environment that supports learning, using several learning media, having reference benchmarks for attitudes and learning motivation, and non-written test assessments Characters Building in Cambridge Curriculum ✓ Applying attitudes to Cambridge Learner Attributes, such as responsibility, confidence, reflective, innovative, and engagaed Characters Building in National Curriculum (Profil Pelajar Pancasila) ✓ Applying attitudes to the Pancasila Student Profile (Profil Pelajar Pancasila), such as having faith, piety to God Almighty and having noble character, global diversity, mutual cooperation, independence, critical reasoning, and creativity The following describes the results of descriptive data related to the research focus obtained through interviews, documentation, and observation. Results The following table is a summary of the research results obtained from several data collection techniques, both interviews, observation and documentation. Discussion B) Effective Learning Day. It is known that the effective day at SIS Semarang is Monday - Friday, with a duration of 1 lesson hour is 30 minutes. Learning per day in the IGCSE class is 5 lesson hours, while for AS & A Level classes it is 7 lesson hours. This is in accordance with Permendikbud No. 22 of 2016 which states that the maximum standard for face-to-face time allocation for Senior High School is 45 minutes, so it can be concluded that SIS Semarang has fulfilled the learning load according to these regulations. C) Teacher. The results of interviews with teaching staff can be concluded that SIS Semarang provides several trainings for teachers with the aim of improving teaching performance in the classroom. The training is Professional Development, which is given to teachers four to five times a year. The discussion in the training focused on the needs of teachers generated during the previous meeting. In this regard, in Law no. 14 of 2005 explains that there are four competencies that must be possessed by teachers, namely pedagogic, personality, social, and professional competencies. Competency factors are related to teacher quality which ultimately has an impact on the quality of education, for this reason efforts are needed to improve teacher quality through teacher training programs (Hoesny & Darmayanti, 2021). D) Learning Resources. Based on the results of interviews with school principals, Cambridge curriculum coordinators, and Economics teachers, it is known that content and learning resources can be accessed by teachers on the official website of Cambridge International Education. Through the School Support Hubin the on this site, teachers can connect with other Cambridge teachers around the world to share information regarding the learning resources used and follow the development of the subjects being taught. The site also provides a list of recommended textbooks in the Cambridge curriculum. There is information at the back of the textbook on which syllabus the book supports, whether it is for IGCSE or AS & A Levels, as well as the year of the syllabus' first exam. E) Curriculum Activities. Furthermore, the results of research on curriculum activities found that SIS Semarang regularly holds House Games every Thursday and Assembly every Friday outside of class hours. Discussion With learning activities outside the classroom as well, teachers can train students to foster a sense of love for the environment, because by 000012 6 htt //ij t/i d h /ij 2019). 2) Implementation. At the implementation stage which is the core of the implementation of the Cambridge curriculum, the presentation includes the use of English, effective day of learning, teaching staff, learning resources, curriculum activities, and remedial provision. The implementation of Cambridge curriculum-based learning basically emphasizes the processes and experiences of students (Cambridge Assessment International Education, 2021). This is in accordance with the learning model applied by SIS Semarang, namely active learning. A) English Usage. In practice, English has been fully used as the language of instruction in learning. This is in accordance with the provisions for the use of foreign languages in educational units listed in Law no. 20 of 2003 concerning the National Education System Article 33 (3) which reads "Foreign languages can be used as the language of instruction in certain educational units to support students' foreign language skills”. B) Effective Learning Day. It is known that the effective day at SIS Semarang is Monday - Friday, with a duration of 1 lesson hour is 30 minutes. Learning per day in the IGCSE class is 5 lesson hours, while for AS & A Level classes it is 7 lesson hours. This is in accordance with Permendikbud No. 22 of 2016 which states that the maximum standard for face-to-face time allocation for Senior High School is 45 minutes, so it can be concluded that SIS Semarang has fulfilled the learning load according to these regulations. C) Teacher. The results of interviews with teaching staff can be concluded that SIS Semarang provides several trainings for teachers with the aim of improving teaching performance in the classroom. The training is Professional Development, which is given to teachers four to five times a year. The discussion in the training focused on the needs of teachers generated during the previous meeting. In this regard, in Law no. 14 of 2005 explains that there are four competencies that must be possessed by teachers, namely pedagogic, personality, social, and professional competencies. Competency factors are related to teacher quality which ultimately has an impact on the quality of education, for this reason efforts are needed to improve teacher quality through teacher training programs (Hoesny & Darmayanti, 2021). D) Learning Resources. Discussion Implementation of the Cambridge Curriculum in Economics Learning at SIS Semarang 000012 - 5 https://ijoerar.net/index.php/ijoerar Implementation of the Cambridge Curriculum in Economics Learning at SIS Semarang The process of implementing the Cambridge curriculum in Economics learning at SIS Semarang is divided into three stages, namely planning, implementation and evaluation. 1) Planning. At the planning stage, it is explained through the preparation of a Unit Plan which refers to the Cambridge syllabus, Management in planning and the parties involved. Learning planning is standard content that is programmed to achieve educational goals (Iskandar, 2019). The Economics study plan at SIS Semarang is based on the syllabus and schemes of work obtained from Cambridge International Education. The results showed that the parties involved in the interview included the school principal, the Cambridge curriculum coordinator who were responsible as coaches and advisors and the Economics teacher as executor. This is in accordance with the theory that the parties involved in curriculum planning are educator administrators, curriculum experts, teachers, parents, and school committees (Sukmadinata, 2006). Conceptually, the Unit Plan document has fulfilled the components in the preparation of learning tools such 000012 - 5 000012 - 5 Meta-Analysis of Problem Based Learning Models Impact towards of Problem Solving Skills in Physics Learning Meta-Analysis of Problem Based Learning Models Impact towards of Problem Solving Skills in Physics Learning as objectives, materials, methods, media, sources and learning assessment (Ananda, 2019). as objectives, materials, methods, media, sources and learning assessment (Ananda, 2019). as objectives, materials, methods, media, sources and learning assessment (Ananda, 2019). 2019). 2) Implementation. At the implementation stage which is the core of the implementation of the Cambridge curriculum, the presentation includes the use of English, effective day of learning, teaching staff, learning resources, curriculum activities, and remedial provision. The implementation of Cambridge curriculum-based learning basically emphasizes the processes and experiences of students (Cambridge Assessment International Education, 2021). This is in accordance with the learning model applied by SIS Semarang, namely active learning. A) English Usage. In practice, English has been fully used as the language of instruction in learning. This is in accordance with the provisions for the use of foreign languages in educational units listed in Law no. 20 of 2003 concerning the National Education System Article 33 (3) which reads "Foreign languages can be used as the language of instruction in certain educational units to support students' foreign language skills”. https://ijoerar.net/index.php/ijoerar Discussion The remedial provision is in accordance with the intent and purpose of the activity, namely the remedial program is an effort to improve student learning completeness, in the form of providing guidance and training to students in order to achieve the expected learning mastery (Hikmasari et al., 2018). y ( ) 3) Evaluation. The last stage, namely the evaluation stage, is explained through information related to the characteristics of students, completeness of facilities and infrastructure, teacher readiness, learning materials, environmental conditions, learning strategies, teacher performance, effectiveness of learning media, attitudes and motivation of students, and evaluation of learning outcomes. In this study, the educational evaluation model by Robert Stake (1967) was applied. Evaluation by Stake has two basic things, namely description and consideration, and consists of three program stages, namely: input, process and output. A) Input Evaluation. Input evaluation, also known as input evaluation, is an assessment of matters relating to the use of available resources in achieving educational goals. The indicators in the input evaluation consist of the characteristics of the students, which is shown by how the school ensures students are ready to learn with the Cambridge curriculum standards through providing trial classes for prospective students. This is in accordance with the theory of school readiness which states that school readiness that is important to pay attention to includes not only academic readiness, but also physical readiness, cognitive readiness, emotional readiness, social readiness and mental readiness (Hurlock, 1999). In terms of facilities and infrastructure, SIS Semarang provides adequate facilities as a place to study. These facilities and infrastructure include things that are basic in nature, as well as aimed at supporting the quality of learning. The explanation of these facilities and infrastructure is in accordance with the definition of learning facilities, which are things that are needed in the learning process, both mobile and immovable in nature, and have the benefit of achieving educational goals smoothly, effectively and efficiently (Arikunto & Yuliana, 2008). Aspects of teacher readiness are observed when the teacher begins to develop plans and prepare classroom tools before learning. This is in accordance with the theory of teacher preparation where the teacher is said to be ready if he has carried out the planning, learning process and actualizing the learning method (Sagala, 2009). The Economics material developed by SIS Semarang is in accordance with the syllabus in the Cambridge curriculum. Discussion Based on the results of interviews with school principals, Cambridge curriculum coordinators, and Economics teachers, it is known that content and learning resources can be accessed by teachers on the official website of Cambridge International Education. Through the School Support Hubin the on this site, teachers can connect with other Cambridge teachers around the world to share information regarding the learning resources used and follow the development of the subjects being taught. The site also provides a list of recommended textbooks in the Cambridge curriculum. There is information at the back of the textbook on which syllabus the book supports, whether it is for IGCSE or AS & A Levels, as well as the year of the syllabus' first exam. E) Curriculum Activities. Furthermore, the results of research on curriculum activities found that SIS Semarang regularly holds House Games every Thursday and Assembly every Friday outside of class hours. With learning activities outside the classroom as well, teachers can train students to foster a sense of love for the environment, because by 000012 - 6 000012 - 6 000012 - 6 https://ijoerar.net/index.php/ijoerar Meta-Analysis of Problem Based Learning Models Impact towards of Problem Solving Skills in Physics Learning -Analysis of Problem Based Learning Models Impact towards of Problem Solving Skills in Physi i Meta-Analysis of Problem Based Learning Models Impact towards of Problem Solving Skills in Physics Learning observing objects around them directly, students will realize how to protect the environment and be grateful for God's creation (Widiasworo, 2017). F) Remedial. Remedial provision is not only carried out through repetition of assessments, but the Economics teacher also routinely repeats material for students who experience learning difficulties, with the hope that this will improve students' abilities. The remedial provision is in accordance with the intent and purpose of the activity, namely the remedial program is an effort to improve student learning completeness, in the form of providing guidance and training to students in order to achieve the expected learning mastery (Hikmasari et al., 2018). observing objects around them directly, students will realize how to protect the environment and be grateful for God's creation (Widiasworo, 2017). F) Remedial. Remedial provision is not only carried out through repetition of assessments, but the Economics teacher also routinely repeats material for students who experience learning difficulties, with the hope that this will improve students' abilities. https://ijoerar.net/index.php/ijoerar Discussion The learning material is developed from the Unit Plan which is arranged according to the topics in the syllabus. The applied material is contained in the Cambridge curriculum reference book used in learning. This is in accordance with the explanation of learning materials in Permendikbud No. 22 of 2016 which states that learning materials can come from textbooks, or other learning resources in the form of local content, the internet, enrichment and remedial. Findings about the state of the 000012 - 7 000012 - 7 https://ijoerar.net/index.php/ijoerar nalysis of Problem Based Learning Models Impact towards of Problem Solving Skills in Physics Meta-Analysis of Problem Based Learning Models Impact towards of Problem Solving Skills in Physics Learning Meta-Analysis of Problem Based Learning Models Impact towards of Problem Solving Skills in Physics Learning school, it is known that SIS Semarang fully supports a positive school environment. This is manifested in the formation of discipline for students, including by getting used to greeting teachers, staff and fellow students, as well as maintaining cleanliness and tidiness of themselves and the environment. Based on the theory of several experts on the school environment, it is known that schools that provide a positive environment have formed good behavior habits for students. This environment includes the physical environment in the form of facilities and infrastructure and non-physical in the form of norms and noble values (Amri, 2011). Active learning learning strategy combined with student centered learning. The learning approach was then applied to lecture, question and answer, recitation and experiment learning methods. The application of these learning strategies is intended to build the involvement and activeness of students in learning. This is in line with the Cambridge curriculum which emphasizes the importance of process in achieving learning objectives. B) Process Evaluation. The next evaluation is process evaluation, which consists of aspects of teacher performance, the effectiveness of learning media, and the attitudes and motivation of students. Teacher performance is related to all the actions experienced by educators in dealing with a task, the answers they compose, in order to provide goals (Yamin & Maisah, 2010). From the results of the research it is known that the school principal regularly supervises to determine teacher performance and also to improve the quality of learning in schools. Judging from the object, there are three kinds of supervision, namely academic supervision, administrative supervision and institutional supervision (Suhardan, 2010). https://ijoerar.net/index.php/ijoerar Discussion Implementation of the Cambridge Curriculum on Character Education Aspects in Economics Learning at SIS Semarang Character development in this study is a combination of the five characters developed in the Cambridge curriculum or also called Cambridge Attributes (responsibility, confidence, reflective, innovative, and participative) with six Pancasila Student Profile indicators (noble character, global diversity, mutual cooperation, independent, critical reasoning, and creative) applied at SIS Semarang. 1) Responsibility. The results of the study show that the habituation of an attitude of responsibility is carried out by the teacher by giving a target time for completing assignments, with the hope that students can have a sense of responsibility to complete school assignments on time. Besides that, the attitude of responsibility of students is also trained through honest examination work. In practice, schools provide strict rules and sanctions when students take exams. This is in accordance with the statement that one of the directions and understanding of students regarding matters that must be considered in studying at school is the attitude of responsibility towards assignments (Siregar, 2017). In addition, among these responsible attitudes include setting targets, having intellectual integrity. 1) Responsibility. The results of the study show that the habituation of an attitude of responsibility is carried out by the teacher by giving a target time for completing assignments, with the hope that students can have a sense of responsibility to complete school assignments on time. Besides that, the attitude of responsibility of students is also trained through honest examination work. In practice, schools provide strict rules and sanctions when students take exams. This is in accordance with the statement that one of the directions and understanding of students regarding matters that must be considered in studying at school is the attitude of responsibility towards assignments (Siregar, 2017). In addition, among these responsible attitudes include setting targets, having intellectual integrity. 2) Confident. Students' self-confidence is integrated into the learning process. Through the application of several learning methods, students are trained to have the courage to ask questions to the teacher and are confident in communicating opinions and ideas in front of the class. This is in accordance with the theory that self-confidence is based on the ability to face a challenging wider life and confidence in decisions and opinions (Novyanti & Alinurdin, 2019). Discussion Further information regarding the effectiveness of learning media. The results of the study show that in every implementation of learning, it requires the use of learning media. When the writer made observations, the teacher used Powerpoint to explain the material on the aggregate demand and supply curve. In addition, the teacher also uses media images to classify public and private goods and wordwall media to explain economic terms in English. This is in accordance with the explanation that the benefits of learning media are to attract the attention of students so that they can foster learning motivation, the meaning of learning materials can be more clearly understood, allowing variations in teaching methods, as well as more activities that can increase student activity (Sudjana & Rival, 2010). Indicators of students' attitudes and motivation can be seen in the implementation of learning at SIS Semarang which is carried out by increasing the attitudes, motivation and also the competence of students. In accordance with the results of observations that the attitudes shown by students in the class were active asking questions, giving opinions, and being independent in doing assignments. In addition, it is known from the results of interviews with several student representatives, that they have high motivation to get good grades and continue their education to a higher level. This is in accordance with the theory of attitude objects that need to be assessed in the learning process, namely attitudes towards learning material, attitudes towards teachers, attitudes in the learning process. C) Output Evaluation. The final evaluation is the output evaluation. Output evaluation or also called evaluation of 000012 - 8 000012 - 8 000012 - 8 https://ijoerar.net/index.php/ijoerar Meta-Analysis of Problem Based Learning Models Impact towards of Problem Solving Skills in Physics Learning Cambridge assessment at SIS Semarang was carried out through written tests, oral tests, and project-based assignments. The written test in the Cambridge curriculum is called a Paper. Paper 1 and Paper 2 for IGCSE and Papers 1, 2, 3 and 4 for AS & A Level. In Papers 1 and 3 the questions posed are in the form of multiple choice, while in Papers 2 and 4 are response data and essays. Oral test, conducted incidentally by the Economics teacher. An example of an oral test is a quiz, whereas project-based assignments are generally aimed at increasing collaboration and creativity. https://ijoerar.net/index.php/ijoerar Discussion The frequency of worship by students while at school was known when the author made observations and interviews with several student representatives. In general, Muslim students carry out the midday and afternoon prayers at the school prayer room, while on Friday the male students will be escorted by the school to the mosque to perform Friday prayers. Meanwhile, Christian, Catholic, Buddhist and Hindu students are known to carry out worship with their parents at least once a week or in accordance with the guidance of their respective beliefs. In addition to the element of piety to God Almighty, based on observations it is known that most students highly uphold morals towards humans. This attitude is reflected in the way students interact, namely by not discriminating between friends who have different beliefs and helping each other between friends of different religions. This is in accordance with the explanation of this character development element, where religious morality is shown by carrying out God's commands, experiencing God's attributes, and continuing to explore in order to understand deeply the teachings of the religion one adheres to. Meanwhile, morality towards humans is realizing that all humans are equal before God, loving fellow God's creations, and respecting differences with other people (Admin, 2022). 6) Have Faith, Fear of God Almighty, and Have Noble Characters. Schools require students to attend their religious subjects at least once a week. The frequency of worship by students while at school was known when the author made observations and interviews with several student representatives. In general, Muslim students carry out the midday and afternoon prayers at the school prayer room, while on Friday the male students will be escorted by the school to the mosque to perform Friday prayers. Meanwhile, Christian, Catholic, Buddhist and Hindu students are known to carry out worship with their parents at least once a week or in accordance with the guidance of their respective beliefs. In addition to the element of piety to God Almighty, based on observations it is known that most students highly uphold morals towards humans. This attitude is reflected in the way students interact, namely by not discriminating between friends who have different beliefs and helping each other between friends of different religions. Discussion Confidence in learning includes communicating his opinions in a structured, critical and analytical manner and respect the opinions of others (Cambridge Assessment International Education, 2021). 3) Reflective. The habituation of a reflective attitude is formed through the implementation of learning, namely by carrying out the process of repeating material that has been previously taught and also directing students to build learning strategies that suit themselves. Habituation of this reflective attitude in the learning process according to the function of reflective thinking is to interpret, formulate relationships between experience and creating continuity (Choy et al., 2017). This is also in accordance with the reflective meaning of Cambridge students, that is, a reflective attitude is manifested by caring about their learning processes and outcomes and developing awareness to become lifelong learners (Cambridge Assessment International Education, 2021). 000012 - 9 https://ijoerar.net/index.php/ijoerar 000012 - 9 000012 - 9 Meta-Analysis of Problem Based Learning Models Impact towards of Problem Solving Skills in Physics Learning 4) Innovative. Students' innovative attitudes are formed through the application of their knowledge to solve problems and practice their adaptability to learning situations, teachers and peers. In the learning process, the problem solving application is applied by giving Economics practice questions in the middle of a learning break. This is in accordance with the explanation that innovative learning at school itself can be done, one of which is by measuring the absorption of each student's knowledge (Purwadhi, 2019). Thus, the attitude of accepting new challenges and facing them creatively and imaginatively can shape ways of thinking and adapting flexibly (Cambridge Assessment International Education, 2021). 5) Engaged. Character development by the last Cambridge, namely engaged. Formed by improving students' social skills, including by forming students in groups to do assignments and also inviting students to always be actively involved in school activities outside the classroom. This is appropriate that the participation of students can create an atmosphere of openness between teachers and students, so that if there are obstacles in learning faced by students they can be overcome more quickly (Dewi et al., 2019). Apart from being carried out in classroom learning, the participatory attitude of Cambridge students is also manifested in social life and sharing knowledge and skills for the wider community (Cambridge Assessment International Education, 2021). 6) Have Faith, Fear of God Almighty, and Have Noble Characters. Schools require students to attend their religious subjects at least once a week. https://ijoerar.net/index.php/ijoerar Discussion This is in accordance with the explanation of this character development element, where religious morality is shown by carrying out God's commands, experiencing God's attributes, and continuing to explore in order to understand deeply the teachings of the religion one adheres to. Meanwhile, morality towards humans is realizing that all humans are equal before God, loving fellow God's creations, and respecting differences with other people (Admin, 2022). p g p p ( ) 7) Global Diversity. The formation of an attitude of global diversity in this case is demonstrated by promoting cultural exchange through activities organized by schools such as the United Nation, the Indonesian Cultural Festival and Diwali. In these activities, students are also actively involved to help the smooth running of the event. Besides that, the existence of Indonesian and Civics courses provided by schools has encouraged the interest of many foreign students to study them. This is in accordance 7) Global Diversity. The formation of an attitude of global diversity in this case is demonstrated by promoting cultural exchange through activities organized by schools such as the United Nation, the Indonesian Cultural Festival and Diwali. In these activities, students are also actively involved to help the smooth running of the event. Besides that, the existence of Indonesian and Civics courses provided by schools has encouraged the interest of many foreign students to study them. This is in accordance 000012 - 10 000012 - 10 Meta-Analysis of Problem Based Learning Models Impact towards of Problem Solving Skills in Physics Learning Meta-Analysis of Problem Based Learning Models Impact towards of Problem Solving Skills in Physics Learning -Analysis of Problem Based Learning Models Impact towards of Problem Solving Skills in Physi with the theory about the key elements of the Pancasila Student Profile which are globally diverse, namely education can encourage students to get to know their history and culture, appreciate the uniqueness of each existing culture, and have the ability to reflect on the process of utilizing awareness and experience of diversity (Jamalulail et al., 2023). with the theory about the key elements of the Pancasila Student Profile which are globally diverse, namely education can encourage students to get to know their history and culture, appreciate the uniqueness of each existing culture, and have the ability to reflect on the process of utilizing awareness and experience of diversity (Jamalulail et al., 2023). Discussion p g p y ( ) 8) Worked together. The cooperative attitude of students, in this case, is developed by the school through social service activities every year at the Bhakti Asih Disabled Orphanage. Information was also obtained from the chairman of the Student Council, that in 2018, a fundraising event was held for victims of natural disasters in Palu City. Within the school itself, the development of a mutual cooperation attitude is also carried out through socialization on topics relevant to the SDGs and current conditions through Assembly activities every Friday. These activities always involve students actively and also collaboration between teachers and students. This is in accordance with the explanation of the key elements in the mutual cooperation indicator in the Pancasila Student Profile. 9) Independent. The results of the study show that the independent attitude of students is reflected when students are able to control their emotions when faced with challenging learning situations, such as quizzes and the use of wordwall media. Besides that, schools also develop an independent attitude by directing students to set learning goals, designing self-development strategies to achieve achievements and ideals to be realized in the future. This is in accordance with the statement that the meaning of independence in this Pancasila Student Profile is being responsible for a process and also for their learning outcomes. The key elements of this indicator are self-awareness and the situation at hand (Rusnaini et al., 2021). 10) Critical Reasoning. Through the learning process, in this case the Economics subject, the application of this critical reasoning attitude is shown through the teacher by training students to find sources of information on Economics content not only through books provided by the school, but also through credible websites and articles. Besides that, in the process of learning Economics with the discussion method, students are also trained to rethink the views of other people who may be contrary to their views and to make conclusions on these views. This is in accordance with the explanation of indicators of critical reasoning in the Pancasila Student Profile, where critical reasoning is meant that students are able to objectively process, analyze, evaluate and draw conclusions from information (Rusnaini et al., 2021). 11) Creative. Creative character development in this case is found in the form of giving assignments to students. CONCLUSION The findings of this study confirm that the establishment of international schools in Indonesia as a form of globalization in the field of education providing good character education and in accordance with the noble values of the Indonesian nation. For educators, it is hoped that they will continue to maintain and develop in organizing an effective learning in improving the competence, abilities and character of students. For future researchers, it is hoped that they can develop this research even better by using other variables. Discussion In the sub-element, generate diverse ideas by considering many perspectives, embodied in a project by students in the form of a podcast about the impact of the pandemic on the economy which is accompanied by interviews with 1-2 sources to provide their responses. Whereas in the sub-element exploring and expressing thoughts in the form of works or actions, it is found in the form of making mind maps by students which contain exploration of their understanding and opinions about Gross Domestic Product (GDP), inflation, and trade balances in several countries, accompanied by cause for the changes. This is in accordance with the theory that the creativity referred to in the 11) Creative. Creative character development in this case is found in the form of giving assignments to students. In the sub-element, generate diverse ideas by considering many perspectives, embodied in a project by students in the form of a podcast about the impact of the pandemic on the economy which is accompanied by interviews with 1-2 sources to provide their responses. Whereas in the sub-element exploring and expressing thoughts in the form of works or actions, it is found in the form of making mind maps by students which contain exploration of their understanding and opinions about Gross Domestic Product (GDP), inflation, and trade balances in several countries, accompanied by cause for the changes. This is in accordance with the theory that the creativity referred to in the 000012 - 11 000012 - 11 000012 - 11 https://ijoerar.net/index.php/ijoerar Meta-Analysis of Problem Based Learning Models Impact towards of Problem Solving Skills in Physics Learning Pancasila Student Profile here is that students are able to modify and produce something original, meaningful, useful, and impactful. Originality in this indicator is very important, because the act of imitating the work of others can form negative habits and behavior (Rusnaini et al., 2021). REFERENCES Adha, M. M., Ulpa, E. P., Johnstone, J. M., & Cook, B. L. (2019). 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https://openalex.org/W4362627830	https://figshare.com/articles/journal_contribution/Figure_S2_from_Cell_of_Origin_Influences_Pancreatic_Cancer_Subtype/22535633/1/files/39998909.pdf	English	null	Figure S1 from Cell of Origin Influences Pancreatic Cancer Subtype	null	2,023	cc-by	1,951	C. tdTomato H&E Lung Metastasis Peritoneum Metastasis F. E. Primary Liver Bright-field Fluorescent Peritoneum Bowel Obstruction Ascites Jaundice Analysis of p53 missense mutants in acinar cell-derived pancreatic cancer development comparing pancreatic cancer-free survival of two different models of p53 deficiency. Pancreatic cancer incidence in KT;Ptf1aCreER;Trp53fl/fl mice (n = 23) and = 23), based on the log-rank test. Not significant = ns. (B) Kaplan-Meier analysis of pancreatic cancer-free survival of KT;Ptf1aCreER;Trp53fl/x (where x represents rts listed in Figure 2A and stratified by sex. Pancreatic cancer incidence in KT;Ptf1aCreER;Trp53fl/- female mice (n = 11) and male mice (n = 12) is similar based on the ncer incidence in KT;Ptf1aCreER;Trp53fl/LSL-R172H female mice (n = 9) and male mice (n = 16) is similar based on the log-rank test. Pancreatic cancer incidence in emale mice (n = 13) and male mice (n = 15) is similar based on the log-rank test. Not significant = ns. (C) Representative images of mice presenting with ascites, n in each cohort. (D) Representative histological image of a liver metastases in a KT;Ptf1aCreER;Trp53fl/LSL-R172H and a KT;Ptf1aCreER;Trp53fl/LSL-R270H mouse analyzed by stochemistry for tdTomato and CK19. Scale Bar = 100 μm. (E) Representative bright-field and fluorescence dissecting scope images of a primary acinar cell-derived um with macroscopic metastases. Scale Bar = 2 cm (F) Representative histological images of a lung metastasis in a KT;Ptf1aCreER;Trp53fl/LSL-R270H mouse and a perito- 1aCreER;Trp53fl/- mouse analyzed by H&E staining and tdTomato immunostaining. Scale Bar = 100 μm. tdTomato CK19 B. H&E tdTomato 100 150 200 250 ays Post-Tamoxifen Trp53fl/fl n=23 Trp53fl/- n=23 ns 0 50 100 150 200 250 300 0 20 40 60 80 100 Days Post-Tamoxifen Pancreatic Cancer Free Survival Trp53fl/- females Trp53fl/-males n=12 Trp53fl/LSL-R172H females Trp53fl/LSL-R172H males Trp53fl/LSL-R270H females Trp53fl/LSL-R270H males n=11 n=16 n=9 n=15 n=13 ns ns ns C. tdTomato H&E Lung Metastasis Peritoneum Metastasis F. E. Primary Liver Bright-field Fluorescent Peritoneum Bowel Obstruction Ascites Jaundice Analysis of p53 missense mutants in acinar cell-derived pancreatic cancer development comparing pancreatic cancer-free survival of two different models of p53 deficiency. Pancreatic cancer incidence in KT;Ptf1aCreER;Trp53fl/fl mice (n = 23) and = 23), based on the log-rank test. Not significant = ns. (B) Kaplan-Meier analysis of pancreatic cancer-free survival of KT;Ptf1aCreER;Trp53fl/x (where x represents rts listed in Figure 2A and stratified by sex. Pancreatic cancer incidence in KT;Ptf1aCreER;Trp53fl/- female mice (n = 11) and male mice (n = 12) is similar based on the ncer incidence in KT;Ptf1aCreER;Trp53fl/LSL-R172H female mice (n = 9) and male mice (n = 16) is similar based on the log-rank test. Pancreatic cancer incidence in emale mice (n = 13) and male mice (n = 15) is similar based on the log-rank test. Not significant = ns. (C) Representative images of mice presenting with ascites, n in each cohort. (D) Representative histological image of a liver metastases in a KT;Ptf1aCreER;Trp53fl/LSL-R172H and a KT;Ptf1aCreER;Trp53fl/LSL-R270H mouse analyzed by stochemistry for tdTomato and CK19. Scale Bar = 100 μm. (E) Representative bright-field and fluorescence dissecting scope images of a primary acinar cell-derived um with macroscopic metastases. Scale Bar = 2 cm (F) Representative histological images of a lung metastasis in a KT;Ptf1aCreER;Trp53fl/LSL-R270H mouse and a perito- 1aCreER;Trp53fl/- mouse analyzed by H&E staining and tdTomato immunostaining. Scale Bar = 100 μm. tdTomato CK19 B. H&E tdTomato 100 150 200 250 ays Post-Tamoxifen Trp53fl/fl n=23 Trp53fl/- n=23 ns 0 50 100 150 200 250 300 0 20 40 60 80 100 Days Post-Tamoxifen Pancreatic Cancer Free Survival Trp53fl/- females Trp53fl/-males n=12 Trp53fl/LSL-R172H females Trp53fl/LSL-R172H males Trp53fl/LSL-R270H females Trp53fl/LSL-R270H males n=11 n=16 n=9 n=15 n=13 ns ns ns C. tdTomato H&E Lung Metastasis Peritoneum Metastasis F. E. Primary Liver Bright-field Fluorescent Peritoneum Bowel Obstruction Ascites Jaundice Analysis of p53 missense mutants in acinar cell-derived pancreatic cancer development comparing pancreatic cancer-free survival of two different models of p53 deficiency. Pancreatic cancer incidence in KT;Ptf1aCreER;Trp53fl/fl mice (n = 23) and n = 23), based on the log-rank test. Not significant = ns. (B) Kaplan-Meier analysis of pancreatic cancer-free survival of KT;Ptf1aCreER;Trp53fl/x (where x represents rts listed in Figure 2A and stratified by sex. C. tdTomato H&E Lung Metastasis Peritoneum Metastasis F. E. Primary Liver Bright-field Fluorescent Peritoneum Bowel Obstruction Ascites Jaundice Analysis of p53 missense mutants in acinar cell-derived pancreatic cancer development comparing pancreatic cancer-free survival of two different models of p53 deficiency. Pancreatic cancer incidence in KT;Ptf1aCreER;Trp53fl/fl mice (n = 23) and = 23), based on the log-rank test. Not significant = ns. (B) Kaplan-Meier analysis of pancreatic cancer-free survival of KT;Ptf1aCreER;Trp53fl/x (where x represents rts listed in Figure 2A and stratified by sex. Pancreatic cancer incidence in KT;Ptf1aCreER;Trp53fl/- female mice (n = 11) and male mice (n = 12) is similar based on the ncer incidence in KT;Ptf1aCreER;Trp53fl/LSL-R172H female mice (n = 9) and male mice (n = 16) is similar based on the log-rank test. Pancreatic cancer incidence in emale mice (n = 13) and male mice (n = 15) is similar based on the log-rank test. Not significant = ns. (C) Representative images of mice presenting with ascites, n in each cohort. (D) Representative histological image of a liver metastases in a KT;Ptf1aCreER;Trp53fl/LSL-R172H and a KT;Ptf1aCreER;Trp53fl/LSL-R270H mouse analyzed by stochemistry for tdTomato and CK19. Scale Bar = 100 μm. (E) Representative bright-field and fluorescence dissecting scope images of a primary acinar cell-derived um with macroscopic metastases. Scale Bar = 2 cm (F) Representative histological images of a lung metastasis in a KT;Ptf1aCreER;Trp53fl/LSL-R270H mouse and a perito- 1aCreER;Trp53fl/- mouse analyzed by H&E staining and tdTomato immunostaining. Scale Bar = 100 μm. tdTomato CK19 B. H&E tdTomato 100 150 200 250 ays Post-Tamoxifen Trp53fl/fl n=23 Trp53fl/- n=23 ns 0 50 100 150 200 250 300 0 20 40 60 80 100 Days Post-Tamoxifen Pancreatic Cancer Free Survival Trp53fl/- females Trp53fl/-males n=12 Trp53fl/LSL-R172H females Trp53fl/LSL-R172H males Trp53fl/LSL-R270H females Trp53fl/LSL-R270H males n=11 n=16 n=9 n=15 n=13 ns ns ns Pancreatic cancer incidence in KT;Ptf1aCreER;Trp53fl/- female mice (n = 11) and male mice (n = 12) is similar based on the ncer incidence in KT;Ptf1aCreER;Trp53fl/LSL-R172H female mice (n = 9) and male mice (n = 16) is similar based on the log-rank test. Pancreatic cancer incidence in emale mice (n = 13) and male mice (n = 15) is similar based on the log-rank test. Not significant = ns. (C) Representative images of mice presenting with ascites, on in each cohort. (D) Representative histological image of a liver metastases in a KT;Ptf1aCreER;Trp53fl/LSL-R172H and a KT;Ptf1aCreER;Trp53fl/LSL-R270H mouse analyzed by stochemistry for tdTomato and CK19. Scale Bar = 100 μm. (E) Representative bright-field and fluorescence dissecting scope images of a primary acinar cell-derived um with macroscopic metastases. Scale Bar = 2 cm (F) Representative histological images of a lung metastasis in a KT;Ptf1aCreER;Trp53fl/LSL-R270H mouse and a perito- 1aCreER;Trp53fl/- mouse analyzed by H&E staining and tdTomato immunostaining. Scale Bar = 100 μm. tdTomato CK19 B. H&E tdTomato 100 150 200 250 ays Post-Tamoxifen Trp53fl/fl n=23 Trp53fl/- n=23 ns 0 50 100 150 200 250 300 0 20 40 60 80 100 Days Post-Tamoxifen Pancreatic Cancer Free Survival Trp53fl/- females Trp53fl/-males n=12 Trp53fl/LSL-R172H females Trp53fl/LSL-R172H males Trp53fl/LSL-R270H females Trp53fl/LSL-R270H males n=11 n=16 n=9 n=15 n=13 ns ns ns C. Bowel Obstruction Ascites Jaundice B. 0 50 100 150 200 250 300 0 20 40 60 80 100 Days Post-Tamoxifen Pancreatic Cancer Free Survival Trp53fl/- females Trp53fl/-males n=12 Trp53fl/LSL-R172H females Trp53fl/LSL-R172H males Trp53fl/LSL-R270H females Trp53fl/LSL-R270H males n=11 n=16 n=9 n=15 n=13 ns ns ns C B. 0 50 100 150 200 250 300 0 20 40 60 80 100 Days Post-Tamoxifen Pancreatic Cancer Free Survival Trp53fl/- females Trp53fl/-males n=12 Trp53fl/LSL-R172H females Trp53fl/LSL-R172H males Trp53fl/LSL-R270H females Trp53fl/LSL-R270H males n=11 n=16 n=9 n=15 n=13 ns ns ns A. 0 50 100 150 200 250 0 20 40 60 80 100 Days Post-Tamoxifen Pancreatic Cancer Free Survival Trp53fl/fl n=23 Trp53fl/- n=23 ns B. C. A. Ascites tdTomato H&E Lung Metastasis Peritoneum Metastasis F. E. Primary Liver Bright-field Fluorescent Peritoneum e mutants in acinar cell-derived pancreatic cancer development ncer-free survival of two different models of p53 deficiency. Pancreatic cancer incidence in KT;Ptf1aCreER;Trp53fl/fl mice (n = 23) and rank test. Not significant = ns. C. tdTomato H&E Lung Metastasis Peritoneum Metastasis F. E. Primary Liver Bright-field Fluorescent Peritoneum Bowel Obstruction Ascites Jaundice Analysis of p53 missense mutants in acinar cell-derived pancreatic cancer development comparing pancreatic cancer-free survival of two different models of p53 deficiency. Pancreatic cancer incidence in KT;Ptf1aCreER;Trp53fl/fl mice (n = 23) and = 23), based on the log-rank test. Not significant = ns. (B) Kaplan-Meier analysis of pancreatic cancer-free survival of KT;Ptf1aCreER;Trp53fl/x (where x represents rts listed in Figure 2A and stratified by sex. Pancreatic cancer incidence in KT;Ptf1aCreER;Trp53fl/- female mice (n = 11) and male mice (n = 12) is similar based on the ncer incidence in KT;Ptf1aCreER;Trp53fl/LSL-R172H female mice (n = 9) and male mice (n = 16) is similar based on the log-rank test. Pancreatic cancer incidence in emale mice (n = 13) and male mice (n = 15) is similar based on the log-rank test. Not significant = ns. (C) Representative images of mice presenting with ascites, n in each cohort. (D) Representative histological image of a liver metastases in a KT;Ptf1aCreER;Trp53fl/LSL-R172H and a KT;Ptf1aCreER;Trp53fl/LSL-R270H mouse analyzed by stochemistry for tdTomato and CK19. Scale Bar = 100 μm. (E) Representative bright-field and fluorescence dissecting scope images of a primary acinar cell-derived um with macroscopic metastases. Scale Bar = 2 cm (F) Representative histological images of a lung metastasis in a KT;Ptf1aCreER;Trp53fl/LSL-R270H mouse and a perito- 1aCreER;Trp53fl/- mouse analyzed by H&E staining and tdTomato immunostaining. Scale Bar = 100 μm. tdTomato CK19 B. H&E tdTomato 100 150 200 250 ays Post-Tamoxifen Trp53fl/fl n=23 Trp53fl/- n=23 ns 0 50 100 150 200 250 300 0 20 40 60 80 100 Days Post-Tamoxifen Pancreatic Cancer Free Survival Trp53fl/- females Trp53fl/-males n=12 Trp53fl/LSL-R172H females Trp53fl/LSL-R172H males Trp53fl/LSL-R270H females Trp53fl/LSL-R270H males n=11 n=16 n=9 n=15 n=13 ns ns ns (B) Kaplan-Meier analysis of pancreatic cancer-free survival of KT;Ptf1aCreER;Trp53fl/x (where x represents K19 H&E tdTomato 50 0 50 100 150 200 250 300 0 Days Post-Tamoxifen P tdTomato H&E Lung Metastasis Peritoneum Metastasis F. E. Primary Liver Bright-field Fluorescent Peritoneum H&E tdTomato y H&E tdTomato CK19 Trp53fl/LSL-R270H Trp53fl/LSL-R172H D. E. tdTomato H&E Lung Metastasis Peritoneum Metastasis F. H&E tdTomato F. D. lementary Figure 2. Analysis of p53 missense mutants in acinar cell-derived pancreatic cancer development aplan-Meier analysis comparing pancreatic cancer-free survival of two different models of p53 deficiency. Pancreatic cancer incidence in KT;Ptf1aCreER;Trp53fl/fl mice (n = 23) and f1aCreER;Trp53fl/- mice (n = 23), based on the log-rank test. Not significant = ns. (B) Kaplan-Meier analysis of pancreatic cancer-free survival of KT;Ptf1aCreER;Trp53fl/x (where x represents ent Trp53 alleles) cohorts listed in Figure 2A and stratified by sex. Pancreatic cancer incidence in KT;Ptf1aCreER;Trp53fl/- female mice (n = 11) and male mice (n = 12) is similar based on the nk test. Pancreatic cancer incidence in KT;Ptf1aCreER;Trp53fl/LSL-R172H female mice (n = 9) and male mice (n = 16) is similar based on the log-rank test. Pancreatic cancer incidence in f1aCreER;Trp53fl/LSL-R270H female mice (n = 13) and male mice (n = 15) is similar based on the log-rank test. Not significant = ns. (C) Representative images of mice presenting with ascites, ice, or bowel obstruction in each cohort. (D) Representative histological image of a liver metastases in a KT;Ptf1aCreER;Trp53fl/LSL-R172H and a KT;Ptf1aCreER;Trp53fl/LSL-R270H mouse analyzed by staining and immunohistochemistry for tdTomato and CK19. Scale Bar = 100 μm. (E) Representative bright-field and fluorescence dissecting scope images of a primary acinar cell-derived and liver and peritoneum with macroscopic metastases. Scale Bar = 2 cm (F) Representative histological images of a lung metastasis in a KT;Ptf1aCreER;Trp53fl/LSL-R270H mouse and a perito- metastasis in a KT;Ptf1aCreER;Trp53fl/- mouse analyzed by H&E staining and tdTomato immunostaining. Scale Bar = 100 μm.
https://openalex.org/W3032586084	https://vtechworks.lib.vt.edu/bitstream/10919/98777/1/PatoliaFiltration.pdf	English	null	Filtration evaluation and clinical use of expired elastomeric P-100 filter cartridges during the COVID-19 pandemic	Infection control and hospital epidemiology	2,020	cc-by	1,299	Filtration evaluation and clinical use of expired elastomeric P-100 filter cartridges during the COVID-19 pandemic Harsh H. Patolia MD1,a , Jin Pan MS2,a, Charbel Harb BE2, Linsey C. Marr PhD2 and Anthony W. Baffoe-Bonnie MD1,3 1Virginia Tech Carilion School of Medicine, Roanoke, Virginia, 2Civil and Environmental Engineering, Virginia Tech, Blacksburg, Virginia and 3Carilion Clinic Roanoke Memorial Hospital, Roanoke, Virginia under investigation that we received from the strategic national stockpile. P100 respirators are designed to filter out at least 99.97% of airborne particles. Although data regarding the perfor- mance of P100 respirators for viral aerosols are scarce, clear evi- dence has demonstrated their filtration superiority compared to N95 respirators.4 During our initial distribution of P100 elasto- meric respirators, we observed that our supply of filter cartridges from the strategic national stockpile had surpassed their 5-year shelf life; they had expired in 2014. They were well kept in unopened packages. Based on collected feedback from frontline providers, many expressed concern and alarm regarding the effectiveness of these devices and their filtering efficiency. In an effort to rapidly verify the filtering efficiency of our elastomeric res- pirator supply and to ensure the safety of our provider staff, we evaluated a sample of our P100 filter cartridges using quantitative methods. To the Editor—The limited supply of more conventional dispos- able personal protective equipment (PPE), namely single-use N95 filtering facepiece respirators (FFRs), among hospital systems in the United States during the COVID-19 pandemic has been alle- viated with the adoption of extended use and reuse policies by the Centers for Disease Control and Prevention (CDC).1 These measures, along with a variety of implemented decontamination methodologies (eg, ultraviolet germicidal irradiation, vaporized hydrogen peroxide, etc), have prolonged PPE supplies during pressing times. Another strategy adopted by the CDC and health systems to protect healthcare providers caring for COVID-19 patients and patients under investigation in limited resource set- tings includes the use of elastomeric FFRs with reusable cartridges. Although elastomeric respirators have not been approved by the Food and Drug Administration for fluid resistance, they have been endorsed by the CDC as reasonable alternatives for N95 FFRs dur- ing the COVID-19 pandemic due to their filtration approval by the National Institutes for Occupational and Safety Health (NIOSH).2 Subsequently, elastomeric respirators have formed a major arm of the COVID-19 pandemic response strategy in many hospital systems. Filtration evaluation and clinical use of expired elastomeric P-100 filter cartridges during the COVID-19 pandemic Filtration efficiency was evaluated by challenging the cartridges with aerosols generated from a 2% sodium chloride solution using a Collison 3-jet nebulizer (BGI MRE-3) in a 280-L polyethylene chamber (Sigma AtmosBag). The concentration and size distribu- tion of the aerosols were measured using a scanning mobility par- ticle sizer (TSI SMPS 3936). For testing, the cartridge was sealed inside a cardboard box, and the cartridge and box were further covered with electrical tape, except for the opening of the cartridge, to prevent leakage through the box itself. A vacuum line was con- nected to the box, and the challenge aerosol was pulled through the cartridge at a flow rate of 15 L per minute. Figure 1 shows the filtration efficiency of the cartridge compared to that of an N95 that was measured using the same experimental setup. The cartridge removed at least 90% of aerosols over the size range of 0.04–1 μm, including the most penetrating size of 0.1–0.3 μm where it demon- strated an efficiency comparable to the N95 respirator. y Elastomeric FFRs contain plastic components that can be easily decontaminated for repeated use. Those elastomeric respirators containing a full facepiece can afford greater protection to wearers by diminishing face seal leakage and provide eye protection. Although various types of elastomeric FFRs exist, their filtration mechanism is universally housed in exchangeable cartridge filters. The replacement of these filters is determined by their respective expiration dates indicated by the manufacturers as well as the pres- ence of visible soiling, contamination, and/or damage. The stra- tegic national stockpile was not significantly restocked after it had been mobilized for the 2009 influenza pandemic.3 Thus, car- tridges for elastomeric respirators in the strategic national stockpile may have surpassed their expiration dates, which voids NIOSH approval regarding filtration efficiency. Our studies demonstrated that the filtration efficiency of P100 filter cartridges past their 5-year expiration date was not significantly different from that of an N95 respirator (3M 1860) (P > .05), although it was <99.97%. These studies are preliminary, but they suggest the possibility of safely implementing expired fil- ter cartridges in elastomeric respirators during limited resource scenarios and patient surges, particularly during the significant logistical challenge presented by the COVID-19 pandemic. With considerable disruptions in global supply chains for N95 respira- tors, hospital systems may consider adopting guidelines that conditionally endorse the use of expired filter cartridges in elasto- meric FFRs. Infection Control & Hospital Epidemiology (2020), 1–2 Infection Control & Hospital Epidemiology (2020), 1–2 Infection Control & Hospital Epidemiology (2020), 1–2 doi:10.1017/ice.2020.257 Infection Control & Hospital Epidemiology (2020), 1–2 doi:10.1017/ice.2020.257 Author for correspondence: Anthony W. Baffoe-Bonnie, E-mail: abaffoe6@vt.edu aAuthors of equal contribution. Cite this article: Patolia HH, et al. (2020). Filtration evaluation and clinical use of expired elastomeric P-100 filter cartridges during the COVID-19 pandemic. Infection Control & Hospital Epidemiology, https://doi.org/10.1017/ice.2020.257 © 2020 by The Society for Healthcare Epidemiology of America. All rights reserved. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. Acknowledgments. None. Acknowledgments. None. 2. Elastomeric respirators: strategies during conventional and surge demand situations. Centers for Disease Control and Prevention website. https:// www.cdc.gov/coronavirus/2019-ncov/hcp/elastomeric-respirators-strategy/ index.html. Accessed April 23, 2020. 2. Elastomeric respirators: strategies during conventional and surge demand situations. Centers for Disease Control and Prevention website. https:// www.cdc.gov/coronavirus/2019-ncov/hcp/elastomeric-respirators-strategy/ index.html. Accessed April 23, 2020. Financial support. No financial support was provided relevant to this article. Financial support. No financial support was provided relevant to this article. Conflicts of interest. All authors report no conflicts of interest relevant to this article. Conflicts of interest. All authors report no conflicts of interest relevant to this article. 3. Reinhard B, Brown E. Face masks in national stockpile of medical supplies have not been substantially replenished since 2009. The Washington Post website. https://www.washingtonpost.com/investigations/face-masks- in-national-stockpile-have-not-been-substantially-replenished-since-2009/ 2020/03/10/57e57316-60c9-11ea-8baf-519cedb6ccd9_story.html. Accessed May 11, 2020. 4. Reusable elastomeric respirators in health care. Reusable elastomeric respi- rators in health care. National Academies Press; 2019. Filtration evaluation and clinical use of expired elastomeric P-100 filter cartridges during the COVID-19 pandemic At our institution, we hoped to distribute the P100 elastomeric respirators to providers caring for COVID-19 patients and patients © 2020 by The Society for Healthcare Epidemiology of America. All rights reserved. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. 2 2 Harsh H. Patolia et al Fig. 1. Filtration efficiency of the P100 filter cartridge and 3M 1860 (N95) for salt (NaCl) particles 0.04–1.0 μm in aerodynamic diameter. Error bars represent standard deviations across 2 car- tridges, each tested twice. 2 Harsh H. Patolia et al c - Fig. 1. Filtration efficiency of the P100 filter cartridge and 3M 1860 (N95) for salt (NaCl) particles 0.04–1.0 μm in aerodynamic diameter. Error bars represent standard deviations across 2 car- tridges, each tested twice. References 1. Recommended guidance for extended use and limited reuse of N95 filtering facepiece respirators in healthcare settings. Centers for Disease Control and Prevention website. https://www.cdc.gov/niosh/topics/hcwcontrols/ recommendedguidanceextuse.html. Accessed April 2, 2020. 4. Reusable elastomeric respirators in health care. Reusable elastomeric respi- rators in health care. National Academies Press; 2019. 4. Reusable elastomeric respirators in health care. Reusable elastomeric respi- rators in health care. National Academies Press; 2019.
https://openalex.org/W2072096048	https://link.springer.com/content/pdf/10.1007/s13437-014-0067-8.pdf	English	null	Development of a prototype tool for ballast water risk management using a combination of hydrodynamic models and agent-based modeling	WMU journal of maritime affairs	2,014	cc-by	11,286	WMU J Marit Affairs (2015) 14:219–245 DOI 10.1007/s13437-014-0067-8 WMU J Marit Affairs (2015) 14:219–245 DOI 10.1007/s13437-014-0067-8 ARTICLE Electronic supplementary material The online version of this article (doi:10.1007/s13437-014-0067-8) contains supplementary material, which is available to authorized users. 1 Introduction Transfers of non-indigenous species may potentially pose a threat to the receiving ecosystem and to the society. Organisms that mass-reproduce when they are transported and released in new environments by humans are “non-indigenous invasive species”, sometimes referred to as “alien species” (definitions and discussions of these concepts can be found in Ruiz and Carlton 2003). Ultimately, they may have serious impact on ecosystems both ecologically and economically. Ships’ ballast water is a main source of non-indigenous marine organisms and, when released, some of these species have caused dramatic and permanent damage to coastal ecosystems around the world (e.g., Bax et al. 2003; Leppäkoski et al. 2003). Since it has been impossible in practise to control and mitigate the spreading of established invasive species in “new” marine environments, efforts should focus on the prevention of introductions. The trend in biological invasion shows an exponential increase during the last 200 years (Leppäkoski et al. 2003). Nevertheless, the problem of marine bio-invasion and the resulting environmental consequences has taken time to be recognized. One of many reasons is that unlike terrestrial ecosystems, bioinvasions in the marine environ- ment are poorly understood since their consequences may be difficult to observe. Many bioinvasions are considered a threat to the ecosystems, to human health as well as to the economy. The invaded ecosystems are threatened as exotic species may alter ecosystem functions and community structure. In some cases, non-indigenous species cause a dramatic decline in important marine resources by competing with the native species for food, habitat, by predation/parasitism, or due to other indirect interactions. Biological invasions may also interact in combination with other natural and anthro- pogenic environmental factors such as climate change, habitat destruction and pollution to jeopardize the integrity of the ecosystem. This means that the impacts of future introductions are uncertain as well as unpredictable and current invasions may evolve into unforeseen intricate patterns. More challenges are forecast to come in handling introductions and efforts should be focused on preventing new establishments in order to protect the native biota and its diversity by means of managing human activity. Development of a prototype tool for ballast water risk management using a combination of hydrodynamic models and agent-based modeling Flemming T. Hansen & Michael Potthoff & Thomas Uhrenholdt & Hong D. Vo & Olof Linden & Jesper H. Andersen Received: 13 August 2013 /Accepted: 7 July 2014 /Published online: 14 August 2014 # World Maritime University 2014 Abstract We report the development of a prototype tool for modeling the risks of spreading of non-indigenous invasive species via ballast water. The tool constitutes of two types of models: a 3D hydrodynamical model calculates the currents in the North Sea and Danish Straits, and an agent-based model estimates the dispersal of selected model organisms with the prevailing currents calculated by the 3D hydrodynamical model. The analysis is concluded by a postprocessing activity, where scenarios of dispersal are combined into an interim estimate of connectivity within the study area. The latter can be used for assessment of potential risk associated with intentional or unintentional discharges of ballast water. We discuss how this prototype tool can be used for ballast water risk management and outline other functions and uses, e.g., in regard to ecosystem-based management and the implementation of the EU Marine Strategy Framework Directive. Keywords Invasive species . non-indigenous species . ballast water. agent-based modelling . individual-based modelling . ecological connectivity. risk assessment . water framework directive . WFD . MIKE 3 . ECO Lab . ABM Lab Electronic supplementary material The online version of this article (doi:10.1007/s13437-014-0067-8) contains supplementary material, which is available to authorized users. F. T. Hansen (): M. Potthoff: T. Uhrenholdt DHI, Hørsholm, Denmark e-mail: fth@dhigroup.com F. T. Hansen (): M. Potthoff: T. Uhrenholdt DHI, Hørsholm, Denmark e-mail: fth@dhigroup.com O. Linden World Maritime University, Malmø, Sweden O. Linden World Maritime University, Malmø, Sweden J. H. Andersen Aarhus University, Aarhus, Denmark Present Address: H. D. Vo Queensland, Australia 220 F.T. Hansen et al. F.T. Hansen et al. 1 Introduction The scope of this work is to demonstrate the usefulness of the combination of hydrodynamical (HD) and agent-based models (ABM) as a tool for understanding the ecological connectivity of marine areas of the North Sea, Kattegat, and the inner Danish straits and their application for risk assessment of invasive species brought to the region (or transported within the region) by ballast water from sea vessels. Ecological connec- tivity mapping is proposed to provide a theoretical basis for identifying areas where ballast water could be released with the least potential risk for species to spread extensively within the region. In addition, connectivity mapping can provide knowledge on how well each specific part of the region is connected with other parts of the region. 2 Methods 2.1 Study area The study area is the North Sea, located on the continental shelf of north-western Europe and bordered by England, Scotland, Sweden, Norway, Denmark, Germany, the Development of a prototype tool for ballast water risk management 221 Netherlands, Belgium, and France. It opens out to the Atlantic Ocean, the English Channel, and towards the less saline Baltic Sea. Netherlands, Belgium, and France. It opens out to the Atlantic Ocean, the English Channel, and towards the less saline Baltic Sea. The Greater North Sea (as defined by OSPAR) has a surface area of 750,000 km2 and a volume of 94,000 km3 and is separated into various areas, the relatively shallow southern North Sea (the Southern Bight and the German Bight), the central North Sea, the northern North Sea, the Norwegian Trench, and the Skagerrak, which is a shallow transition zone between the Baltic and the North Sea (OSPAR 2010). In the eastern part of the North Sea, along the coasts of Denmark and Germany, mean winter sea surface temperature (SST) is usually less than 3 °C. The summer mean SST is 18 °C and declines to 13 °C northwards along eastern Britain (Hayward and Ryland 1995). The annual freshwater river input is ca 300 km3, about one third of that comes from the snow-melt waters of Norway and Sweden and the rest from major rivers while the main source of fresh water supply is through the Baltic Sea (Hayward and Ryland 1995). The most prominent current circulation of the North Sea is a roughly anticlockwise flow where residual currents moves southwards along the east coast of the UK and northwards along the Western European coast. Saline water enters the Baltic Sea through Kattegat at depth while surface flow of brackish water from the Baltic Sea enters the Kattegat and North Sea (OSPAR 2010). The North Sea, which is an economically and ecologically important marine region, is sensitive to a range of human activities. Key issues are nutrient enrichment and eutrophication, contamination with hazardous substances, overfishing, physical modi- fication and an unfavorable biodiversity status (OSPAR 2010; HELCOM 2010). Introduction of non-indigenous species has been identified as an emerging issue. Many areas in the North Sea are valuable habitat for marine life as well as of economic importance for the surrounding states. 2 Methods The shallow and productive North Sea, while being of one of the busiest seas in the world in seaborne trading, is also heavily exploited for its natural resources. Activities such as fishing, dredging, oil and gas exploration, shipping, discharges of nutrients and contaminants have polluted as well as depleted reserves in the area. Increases in awareness for the protection of the environment and resource management in the North Sea have surged during the last decades (Misund and Skjoldal 2005), and one significant issue that has been identified relatively recently is the ramifications of species introduction via ship transportation and accidental releases from aquaculture. The most up-to-date inventory of alien species in the North Sea region lists alien aquatic species of 167 taxa (Gollasch et al. 2009). This calls for a precautionary approach to prevent future arrivals of new species. One of the key vectors in moving aquatic alien species is shipping, e.g., ballast water-mediated species introductions from ships prevail in many regions world-wide. 2.3 Model setup A 3D North Sea hydrodynamic model is applied as the basis for the agent-based modeling (ABM) and subsequent connectivity modeling. A period of a full year (2005) has been modeled in order to capture the seasonal and higher frequency variability of the North Sea circulation. The model represents the overall circulation patterns in the North Sea and the Belt Sea comprising of tide, meteorologically and density-driven circulation, freshwater inputs, and stratification. A local 3D hydrody- namic model resolving the Belt Sea in a higher resolution has also been applied. The model domain includes the major part of the North Sea, the Belt Sea, and the Baltic Sea. The model applies a Cartesian grid in UTM-32 projection with a horizontal resolution of 3 nautical miles. In the vertical dimension, a 2-m resolution is used, with a maximum of 110 layers depending on the local water depth. However, the surface layer with surface elevation varying with the actual tide has a typical thickness of 5 m. For areas with depths under level −223 m, the rest of the water column is included in the lowest layer. The model domain is shown in Fig. 1. The model has been run for the period 2000–2008, but the period applied for the present purpose is the year 2005. The model runs with a hydrodynamic time step of 300 s. The model results in terms of 3D fields of, for example, current, salinity, and temperature are saved every 1 h. The forcings on the open boundaries towards the Norwegian Sea and the English Channel include: (1) Astronomical tide along boundary (actual values for 2005), (2) salinity distribution in vertical sections (monthly climatologic from ICES), and (3) temperature distribution in vertical sections (monthly climatologic from ICES). Atmospheric forcings (e.g., wind, air pressure, air temperature, cloudiness, and precip- itation (actual 2D maps with 1 h resolution)) were originally delivered by Vejr2, a former meteorological forecast service provider. The wind and air pressure are incor- porated in the momentum equations, and the precipitation is used in the mass equation. Wind is also included in the turbulence module. The heat exchange module, which calculates the sea-air heat exchange, makes use of wind, air temperature and cloudiness. The runoff in terms of discharges of freshwater from land to the model domain is represented in the model by 85 source points. 2.2 Hydrodynamic model The applied hydrodynamic model is based on the MIKE 3 modeling system developed by DHI. The MIKE 3 model is a dynamic time-dependent 3D baroclinic model for free surface flows. The mathematical foundation of the model are the Reynolds-averaged Navier-Stokes equations in three dimensions, including the effects of turbulence and variable density, together with conservation equations for mass, heat, and salt, an 222 F.T. Hansen et al. equation of state for the density, a turbulence module and a heat exchange module. The equations are solved on a Cartesian grid by means of the finite difference techniques. The hydrodynamic model provides a full 3D model representation of the water levels, flows, salinity, temperature, and density within the modeling domain. For more information on the MIKE 3 modeling system, reference is made to DHI (2009) and DHI (2011). 2.3 Model setup The runoff is based on data from SMHI’s operational HBV model and on data from Global Runoff Data Centre (GRDC). It is important to note that the applied sources are lumped sources, which means that they represent the main rivers at each location as well as the non-resolved rivers/inflows in the vicinity of the location. This means that the total runoff to the Baltic Sea/North Sea is correctly represented in the 85 model sources. Initial fields of salinity and temper- ature, i.e., 3D fields of salinity and temperature within the model domain, have been established based on previous model runs. The present hydrodynamic model setup is an updated version of the model used for the BANSAI project (SMHI 2006). Development of a prototype tool for ballast water risk management 223 Fig. 1 Model domain and location of selected measurement stations. The thick black lines indicate the open boundaries of the model Fig. 1 Model domain and location of selected measurement stations. The thick black lines indicate the ope boundaries of the model Since the North Sea hydrodynamic model is relatively coarse in the Belt Sea (3 nautical miles cell size), a local hydrodynamic model covering the Belt Sea and the Baltic Sea was also applied. This model has a finer resolution (down to 1 km cell size) in the Belt Sea as illustrated in Fig. 2 and thus represents the flow here in more detail. The model was developed for another purpose, but has been made available for the present purpose (FEHY 2012). 2.4 Agent-based modeling Agent-based models (ABM) have been widely applied in recent years for simulating a variety of phenomena within very diverse disciplines such as biology and ecology, social sciences, industrial process optimization, traffic infrastructure planning, and the financial sector just to mention a few examples. In ecology, ABMs aim at describing the behavior and state of discrete entities such as individual organisms or groups of organisms (∼superindividuals). One key element of ABMs is the ability to simulate how individuals respond (in terms of behavior and state) to a spatially and temporarily varying environment. When studying the aquatic environment and how small individ- uals spread within an aquatic system, information on water movement is a fundamental need. Here, 3D hydrodynamical models describing the water currents in high temporal and spatial resolution can provide a very detailed basis for these types of studies. By defining agents as discrete entities with an explicit x-y-z coordinate at any given point in time it is possible to link this type of ABM with a hydrodynamical model and thus combine the current driven movement of agents (=advection/dispersion) with F.T. Hansen et al. 224 Fig. 2 Section of the computational mesh for the hydrodynamic model covering the inner Danish waters. Colors indicate depth intervals in meters Fig. 2 Section of the computational mesh for the hydrodynamic model covering the inner Danish water Colors indicate depth intervals in meters biological movement behavior processes such as horizontal swimming, vertical migra- tion, and age-induced settling. This type of ABM is often referred to as a Lagrangian type ABM and has been applied frequently, e.g., in studies examining the spreading and migration of pelagic larvae and fish (e.g., Goodwin et al. 2001; Humston et al. 2000; Cowen et al. 2006). To simulate the potential spread of marine invasive species deriving from ballast water an (Lagrange type) ABM was developed and applied in combination with the hydrodynamical model as described in the previous section. The ABM framework applied is an integrated part of the ecological modeling software ECO Lab, which is an open equation solver for building and executing biological and ecological models of aquatic systems. The developed ABM is applied here to simulate the spread of agents (or organisms) in the entire model area primarily driven by advective processes predicted by the hydrodynamical model. 2.4 Agent-based modeling Three model organisms are chosen to represent examples of major groups of marine organisms likely to be introduced as invasive species through the release of ballast water within the North Sea region. Here “groups of marine organisms” refer to organisms which exhibit common behavioral characteristics. The groups of organisms considered here include representatives of a: (1) planktonic species (purely passive drifters), (2) pelagic larvae of a benthic invertebrate species (passive drifters in combination with settling), and (3) fish species (passive drifters in combi- nation with active swimming activity). We mimic the spread of these three types of Development of a prototype tool for ballast water risk management 225 model organisms in a very simplistic way by: (1) simulating passive drift in combina- tion with a constant mortality rate, (2) simulating passive drift in combination with active settling activity triggered as a function of age, and (3) simulating passive drift in combination with active horizontal swimming behavior including mortality rate. In addition to these three simulations, as a reference, we simulate passive drifting only subject to advection/dispersion processes. The approach described here is merely an attempt to address the spreading mech- anisms and potential of small marine organisms in a general way. The approach is deliberately not addressing species-specific spreading. Also, when considering the risk of introducing invasive species in the marine environment species-specific habitat preferences, life histories and environmental tolerances are essential to understand and predict the ability of introduced species to establish a sustainable population. These issues are not addressed here. However, species-specific traits and the implica- tion for establishing sustainable populations can be simulated by extending the current approach combining hydrodynamic modeling and ABM with habitat maps and/or classical concentration-based (Euler type) ecological modeling. The latter describing any necessary dynamical parameter affecting the organism such as salinity, tempera- ture, dissolved oxygen, food abundance, etc. Unlike the hydrodynamical modeling, due to the nature of the phenomena modeled neither calibration nor validation of the ABM is being considered. This means that the modeling exercise here apart from the hydrodynamical modeling is predominantly theoretical. The ABM approach can be seen as an interpreter to describe how common behavioral characteristics and traits may or may not result in significant deviations from passive drift. The results of the model approach should be evaluated as such. 2.5 ABM formulation Selected simplistic functional behaviors and life histories used for formulation of the ABM include: (1) Movement in terms of dispersion and active swimming, (2) settling, (3) mortality, and (4) longevity. The background for and the ABM formulations used in this study are described in Appendix 1 in the Online Supplementary Material. Please note that this appendix includes additional references not cited in this paper. 2.6 Connectivity mapping 2.6 Connectivity mapping The combination of HD modeling and ABMs (or particle tracking models) has been applied in several studies addressing the degree of connectivity between specific habitats or subregions within marine regions (Cowen et al. 2003, 2006; Paris et al 2005; Christensen et al 2008; Berglunda et al. 2012) However, in most cases, the studies have focused on specific species and connectivity between the species-specific habitats, or in more general connectivity in terms of, e.g., dispersal of passively drifting larvae between specific habitats such as coral reefs, spawning sites, etc. A more general approach applying a combination of hydrodynamical modeling and simple particle tracking has been proposed to establish a framework for identifying connectivities of any sub-region within the South-Western shelf region of Australia (Condie et al. 2006). In short, the particle trajectories from the simulations are treated statistically and translated into probability maps describing the “probability of any two regions within 226 F.T. Hansen et al. Fig. 3 Sketch of the differences between the concepts of downstream connectivity and upstream connectivity Fig. 3 Sketch of the differences between the concepts of downstream connectivity and upstream connectivi the model domain being connected by the modeled circulation”. Probabilities were “computed for a range of dispersion times on a 0.1 degree geographical grid” covering the model domain. The connectivity statistics addresses connectivities in discrete points in time and space as well as statistics aggregated for longer periods (months or quarters). As part of the project, a web-based service was developed where users can select a “source area” from a map interface, select start time, and dispersal duration, and as a result get a connectivity probability map for the selected source area and dispersal duration. In this study, the above-outlined approach has been further developed, e.g., by (1) including not only passive particle tracking but also biological processes by applying ABM techniques and by (2) developing an overall connectivity index or indices compiling all connectivity statistics of each local area into a single value. In order to define “connectivity”, we discriminate between two types of connectiv- ities: downstream connectivity and upstream connectivity (Fig. 3). Downstream connectivity we define as connectivity between donor areas (or source areas), and surrounding areas (or receiving areas). Here “areas” does not necessarily refer to computational grid cells but rather any areal division of the model domain into, e.g., a regular grid or a number of management units. 2.7 Connectivity probability All agent trajectories stored every 6 h of the 1-year simulation period are analyzed statistically. The model domain is divided into 25×25 km quadratic grid cells resulting in approximately 1,000 local areas covering the sea area. This is the spatial resolution at which connectivities will be evaluated. For each agent registered in an area, the future and previous location of each agent is registered/tracked considering four dispersal times (forward and backwards in time). This is repeated for every 6 h. For details on the selection of dispersal times, see the next section. To calculate downstream connectivity for the whole simulation period, the numbers of agents remaining in the area as well as appearing in other areas at each of the four dispersal times are counted. Downstream connectivity probabilities are calculated simply by dividing these numbers for each area by the total number of agents registered in the area analyzed. The outcome is a distribution map for each area showing the distribution of probabilities, i.e., the probability of an agent in an area to be registered sometimes in the future in the same area and in each of the surrounding areas. In cases where mortality is included in the scenario, probabilities are weighted according to the likelihood that an agent survives each of the four dispersal times. To calculate upstream connectivity for the whole simulation period for an area, the numbers of agents originating from the same area as well as each of the surrounding areas at each of the four dispersal times backwards in time are counted. Upstream connectivity probabilities are calculated by dividing these numbers by the total number of agents registered in the area analyzed. Also here, the outcome is a distribution map for each area showing the distribution of probabilities, i.e., the probability of an agent in an area having visited each of the surrounding areas including the probability of agents that have remained in the area analyzed during the dispersal times considered. For scenario 1 (passive drifting), scenario 2 (planktonic organism), and scenario 3 (juvenile fish) these procedures are repeated for all 25×25 km areas. For scenario 4, the combination of mortality and settling was not simulated in one simulation. Since mortality is high (0.1 per day) and mean settling age is 30 days, only a very little fraction of introduced agents will reach settling age. 2.6 Connectivity mapping During a simulation, each agent “visiting” an area at any time will have a distinct trajectory forward in time visiting other areas in the model domain. When simulating a large number of agents, the equivalent large number of trajectories forward in time can be statistically analyzed revealing the probability of areas to supply agents to other areas. This we refer to a downstream connectivity probability. Downstream connectivity answers questions such as “where do the agents go from here?” Upstream connectivity we define as connectivity between receiving areas and source areas. Again “areas” can refer to any areal division of the model domain. During a simulation, each agent “visiting” an area at any time of the simulation will have a distinct trajectory backwards in time having visited other areas in the model domain Development of a prototype tool for ballast water risk management 227 prior to the registration of the agent in the area analyzed. When simulating a large number of agents, the equivalent large number of trajectories backwards in time can be statistically analyzed revealing the probability of areas to receive agents from other areas. This we refer to as an upstream connectivity probability. Upstream connectivity answers questions such as “where do the agents come from?” Both upstream and downstream connectivity probabilities can be evaluated at any dispersal time ranging from seconds to years depending on the organism and dispersal phenomenon considered. 2.7 Connectivity probability To achieve sufficient statistical basis for the connectivity analysis, this would require a very large number of agents in the model simulation. For technical reasons, this was not desirable. Instead mortality was taken into account as part of the postprocessing of the model results. For each agent registered in an area every 6 h, the future location where it settles is registered. For all agents registered in each area at any time for the whole simulation period, the numbers of agents settled in each of the surrounding areas, including the area analyzed, are counted and the probabilities are calculated by dividing these 228 F.T. Hansen et al. F.T. Hansen et al. numbers by the total number of agents registered in the area analyzed. To account for mortality (0.1 per day), the number of agents settled in an area is adjusted using the following equation: numbers by the total number of agents registered in the area analyzed. To account for mortality (0.1 per day), the number of agents settled in an area is adjusted using the following equation: N adj ¼ N 1– k ð Þt ð2:1Þ ð2:1Þ Nadj is the adjusted number of settled agents N the number of agents settled in an area k the mortality rate per day (=0.1 per day ∼0.1 daily mortality probability), and t time between the time an agent is registered until it settles. Similarly to the downstream dispersal probabilities, prior to calculating the upstream dispersal probabilities for each area in scenario 4, numbers of agent originating from each of the surrounding areas are adjusted according to the equation above, Nadj now being the adjusted number of agents at the point in time where agents are discharged into the water, and t is the time between the time of discharge until the agents is settled. For downstream connectivity probabilities in scenario 4, since agents are registered every 6 h the same agent will be registered every sixth hour starting at the time of its introduction to the model domain until it settles somewhere (on average 30 days after introduction). Every consecutive 6-h time step that the same agent is registered, the time remaining until settling decreases by 6 h. 2.7 Connectivity probability Table values represent probabilities of agents in one area (=source area) ending up in another area (=receiving area) sum of probabilities will be less than 1, with all probabilities (in case of downstream connectivity) representing the probability of an organism being distributed to other areas a specific point in time ahead. sum of probabilities will be less than 1, with all probabilities (in case of downstream connectivity) representing the probability of an organism being distributed to other areas a specific point in time ahead. Since connectivity probabilities are expected to vary spatially depending on the dispersal time considered, four dispersal times were selected for each analysis in order to cover a range of dispersal situations for a given organism. These four dispersal times were selected for each analysis from three criteria: (1) ecological relevance, (2) a minimum of 10 % of agents at t=0 remaining at any dispersal time, and (3) seasons evenly reflected in calculation results. Here “ecological relevance” refers to, e.g., that the dispersal time should lie within the expected life duration and/or the duration of the pelagic stage. For scenario 1 (passive drifting) upstream and downstream connectivity probabilities are calculated for different sets of four dispersal times: 2, 4, 6, and 8 days, 3, 9, 15, and 21 days, and 7, 14, 21, and 28 days. The different sets of dispersal times are used for comparison with scenarios 2, 3, and 4 (see below), and in order to evaluate how selections of different dispersal times may influence the calculated connectivities. A maximum of 28 days were applied primarily to ensure that months and seasons were evenly reflected in calculation results. For scenario 2 (planktonic species) upstream and downstream connectivity proba- bilities are calculated for four dispersal times: 3, 9, 15, and 21 days. These were selected for the following reasons: since a mortality of 0.1 day−1 is applied, 10 % of the agents at t=0 remains approximately 24 days later. The 24 days were divided evenly into four time periods (0–6 days, 6–12 days, etc.) and the mean day numbers of each of four 6-day periods were selected as the four dispersal times (∼3, 9, 15, and 21 days). For scenario 3 (fish), upstream and downstream connectivity probabilities are calculated for four dispersal times: 1, 2, 3, and 4 weeks. 2.7 Connectivity probability In this way, all the registrations of an agent, each time representing a time and location of release of ballast water, will correspond to the assumption that the distribution of larvae age classes is uniformly distributed in the ballast water at the time of release. Notice that for scenario 4 while downstream connectivity probabilities reflect the assumption that all age classes are evenly represented in the ballast water at time of discharge, upstream connectivity probabilities reflect the case where organisms are age class zero at the time of discharge. The outcome of the connectivity probability mapping for all scenarios as described above consists of connectivity probability matrices equivalent to distance matrices applied, e.g., as a look up table for distances in kilometers between major cities or locations in road atlases. Instead of distances in kilometers, the connectivity matrices include numbers representing probabilities of, in case of downstream connectivity, agents in area A ending up in area B and agents in area B ending up in area A. Contrary to a distance matrix, either direction has different probabilities. A conceptual example is shown in Table 1. The resulting connectivity matrices will consist of approximately two 1,000×1,000 matrices for downstream and upstream connectivities respectively, for each set of dispersal times. Connectivity probabilities for each area can be extracted from the two matrices to produce connectivity probability maps for downstream and upstream connectivities respectively. These approximately 2,000 maps can be referred to a multilayer connectivity maps or MCMs and are available as Online Supplementary Material. Ideally, in cases where all agents simulated as passive drifters (as in scenario 1) and where agents are not subject to mortality, the sum of probability values within each probability map will be 1. However, when mortality is included as part of the ABM, the Development of a prototype tool for ballast water risk management 229 Table 1 Example of a downstream connectivity matrix for 4 areas (1–4). Table values represent probabilities of agents in one area (=source area) ending up in another area (=receiving area) Source area Receiving area 1 2 3 4 Sum 1 0.92 0.04 0.00 0.04 1.00 2 0.01 0.31 0.08 0.60 1.00 3 0.07 0.50 0.29 0.14 1.00 4 0.00 0.15 0.63 0.22 1.00 Sum 1.00 1.00 1.00 1.00 Table 1 Example of a downstream connectivity matrix for 4 areas (1–4). 2.7 Connectivity probability These were selected for the following reasons: since juvenile fish typically has much lower mortality (here, 0.003 day−1) 10 % of agents will remain after approximately 2 years. Thus, to cover the entire time span, this will require a much longer simulation than the 1-year simulation for the current study. In addition, the need to reflect months and seasons evenly results in a maximum of approximately 1 month dispersal time being selected. Based on these considerations, four weekly dispersal times were selected. 230 F.T. Hansen et al. For scenario 4 (pelagic larvae of benthic invertebrate) upstream and downstream connectivity probabilities are calculated for the same four dispersal times as for scenario 2. 2.8 Connectivity indices The primary strength of the connectivity probability matrices described in the previous section is to give detailed information on how connected any two areas are within the modeling domain integrated over time. However, more than 1,000 maps require some kind of simplification in order to provide a more overall measure on how well-connected areas are in general without necessarily providing any information on precisely which areas are interconnected. Within ballast water risk assessment, there is a need to distin- guish between areas more likely to export organisms far away and/or to a larger area than other areas (∼through downstream connectivity). These areas with high dispersal potential can be perceived as high risk zones where the release of invasive species through ballast water may have an increased potential of reaching optimal habitat conditions thereby increasing the likelihood for establishing a population successfully. Here, we propose a methodology to compile all information from the downstream connectivity probability maps into one single map using a simple and transparent approach. Similarly, in terms of upstream connectivity, it is important to identify areas more likely to receive invasive species than others, i.e., acting as sinks. These areas can be identified from upstream connectivity probability matrices as areas with high proba- bilities of receiving agents from far away and/or from a large area. It is clear that the proposed simplification of connectivities through the development of connectivity indices assumes that agents are distributed randomly within the entire modeling domain which is not the case when it comes to invasive species from ballast water. 2.7 Connectivity probability However, indices will give indications on where release of ballast water may be more likely to result in a significant spread of organisms, and which areas will be more likely to receive invasive species than others. This type of information is important from a management point of view. For calculation of connectivity indices with the term “momentum” (M) is intro- duced. Momentum can be calculated in several ways. The momentum can be calculated for each area and its probability map by simply summing the products of connectivity probabilities of each surrounding area and the distances to the areas: M area i ð Þ ¼ ∑Prob area j ð Þ D area j ð Þ ð2:2Þ ð2:2Þ This approach weights the probabilities with distance only. The results presented in this report apply this formula. However, alternative definitions of “Momentum” may be applied to include multiplication of probabilities with areal coverage instead of dis- tance, or a combination of areal coverage and distance, where areal coverage refers to the size of the area covered by say the 90 % fractile of probabilities. 3 Results and discussion The hydrodynamic model has been validated in terms of water level, salinity and temperature in a number of stations. The comparisons generally show a fairly good Development of a prototype tool for ballast water risk management 231 comparison between measurement and model. A few examples of this validation are given in Appendix 2 (see locations in Fig. 1). The comparisons demonstrate that the model is able to reproduce the water temperatures well within the model domain. Both the variability and the absolute values are captured by the model. The annual cycle of the thermal stratification is described well by the model. Also, the salinity conditions are described well by the model. The model captures the salinity stratification and the intrusions of saline North Sea water through the Belt Sea to the Arkona and Bornholm basins and further into the Gotland Deep in the Baltic Proper. The vertical structure of the water column in the northern North Sea is illustrated in Fig. 4b, which shows the annual mean salinity and water temperature in a vertical section between Scotland and Norway. The lower salinities of surface waters along the Norwegian coast represent the outflowing brackish Baltic water. The model includes the tidal currents as well as the meteorologically induced and the baroclinic currents. The modeled annual mean surface currents are shown in Fig. 4a. The mean current may be regarded as the residual currents, which may be expected to be important with respect to connectivity. Significant northward and northeastward residual current is observed in the southeastern and eastern part of the North Sea, whereas the western part displays relatively lower residual currents. In the Belt Sea, an outward (northward) residual surface flow is observed, which transports the brackish Baltic water out into Skagerrak. In Skagerrak, an anticlockwise gyre is observed and a residual flow along the Norwegian coast brings the Baltic surface water into the North Sea and further into the Norwegian Sea. All these features are in accordance with the literature. In the vertical, Fig. 4c shows a marked outward residual current in the upper water column along the Norwegian coast. This represents the outflow of mixed, brackish Baltic water and is consistent with the vertical salinity distribution mentioned above. In other parts of the vertical cross-section, a relatively low, southward residual current is observed. 3 Results and discussion Because the calculation procedures are repeated for each 6-h time step of the 1-year simulation time, each agent will be included multiple times as part of the statistical basis for the analyses. Thus, the total number of agents being analyzed results in a large number of agents as the basis for the statistical probability maps. The statistical analyses of connectivity were done based on results from the ABM simulations carried out for both a regional model for the entire model domain and a local model for the Kattegat, the Belts, and the western part of the Baltic Sea. The statistical basis for the downstream connectivity of scenario 1 is shown in Figs. 5 and 6 for the two models. For the vast majority of the model domain, the statistical basis for each area is more than 1,000 agents. Smaller numbers are found close to the shorelines and in the inner Danish straits. This is partly because a part of the 25×25 km squares along shorelines include land and thus the area covered by water is smaller than 25× 25 km. In addition, the smaller numbers may be a result of more agents being excluded from the simulation because agents closer to land and in shallow areas more likely hit land or seafloor boundaries. As described in the previous sections, these coastal areas are discarded from the statistical analyses. Also in areas located close to the open boundaries of the model domain, the statistical basis is small because of many agents crossing the open boundaries and subsequently not available for statistical analyses. Because of these issues, the robustness of the methodology based on the current simulations is strongest 232 F.T. Hansen et al. Fig. 4 a Modeled annual (2005) mean surface currents. b Modeled annual mean water temperature in a vertical section from Scotland to Norway. c The north-south current component in the same vertical section. See Fig. 1 for the location of the vertical section Fig. 4 a Modeled annual (2005) mean surface currents. b Modeled annual mean water temperature in a vertical section from Scotland to Norway. c The north-south current component in the same vertical section. See Fig. 3 Results and discussion 1 for the location of the vertical section in the open waters of the North Sea, Kattegat, and the eastern Baltic Sea, and may be less robust in some parts of the coastal and shallow waters, and close to the open model boundaries. Robustness can be improved by applying more agents in the inner Danish straits, by improving the model describing the agent trajectories more correctly close to land and seafloor boundaries, and by extending the model domain further out in order to reduce the impact of open model boundaries on connectivity statistics. The statistical basis shown in Figs. 5 and 6 is for scenario 1 for 1 week dispersal time. Similarly, data on statistical basis can be shown for scenarios 2–4 and for each dispersal time. Development of a prototype tool for ballast water risk management 233 Fig. 5 Statistical basis for the downstream connectivity probability mapping of scenario 1. Numbers are numbers of agents available for the statistical analysis of the downstream connectivity using the regional model Fig. 5 Statistical basis for the downstream connectivity probability mapping of scenario 1. Numbers are numbers of agents available for the statistical analysis of the downstream connectivity using the regional model 3.1 Connectivity probability maps An example of downstream connectivity probability maps for scenario 1 (simple drifting) for one selected area in the North Sea (indicated by red arrows) for four different dispersal times: 1, 2, 3, and 4 weeks is shown below (Fig. 7). Values are probability values between 0 and 1, and the sum of probabilities in each map is 1 since no mortality is considered. Only values greater than 0.01 are shown. In all maps, the probability distributions are highly influenced by north-going currents along the west coast of Jutland showing that probability values greater than 0.01 are dominating in the northern and northeastern directions. Maps also indicate that there are significant differences in probability maps depending on the dispersal time considered—the longer the dispersal time considered, the further away agents move. Four probability maps of the four different dispersal times are combined into one probability map (Fig. 8). Because no mortality is included, each dispersal time weight- ed equally and probabilities for the four dispersal times are simply averaged. Fig. 6 Statistical basis for the downstream connectivity probability mapping of scenario 1. Numbers are numbers of agents available for the statistical analysis of the downstream connectivity using the local model Fig. 6 Statistical basis for the downstream connectivity probability mapping of scenario 1. Numbers are numbers of agents available for the statistical analysis of the downstream connectivity using the local model F.T. Hansen et al. 234 Fig. 7 Downstream connectivity probability maps for one selected area in the North Sea (indicated by red arrows) for four dispersal times: 1, 2, 3, and 4 weeks. Only probability values larger than 0.01 are shown Fig. 7 Downstream connectivity probability maps for one selected area in the North Sea (indicated by re arrows) for four dispersal times: 1, 2, 3, and 4 weeks. Only probability values larger than 0.01 are shown All probability maps presented in Figs. 7 and 8 reflect the hydrodynamical variations during 2005 for scenario 1 where simple drifting is simulated. Similar maps for any area within the model domain can be extracted from the downstream connectivity matrices for scenarios 1–4 representing a given dispersal time or a combination of dispersion times. For scenarios where mortality is included, the connectivity probability maps of the combination of the four dispersal times are weighted according to the Fig. 8 Downstream connectivity probability map for one selected area (the same as in Fig. 3.1 Connectivity probability maps 7) in the North Sea (indicated by red arrow) with aggregated probability values for 1, 2, 3, and 4 weeks’ dispersal times Fig. 8 Downstream connectivity probability map for one selected area (the same as in Fig. 7) in the North S (indicated by red arrow) with aggregated probability values for 1, 2, 3, and 4 weeks’ dispersal times Development of a prototype tool for ballast water risk management 235 probability of agents to survive on each of the four dispersal times. This will result in an aggregated probability map where, e.g., 1 week dispersal probabilities will be weighted more than 2-, 3-, and 4-week probability values. Other dispersal times, e.g., 2, 4, 6, and 8 days, will show probability maps with a much narrower distribution of >0.01 probabilities around each area. These are not presented here. Similarly, maps for upstream connectivity probabilities can be extracted for each area representing the probability of agents registered in an area having come from other areas. Results for the upstream analyses are not presented here. Both upstream and downstream probability connectivity maps for scenario 1 aggre- gated over time for every 25×25 km blocks are available on Online Supplementary Material. 3.2 Connectivity indices Downstream momentum was calculated for the four scenarios based on the aggregated connectivity probabilities (=probabilities evaluated based on multiple dispersal times). Notice that momentums for scenario 3 are based on combined 1-, 2-, 3-, and 4-week dispersal times, while for scenarios 2 and 4, momentums are based on combined 3, 9, 15, and 21 days dispersal times. For comparisons, scenario 1 will be evaluated at each of these set of time scales. Downstream momentum for scenario 1 for 1-, 2-, 3-, and 4-weeks’ dispersal times are shown in Figs. 9 and 10. The greatest downstream momentum values are found in the northeastern part of the North Sea between the Danish and Norwegian coasts, along the west coast of the Netherlands and in Kattegat and the Danish belts, while large parts of the western part of the North Sea, German Bight, and the Baltic sea east of Bornholm have low connectivities. Fig. 9 Downstream connectivity indices (momentums) for scenario 1 (passive particle tracking). Indices are based on combined 1-, 2-, 3-, and 4-weeks’ dispersal times Fig. 9 Downstream connectivity indices (momentums) for scenario 1 (passive particle tracking). Indices a based on combined 1-, 2-, 3-, and 4-weeks’ dispersal times F.T. Hansen et al. 236 Fig. 10 Downstream connectivity indices (momentums) for scenario 1 (passive particle tracking) presented as deviation from mean value in percentages for 1-, 2-, 3-, and 4-weeks’ dispersal times Fig. 10 Downstream connectivity indices (momentums) for scenario 1 (passive particle tracking) presented deviation from mean value in percentages for 1-, 2-, 3-, and 4-weeks’ dispersal times Downstream momentum for scenario 2 for 3-, 9-, 15-, 21-days’ dispersal times are shown in Figs. 11 and 12. Downstream momentum values of scenario 2 show a similar distribution pattern as in scenario 1. Notice that dispersal time applied for scenarios 1 and 2 are different. For more accurate comparison, see the following section. Downstream momentum for scenario 3 for 1-, 2-, 3-, 4-weeks’ dispersal times are shown in Figs. 13 and 14. Fig. 11 Downstream connectivity indices (momentums) for scenario 2 (planktonic organisms). Indices are based on combined 3-, 9-, 15-, and 21-days’ dispersal times Fig. 11 Downstream connectivity indices (momentums) for scenario 2 (planktonic organisms). Indices are based on combined 3-, 9-, 15-, and 21-days’ dispersal times Development of a prototype tool for ballast water risk management 237 Fig. 3.2 Connectivity indices 12 Downstream connectivity indices (momentums) for scenario 2 (planktonic organism) presented as deviation from mean value in percentages for 3-, 9-, 15-, and 21-days’ dispersal times Fig. 12 Downstream connectivity indices (momentums) for scenario 2 (planktonic organism) presented deviation from mean value in percentages for 3-, 9-, 15-, and 21-days’ dispersal times Downstream momentum values for scenario 3 show a similar distribution pattern to that in scenario 1. For more accurate comparison see the following section. Downstream momentum values for scenario 3 show a similar distribution pattern to that in scenario 1. For more accurate comparison see the following section. Downstream momentums for scenario 4 for dispersal times corresponding to the time duration of the time between the registration of each organism until it settles are shown in Figs. 15 and 16. Downstream momentum values for scenario 4 show a similar overall distribution pattern to those seen in scenarios 1, 2, and 3, however with some deviation. For more accurate comparison see the following section. Fig. 13 Downstream connectivity indices (momentums) for scenario 3 (juvenile fish). Indices are based on combined 1-, 2-, 3-, and 4-weeks’ dispersal times Fig. 13 Downstream connectivity indices (momentums) for scenario 3 (juvenile fish). Indices are based on combined 1-, 2-, 3-, and 4-weeks’ dispersal times F.T. Hansen et al. 238 Fig. 14 Downstream connectivity indices (momentums) for scenario 3 (juvenile fish) presented as deviation from mean value in percentages for 1, 2, 3, and 4 weeks’ dispersal times Fig. 14 Downstream connectivity indices (momentums) for scenario 3 (juvenile fish) presented as deviation from mean value in percentages for 1, 2, 3, and 4 weeks’ dispersal times 3.3 Importance of dispersal time 3.3 Importance of dispersal time The selection of dispersal time for evaluating connectivity between marine areas is important simply because the longer time we “follow” the dispersal of an organism, the longer distance the organism is likely to travel, given that the organism is still alive. In terms of connectivity indices, this implies that longer dispersal times give greater connectivity indices. However, in addition to the magnitude of the connectivity indices, the spatial pattern of connectivity Fig. 15 Downstream connectivity indices (momentums) for scenario 4 (pelagic larvae). Indices are based on dispersal times corresponding to the time duration until each individual settles Fig. 15 Downstream connectivity indices (momentums) for scenario 4 (pelagic larvae). Indices are based o dispersal times corresponding to the time duration until each individual settles Development of a prototype tool for ballast water risk management 239 Fig. 16 Downstream connectivity indices (momentums) for scenario 4 (pelagic larvae) presented as deviation from mean value in percentages for dispersal times corresponding to the time duration until each individual settles Fig. 16 Downstream connectivity indices (momentums) for scenario 4 (pelagic larvae) presented as deviation from mean value in percentages for dispersal times corresponding to the time duration until each individual settles indices may change. As an example, connectivity indices were calculated for scenario 1 for two sets of dispersal times: 2, 4, 6, 8 days, and 7, 14, 21, 28 days and deviation from the overall mean value were plotted (Fig. 17). Although the overall patterns of the two plots for the two sets of dispersal times are similar, some discrepancies are evident. Some areas show larger deviation from the mean value when evaluated on a longer time span, while others show smaller deviation from the mean value. 3.4 Importance of biological factors 3.4 Importance of biological factors As for the selection of dispersal time when evaluating connectivity between marine areas, biological factors including mortality, settling, etc., are important because the longer an organism lives, the longer we can “follow” the dispersal of an organism and the longer distance the organism is likely to travel if flow conditions are suitable. Thus, when applying connectivity probability maps to predict, for instance, the likelihood that Fig. 17 Downstream connectivity indices (momentums) for scenario 1 (passive particle tracking) presented as deviation from mean value in percentages for 2-, 4-, 6-, and 8-days (left) and 1-, 2-, 3-, and 4-weeks’ (right) dispersal times Fig. 17 Downstream connectivity indices (momentums) for scenario 1 (passive particle tracking) presented as deviation from mean value in percentages for 2-, 4-, 6-, and 8-days (left) and 1-, 2-, 3-, and 4-weeks’ (right) dispersal times F.T. Hansen et al. 240 Fig. 18 Comparison of downstream connectivity indices (momentums) for scenario 1 (passive particle tracking) and scenario 2 (planktonic organism. Left panel Scenario 2 (planktonic organism). Right panel Scenario 1 (passive particle tracking). Presented as deviation from mean value in percentages for 3-, 9-, 15-, 21-days’ dispersal times Fig. 18 Comparison of downstream connectivity indices (momentums) for scenario 1 (passive particle tracking) and scenario 2 (planktonic organism. Left panel Scenario 2 (planktonic organism). Right panel Scenario 1 (passive particle tracking). Presented as deviation from mean value in percentages for 3-, 9-, 15-, 21-days’ dispersal times an introduced organism in an area will spread to other specific areas, biological factors are important. Biological factors may also have an effect when comparing the connectivities between areas. In Figs. 18 and 19 are shown the comparisons for scenarios 1 and 2, and scenarios 1 and 3, respectively, for the calculated deviation from the mean value of the connectivity indices. Comparisons between scenarios 1 and 2 are done for 3-, 9-, 15-, 21-days’ dispersal time, and comparisons between scenarios 1 and 3 are done for 7-, 14-, 21-, 28-days’ dispersal times. Both comparisons show a similar overall pattern in the variability of connectivity indices. However, scenario 2 shows some differences compared to scenario 1. For instance, connectivity indices in the Danish belts show significantly lower deviation from mean than in scenario 1. 3.4 Importance of biological factors This indicates that when evaluating the connectivity of specific marine areas for organisms with high mortality, it may be important to consider their mortality when simulating the spread of organ- isms. Scenario 3 on the contrary shows very little deviation from scenario 1 indicating that swimming behavior simulated as random walk has no major effect on the spatial variability in connectivity. For scenario 4, comparisons are done with scenario 2, not scenario 1, since scenario 4 is not evaluated on selected dispersal time, but rather dispersal times corresponding to the time duration from the time of registration of an agent in an area until it settles. To get an idea how settling affects the momentum, comparison of “deviations from mean” Fig. 19 Comparison of downstream connectivity indices (momentums) for scenario 1 (passive particle tracking) and scenario 3 (juvenile fish). Left panel Scenario 3 (juvenile fish). Right panel Scenario 1 (passive particle tracking). Presented as deviation from mean value in percentages for 1-, 2-, 3-, 4-weeks’ dispersal times Fig. 19 Comparison of downstream connectivity indices (momentums) for scenario 1 (passive particle tracking) and scenario 3 (juvenile fish). Left panel Scenario 3 (juvenile fish). Right panel Scenario 1 (passive particle tracking). Presented as deviation from mean value in percentages for 1-, 2-, 3-, 4-weeks’ dispersal times Development of a prototype tool for ballast water risk management 241 Fig. 20 Comparison of downstream connectivity indices (momentums) for scenario 2 (planktonic organisms) and scenario 4 (pelagic larvae of benthic invertebrate). Left panel Scenario 4 (pelagic species). Right panel Scenario 2 (planktonic organism). Presented as deviation from mean value in percentages. Dispersal times evaluated for scenario 2 is 3, 9, 15, and 21 days and for scenario 4 the time duration until each individual settles Fig. 20 Comparison of downstream connectivity indices (momentums) for scenario 2 (planktonic organisms) and scenario 4 (pelagic larvae of benthic invertebrate). Left panel Scenario 4 (pelagic species). Right panel Scenario 2 (planktonic organism). Presented as deviation from mean value in percentages. Dispersal times evaluated for scenario 2 is 3, 9, 15, and 21 days and for scenario 4 the time duration until each individual settles between scenario 2 and 4 are shown (Fig. 20). Differences between scenarios 2 and 4 are the settling introduced in scenario 4 and that momentums of scenario 2 are evaluated at discrete dispersal times (3, 9, 15 and 21 days). 3.4 Importance of biological factors Here, as for scenarios 2 and 3, the main overall patterns in the variability of connectivity indices are maintained, with some deviations locally. 3.5 Momentum—upstream Upstream connectivity indices are only shortly presented here since the main focus of this study is to identify areas where the release of ballast water may have a high potential for spreading to other parts of the North Sea region. We will not go into detail on how dispersal time or biological factors may affect the outcome of the upstream connectivity analysis. However, in general, the upstream connectivity indices show similar spatial variability between areas, clearly identifying areas more likely to receive Fig. 21 Upstream connectivity indices (momentums) for scenario 1 (passive particle tracking). Indices are based on combined 1-, 2-, 3-, and 4 weeks’ dispersal times Fig. 21 Upstream connectivity indices (momentums) for scenario 1 (passive particle tracking). Indices a based on combined 1-, 2-, 3-, and 4 weeks’ dispersal times 242 F.T. Hansen et al. ballast-water-derived organisms from far away. Figure 21 shows the upstream connec- tivity indices for scenario 1 (passive particle tracking) based on 1-, 2-, 3-, 4-weeks’ dispersal times. ballast-water-derived organisms from far away. Figure 21 shows the upstream connec- tivity indices for scenario 1 (passive particle tracking) based on 1-, 2-, 3-, 4-weeks’ dispersal times. Areas with the highest values of upstream connectivity are primarily areas of the deeper part of the waters between northwestern Jutland and Norway and along the southwestern Norwegian coast. The values are highly distinct from other marine areas and this indicates that these areas serve as major sinks or receiving water bodies of passively transported agents. Again, as for the downstream connectivity, lowest values are found in particular in the western part of the North Sea, in the German Bights and the Baltic Sea east of Bornholm. Intermediate values are found in the eastern part of the North Sea, in Kattegat, the Danish straits including Fermarn Belt, and in the western parts of the Baltic Sea. 4 Conclusions and outlook The analyses carried out for this study show that when evaluating the connectivity of marine waters of the North Sea region including Skagerak, Kattegat, the Danish belts, and the western parts of the Baltic Sea, the hydrodynamics seem to play the most important role when considering small organisms with limited ability to perform a significant autonomic movement behavior. Despite some differences in calculated connectivities due to biological factors and the choice of dispersal time applied for the connectivity analyses, the overall pattern of the variability of connectivity indices show, at least at this preliminary stage of analysis and at a regional level, a rather unambiguous indication that biological factors and dispersal time are only of secondary importance when ranking areas according to their degree of connectivity. At the local level, however, under some conditions, biological factors as well as the choice of dispersal time applied may play an important role. We recommend that additional biological factors that could have a potential impact on connectivity of marine areas should be identified and tested to sustain (or contradict) these conclusions. These may include: (1) simulation of diurnal vertical migration of planktonic species, (2) simulation of oriented swimming behavior of juvenile fish, and (3) analyses of differences in connectivity between seasons. Also, the methodology proposed here for calculating connectivity (as momentums) solely depending on the distance traveled by each simulated organism during a number of dispersal time needs to be consolidated. As an example, the inclusion of the area covered by, e.g., a 90 % fractile of connectivity probabilities could be considered. In addition to connectivity indices, the downstream connectivity probability maps for each individual area have a very high potential for predicting the probability of a ballast-water-derived organism ending up at a specific location, e.g., locations identi- fied as being especially sensitive or at locations providing suitable habitat for specific species. Similarly, upstream connectivity probability maps for a specific area (a given habitat, Marine-Protected Areas, etc.) predict which areas may potentially contribute with ballast-water-derived organisms. The primary aim of the methodology presented here is to demonstrate how to apply a combination of agent-based modeling and hydrodynamical modeling to describe and develop measures for the inter-connectivity of marine ecosystems systems and its Development of a prototype tool for ballast water risk management 243 potential application for ballast water risk assessment. 4 Conclusions and outlook During this work, a number of issues have been identified on how to improve the methodology: (1) the hydrodynam- ical model was not developed specifically to address these issues, in particular near- shore hydrodynamics are important to avoid agents being “captured” in still water which in most cases may be a model resolution artifact rather than a true hydrodynam- ical phenomenon, (2) agent-based models may be further developed to be species- specific including habitat preferences, environmental stresses and population dynamics, and (3) inclusion of water quality modeling will improve prediction of the effects on environmental stressors on agents. In case our prototype tool is further developed, we foresee that it may be useful for at least three other purposes: 1. Data layers for assessments of cumulative human pressures and impacts (sensu Halpern et al. 2008). Mapping of cumulative pressures and impacts relies on ecologically relevant data layers, both for pressures and ecosystem components. The downstream connectivity index presented in this study could potentially be regarded as a pressure layer representing likely dispersal routes of introduced alien species. According to the EU Marine Strategy Framework Directive (Anon 2008), which is based on the Ecosystem Approach to management of human activities, all “marine” EU Member States are required to map cumulative pressures (see examples in Korpinen et al. 2012 and Andersen et al. 2013). The mapping carried out so far in Europe does not, as far as we know, take into account aliens species and their potential dispersal routes. It would in our opinion be an important step forward to full implementation of the Ecosystem Approach as both alien species and dispersal routes are included in the next generation of cumulative impact assessments 2. Marine Spatial Planning, including zoning and site selection, i.e., for future designation and design of Marine-Protected Areas taking the identification of so- called “Blue Corridors” into consideration (Martin et al. 2006). We foresee that the connectivity index can be modified and used for estimation of where species- specific “Blue Corridors” might be located. If so, the index developed and presented in this study could be useful for evidence-based design and designation of networks of Marine-Protected Areas in regional seas or at subregional scale. 3. Ballast water risk assessments, especially in regard to exemptions from the Ballast Water Management Convention (BWMC), adopted in 2004 (IMO 2004). References Andersen JH, Stock A, Heinänen S, Mannerla M, Vinther M. (2013): Human uses, pressures and impacts in the eastern North Sea. Aarhus University, DCE - Danish Centre for Environment and Energy. Technical Report from DCE - Danish Centre for Environment and Energy No. 18. 134 pp. http://www2.dmu.dk/ Pub/TR18.pdf. Assessed 2 May 2014 Anon. (2008) Directive 2008/56/EC of the European Parliament and the Council of 17 June 2008 establishing a framework for community action in the field of marine environmental policy (Marine Strategy Framework Directive). Official Journal of the European Union, Brussels, L 164/19 Bax N, Williamson A, Aguero M, Gonzalez E, Geeves W (2003) Marine invasive alien species: a threat to global biodiversity. Mar Pol 27(4):313–323 Berglunda M, Jacobia MN, Jonsson PR (2012) Optimal selection of marine protected areas based on connectivity and habitat quality. Ecol Mod 240:105–112 Christensen A, Jensen H, Mosegaard H, St. John M, Schrum C (2008) Sandeel (Ammodytes marinus) larval transport patterns in the North Sea from an individual-based hydrodynamic egg and larval model. Can J Fish Aquat Sci 65:1498–1511 Condie S, Andrewartha J, Mansbridge J, Waring J (2006) Modelling circulation and connectivity on Australia’s North West Shelf. North-West Shelf Joint Environmental Management Study, Technical Report no. 6, 71 pp Cowen RK, Paris CB, Olson DB, Fortuna JL (2003) The role of long distance dispersal versus local retention in replenishing marine populations. Gulf and Caribbean Research Supplement. Gulf Caribbean Res 14(2): 129–138 Cowen RK, Paris CB, Srinivasan A (2006) Scaling of connectivity in marine populations. Science 311:522 DHI (2009) MIKE 3 Flow Model, Hydrodynamic Module, Scientific Documentation. MIKE by DHI 2009, 58 pp DHI (2011) MIKE 21 and MIKE 3 Flow Model FM, Hydrodynamic and Transport Module, Scientific Documentation. MIKE by DHI 2011, 58 pp FEHY (2012) Fehmarnbelt Fixed Link EIA. Hydrography of the Fehmarnbelt area – Impact Assessment. Report No. E1TR0058 Volume II, 131 pp Gollasch S, Haydar D, Minchin D, Wolff WJ, Reise K (2009) Introduced aquatic species of the North Sea coasts and adjacent brackish waters. Ecol Stud 204:507–528 Goodwin A, Nestler JM, Loucks DP, Chapman RS (2001) Simulating mobile populations in aquatic ecosystems. 4 Conclusions and outlook Exemption can be granted from ships sailing routinely between specific ports or locations and is based on a risk assessment (RA) using the best available scientific information (MEPC 2007). In our opinion, the approach presented and discussed in this study potentially set a new standard for what can de done when assessing the risk. For example, the modeling setup can be modified to specific local conditions (salinity, temperature, dominating currents, etc.) and target to specific organisms of interests. In conclusion, we have developed a prototype Decision Support Tool for modeling of risks of spreading of introduced alien species via ballast water. The prototype tool reported is based on two types of models and a postprocessing activity: Firstly, a 3D hydrodynamical model calculates the currents in the North Sea and Danish Straits. 244 F.T. Hansen et al. Secondly, an agent-based model estimates the dispersal of selected model organisms with the current regime calculated by the 3D model. Thirdly, scenarios of dispersal are combined into an interim estimate of connectivity within the study area. Secondly, an agent-based model estimates the dispersal of selected model organisms with the current regime calculated by the 3D model. Thirdly, scenarios of dispersal are combined into an interim estimate of connectivity within the study area. The prototype tool should in our opinion be regarded as a platform for further development and testing. However, it can in its present form be used for interim estimates of connectivity and hence as a tool for assessment of potential risk associated to intentional or unintentional discharges of ballast water. The tool can also be used for other purposes, e.g., in regard to ecosystem-based management and the implementation of the EU Marine Strategy Framework Directive. Acknowledgments This study is based on work done under The Ballast Water Opportunity project, which is co-funded by the INTERREG IVB North Sea Region Programme of the European Regional Development Fund (ERDF), the Danish Nature Agency and DHI. The authors would like to thank Johnny Reker, Joachim Raben-Levetzau, Flemming Møhlenberg, and Ulrik Berggren for constructive discussions as well as Ciarán Murray for language checking. References Journal of Water Resources Planning and Management, November/December Halpern BS, Walbridge S, Selkoe KA, Kappel CV, Micheli F, D’Agrosa C, Bruno JF, Casey KS, Ebert C, Fox HE, Fujita R, Heinemann D, Lenihan HS, Madin EMP, Perry MT, Selig ER, Spalding M, Steneck R, Watson R (2008) A global map of human impacts on marine ecosystems. Science 319:948–952 Hayward PJ, Ryland JS (1995) Handbook of the marine fauna of north-west Europe, XI. Oxford University Press, Oxford, 800 pp 245 Development of a prototype tool for ballast water risk management HELCOM (2010) Ecosystem Health of the Baltic Sea. HELCOM Initial Holistic Assessment 2003-2007. Balt. Sea Env. Proc. 122. Helsinki Commission. 63 pp. http://www.helcom.fi/stc/files/Publications/ Proceedings/bsep122.pdf. Accessed 2 May 2014. Humston R, Ault JS, Lutcavage M, Olson DB (2000) Schooling and migration of large pelagic fishes relative to environmental cues. Fish Oceanogr 9(2):136–146 IMO (2004) Adoption of the final act and any instruments, recommendations and resolutions resulting from the work of the conference. International convention for the control and management of ships’ ballast water and sediments, 2004. Adopted 16 February 2004. 36 pp Korpinen S, Meski L, Andersen JH, Laamanen M (2012) Human pressures and their potential impact on the Baltic Sea ecosystem. Ecol Ind 15:105–114 Leppäkoski E, Gollasch S, Olenin S (2003) Invasive aquatic species of Europe. Distribution, impacts and management. Kluwer, Dordrecht Å Martin G, Makinen A, Andersson Å, Dinesen GE, Kotta J, Hansen J, Herkül K, Ockelmann KW, Nilsson P, Korpinen S (2006) Literature review of the “Blue Corridors” concept and it’s applicability to the Baltic Sea. BALANCE Interim report No. 4, 67 pp. http://balance-eu.org/xpdf/balance-interim-report-no-4.pdf. Accessed 2 May 2014 MEPC (2007) Guidelines for risk assessment under regulation A-4 of the BWM convention (G7). Annex 2 adopted 13 July 2007. The Marine Environment Protection Committee, IMO. 16 pp Misund OA, Skjoldal HR (2005) Implementing the ecosystem approach: experiences from the North Sea, ICES, and the Institute of Marine Research. Norway Mar Ecol Prog Ser 300:241–296 OSPAR (2010) Quality Status Report 2010. OSPAR Commission. 175 pp. http://qsr2010.ospar.org/en/index. html. Accessed 2 May 2014 Paris CB, Cowen RK, Claro R, Lindeman KC (2005) Larval transport pathways from Cuban snapper (Lutjanidae) spawning aggregations based on biophysical modelling. Mar Ecol Prog Ser 296:93–106 ( j ) p g gg g p y g g uiz GM, Carlton JT (Eds) (2003) Invasive species: Vectors and management strategies. Island Press, 509 pp SMHI (2006) The year 2005. References An environmental status report of the Skagerrak, Kattegat and the North Sea. The Baltic and North Sea Marine Environmental Modelling Assessment Initiative (BANSAI). SMHI, IMR and DHI. Published by SMHI. 28 pp
https://openalex.org/W2953505341	https://napier-repository.worktribe.com/preview/1833193/Risk-factors%20and%20comorbidity%20of%20ICD-11%20PTSD%20and%20Complex%20PTSD....pdf	English	null	Risk factors and comorbidity of ICD‐11 PTSD and complex PTSD: Findings from a trauma‐exposed population based sample of adults in the United Kingdom	Depression and anxiety	2,019	cc-by	7,616	System, Palo Alto, CA USA 6 Department of Psychiatry and Behavioural Sciences, Stanford University, Stanford, CA, USA. USA. 7Psychology and Psychological Therapies Directorate, Cardiff & Vale University Health Board, Cardiff, UK. 8Division of Psychological Medicine & Clinical Neurosciences, School of Medicine, Cardiff University, Cardiff, UK. 10Ulster University, School of Psychology, Derry, Northern Ireland. Affiliations 1School of Health & Social Care, Edinburgh Napier University, Edinburgh, UK 2NHS Lothian, Rivers Centre for Traumatic Stress, Edinburgh, UK 3Department of Psychology, Maynooth University, Kildare, Ireland 4Centre for Global Health, Trinity College Dublin, Dublin, Ireland 5National Center for PTSD Dissemination and Training Division, VA Palo Alto Health Care System, Palo Alto, CA USA 1Address for Correspondence: Prof. Thanos Karatzias, Edinburgh Napier University, Sighthill Campus, Sighthill Court, Edinburgh EH11 4BN, Scotland UK,Tel. (+44) (0) 131 455 5345, Email.t.karatzias@napier.ac.uk 1 1 Abstract Background: Following the recently published 11th version of the WHO International Classification of Diseases (ICD-11), we sought to examine the risk factors and comorbidities associated with posttraumatic stress disorder (PTSD) and Complex PTSD (CPTSD). Method: Cross-sectional and retrospective design. The sample consisted of 1051 trauma- exposed participants from a nationally representative panel of the UK adult population. Results: 5.3% (95% CI = 4.0%-6.7%) met the diagnostic criteria for PTSD and 12.9% (95% CI = 10.9%-15.0%) for CPTSD. Diagnosis of PTSD was independently associated with being female, being in a relationship, and the recency of traumatic exposure. CPTSD was independently associated with younger age, interpersonal trauma in childhood, and interpersonal trauma in adulthood. Growing up in an urban environment was associated with diagnosis of PTSD and CPTSD. High rates of physical and mental health comorbidity were observed for PTSD and CPTSD. Those with CPTSD were more likely to endorse symptoms reflecting Major Depressive Disorder (OR = 21.85, 95 CI = 12.51 – 38.04) and Generalized Anxiety Disorder (OR = 24.63, 95 CI = 14.77 – 41.07). Presence of PTSD (OR = 3.13, 95 CI = 1.81 – 5.41) and CPTSD (OR = 3.43, 95 CI = 2.37 – 4.70) increased the likelihood of suicidality by more than three times. Nearly half the participants with PTSD and CPTSD reported the presence of a chronic illness. Conclusions: CPTSD is a more common, co- morbid, debilitating condition compared PTSD. Further research is now required to identify effective interventions for its treatment. Keywords: ICD 11 PTSD, CPTSD, risk factors, comorbidities 2 2 Introduction Furthermore, CPTSD is more strongly associated with symptoms of 3 depression, anxiety, borderline personality disorder, and suicidality than PTSD (Hyland et al., 2018). Identifying risk factors and comorbidities associated with these disorders has important implications for diagnosis and subsequent treatment, as well as for prevention and early intervention. If clinicians know who is more likely to be adversely affected by a particular disorder following traumatic exposure, they can monitor individuals with a view to providing interventions before their problems become chronic. Especially in the case of CPTSD, an understanding of the risk factors will contribute to the development of theoretical models to explain its onset and course, which could ultimately lead to more focused interventions and better treatment outcomes. depression, anxiety, borderline personality disorder, and suicidality than PTSD (Hyland et al., 2018). Identifying risk factors and comorbidities associated with these disorders has important implications for diagnosis and subsequent treatment, as well as for prevention and early intervention. If clinicians know who is more likely to be adversely affected by a particular disorder following traumatic exposure, they can monitor individuals with a view to providing interventions before their problems become chronic. Especially in the case of CPTSD, an understanding of the risk factors will contribute to the development of theoretical models to explain its onset and course, which could ultimately lead to more focused interventions and better treatment outcomes. Introduction In 2018, the World Health Organization published the 11th version of the International Classification of Diseases (ICD-11); the official diagnostic system used worldwide. Substantial revisions were made to the trauma-related diagnoses in ICD-11 that are located within the parent category of ‘Disorders Specifically Associated with Stress’. The symptom profile and diagnostic requirements for Posttraumatic Stress Disorder (PTSD) were narrowed relative to the ICD-10 and the fifth edition of the Diagnostic and Statistical Manual (DSM-5: American Psychiatric Association [APA], 2013), and Complex PTSD (CPTSD) was introduced to the diagnostic nomenclature for the first time (see Table 1; Maercker et al., 2013). A multi-national field survey found that clinicians demonstrated accuracy in differential diagnoses between PTSD and CPTSD formulations (Keeley et al., 2016), and a review of the extant research indicated support for the construct validity of PTSD and CPTSD (Brewin et al., 2017). CPTSD has been shown to occur more frequently following exposure to traumatic events that are of an interpersonal nature, that are difficult or impossible to escape from, and particularly when occurring during early development (e.g., childhood sexual abuse, torture, captivity; see Karatzias et al., 2018a). CPTSD is also a more impairing condition than PTSD (Karatzias et al., 2017) and may require enhanced interventions which are quantitatively and qualitatively different to those currently offered for PTSD (Karatzias et al., 2018b). Little is currently known about the risk factors that differentiate PTSD and CPTSD following CPTSD has been shown to occur more frequently following exposure to traumatic events that are of an interpersonal nature, that are difficult or impossible to escape from, and particularly when occurring during early development (e.g., childhood sexual abuse, torture, captivity; see Karatzias et al., 2018a). CPTSD is also a more impairing condition than PTSD (Karatzias et al., 2017) and may require enhanced interventions which are quantitatively and qualitatively different to those currently offered for PTSD (Karatzias et al., 2018b). Little is currently known about the risk factors that differentiate PTSD and CPTSD following traumatic exposure, or, how frequently other mental and physical health disorders co-occur with PTSD and CPTSD. Emerging evidence, primarily drawn from clinical samples, suggests that childhood sexual abuse, negative trauma-related cognitions, distress intolerance, and unemployment status are associated with an increased risk of CPTSD (Hyland et al., 2017; Hyland et al., 2018). TABLE 1 HERE With this in mind, the current study was undertaken to: (a) identify demographic (gender, age, urbanicity, history of emigration, relationship status, number of children, 4 (gender, age, urbanicity, history of emigration, relationship status, number of children, employment status, and education status) and trauma-related (time passed since traumatic exposure, childhood non-interpersonal trauma, childhood interpersonal trauma, adulthood non-interpersonal trauma, and adulthood interpersonal trauma) risk factors associated with PTSD and CPTSD diagnosis; and (b) determine the comorbidity rates for PTSD and CPTSD with Major Depressive Disorder (MDD), Generalized Anxiety Disorder (GAD), Alcohol Use Disorder (AUD), suicidality, and the presence of a chronic illness. Based on the existing literature of the risk factors for ICD-11 PTSD and CPTSD, as well as the wider trauma literature, we expected that PTSD would be would associated with being female (Tolin & Foa, 2006) and that CPTSD would be associated with exposure to an increased number interpersonal trauma types during childhood (Hyland et al., 2017). Although not yet examined in relation to ICD-11 PTSD and CPTSD, urban residence has been shown to increase the risk for multiple psychiatric disorders including mood and anxiety disorders independent of other covariates (Peen, Schoevers, Beekman, & Dekker, 2010), therefore, we 4 4 expected that growing up in an urban area would be associated with an increased risk for both PTSD and CPTSD. Finally, based on data from clinical (Hyland et al., 2018) and internally displaced (Shevlin et al., 2018) samples, we expected that PTSD and CPTSD would be highly comorbid with the other psychiatric disorders measured in this study. 2.1 Participants and procedures A trauma-exposed adult sample of the United Kingdom (UK) population was used in the current study. Sample participants were drawn from an existing online research panel that was representative of the UK adult population. Panel members were randomly recruited through probability-based sampling. Inclusion criteria for the current sample were that respondents were (a) born in the UK, (b) aged 18 years or older at the time of the survey, and (c) screened positive for at least one traumatic event in their lifetime (assessed using the Life Events Checklist, described below). Ethical approval was granted by the Research Ethics Committee of the institution to which the second author was affiliated at the time of the survey. No inducements or incentives were offered for participation. In total, 2,653 panel members were contacted, and 1,051 people qualified for inclusion on the basis of meeting the inclusion criteria (selection rate = 39.6%). The mean age of the sample was 47.18 years (SD = 15.00, range = 18-90 years), and 68.4% (n = 719) of participants were female. The majority of respondents indicated that they did not grow up in an urban area (54.7%, n = 574), had not emigrated in their lifetime (82.2%, n = 864), were currently in a committed relationship (70.4%, n = 740), did not have any children under the age of 16 years (67.5%, n = 709), were in full- or part-time employment (58.5%, n = 615), and had completed a college or university education (62.7%, n = 659). The mean age of the sample was 47.18 years (SD = 15.00, range = 18-90 years), and 68.4% (n = 719) of participants were female. The majority of respondents indicated that they did not grow up in an urban area (54.7%, n = 574), had not emigrated in their lifetime (82.2%, n = 864), were currently in a committed relationship (70.4%, n = 740), did not have any children under the age of 16 years (67.5%, n = 709), were in full- or part-time employment (58.5%, n = 615), and had completed a college or university education (62.7%, n = 659). = 659). 5 5 2.2. Measures 2.2. Measures 2.2.1 Traumatic Exposure: A revised version of the Life Events Checklist for DSM- 5 (LEC; Weathers et al., 2013) was used to assess for childhood (“before the age of 18”) and adulthood (“at or after the age of 18”) exposure to 16 different traumatic events. Participants indicated on a ‘Yes’ (1) or ‘No’ (0) basis if they had directly experienced each traumatic event during both developmental periods. Following Ehring and Quack’s (2010) approach, we calculated summed scores for non-interpersonal trauma (natural disaster, fire or explosion, transportation accident, serious accident at work or home or during recreational activity, exposure to toxic substance, life-threatening illness or injury, severe human suffering, sudden and violent death, sudden and unexpected death of someone close to you) and interpersonal trauma (physical assault, assault with a weapon, sexual assault, other unwanted or uncomfortable sexual experiences, combat or exposure to a war-zone, captivity, serious injury and/or harm and/or death you caused to someone else) in childhood and adulthood. Scores for non-interpersonal trauma (in childhood and in adulthood) ranged from 0-9, and scores for interpersonal trauma (in childhood and in adulthood) ranged from 0-7. 2.2.2 ICD 11 PTSD and CPTSD: The International Trauma Questionnaire (ITQ: Cloitre et al., 2018) is a self-report measure of ICD-11 PTSD and CPTSD. The ITQ assess the respondent’s most distressing traumatic event and the time passed since the trauma occurred (response options: < 6 months ago, 6-12 months ago, 1-5 years ago, 5-10 years ago, 10 to 20 years ago, and > 20 years ago). The ITQ contains 12 items measuring the six PTSD ( = .91) and six Disturbances in Self-Organisation (DSO) symptoms ( = .92). There are three items measuring functional impairment (in the domains of social-life, work-life, and other important areas of life) associated with both sets of symptoms. Individuals respond to the PTSD items in terms of how much they have been bothered by each symptom over the past month, and respond to the DSO items in terms of how they typically feel, think about 6 6 themselves, and relate to others. All items are measured using a five-point Likert scale ranging from 0 (“Not at all”) to 4 (“Extremely”). Diagnostic requirements are presented in Table 1, and a symptom was considered present based on a score of ≥ 2 (“Moderately”). Importantly, the ICD-11 diagnostic rules only permit a diagnosis of PTSD or CPTSD, not both. 2.2. Measures The psychometric properties of the ITQ, and the diagnostic rates of PTSD and CPTSD, were demonstrated in a prior study based on this exact sample (Cloitre et al., 2018). The diagnostic rates are presented below in the results section. 2.2.3 Major Depressive Disorder (MDD): The Patient Health Questionnaire-9 (PHQ-9; Kroenke, Spitzer, & Williams, 2001) is a nine-item, self-report measure of MDD that aligns to the diagnostic criteria of the DSM-5 (APA, 2013). Individuals are instructed to respond to each item in terms of how much they have been bothered by that symptom over the past two weeks using a four-point Likert scale ranging from 0 (“Not at all”) to 3 (“Nearly every day”). The PHQ-9 has demonstrated strong psychometric properties (Manea, Gilbody & McMillan, 2015) and the internal reliability among the current sample was excellent ( = .94). Scores range from 0 to 27 and scores ≥ 10 are indicative of a diagnosis of MDD (Kroenke et al., 2009). 2.2.4 Generalized Anxiety Disorder (GAD): The Generalized Anxiety Disorder 7- Item Scale (GAD-7; Spitzer, Kroenke, Williams & Lowe 2006) is a seven-item, self-report measure of GAD. Individuals are instructed to respond to each item in terms of how much they have been bothered by each symptom over the past two weeks using a four-point Likert scale ranging from 0 (“Not at all”) to 3 (“Nearly every day”). The GAD-7 has demonstrated good psychometric properties (Kertz, Bigda-Peyton & Bjorgvinsson, 2013) and the internal reliability among the current sample was excellent (α = .95). Scores range from 0 to 21 and scores ≥ 10 are indicative of a diagnosis of GAD (Spitzer et al., 2006). 7 7 2.2.5 Alcohol Use Disorder (AUD): The presence of an AUD was measured using the AUDIT-C, a brief self-report measure comprised of the first three questions of the Alcohol Use Disorders Identification Test (Saunders, Aasland, Babor & de la Fuente, 1993). The clinical utility of the AUDIT-C has been demonstrated in multiple sample including the general population (Nayak, Bond, Cherpitel, Patel & Greenfield 2009), military veterans (Bradley et al., 2003), and hospitalised patients (Kelly, Donovan, Chung, Bukstein & Cornelius 2009). Scores on the AUDIT-C range from 0-12, and based on a nationally representative sample of adults from the United States, scores ≥ four effectively capture a DSM-5 diagnosis of AUD (Dawson, Smith, Saha, Rubinsky, & Grant 2012). 2.2. Measures 2.2.6 Suicidality / Self-harm: Suicidality was measured using two items taken from a self-report measure of Borderline Personality Disorder symptoms. These questions asked respondents: “Have you tried to hurt or kill yourself or threatened to do so?” and “Have you ever cut, burned, or scratched yourself on purpose?” These items were responded to using a ‘Yes’ (1) or ‘No’ (0) response format. A person was deemed to screen positive for lifetime suicidality if they answered ‘Yes’ to either question. 2.2.7 Chronic Illness: The presence of a chronic illness was assessed using the first item of the Charlson Comorbidity Index (Charlson, Pompei, Ales & MacKenzie, 1987). This item asks the respondent “Do you suffer from any chronic illness?” using a ‘Yes’ (1) or ‘No’ (0) response scale. Respondents are subsequently asked to indicate the specific type of chronic illness they have been diagnosed with. 2.3 Data Analysis Hierarchical binary logistic regression analysis was used to examine the associations between each risk factor and meeting diagnostic criteria for PTSD and CPTSD, respectively. Eight demographic variables were entered into the model at step 1 and these were: sex (0 = Male, 1 = Female), age (measured years), having grown up in an urban area (0 = No, 1 = 8 8 Yes), history of emigration (0 = No, 1 = Yes), relationship status (0 = Not in a committed relationship, 1 = In a committed relationship), number of children under the age of 16, employment status (0 = Employed, 1 = Unemployed), and attendance of college/university (0 = No, 1 = Yes). Five trauma-related variables were entered into the model at step 2 and these included: the time passed since trauma (1. < 6 months ago, 2. 6-12 months ago, 3. 1-5 years ago, 4. 5-10 years ago, 5. 10 to 20 years ago, 6. > 20 years ago), childhood non-interpersonal trauma, childhood interpersonal trauma, adulthood non-interpersonal trauma, and adulthood interpersonal trauma. The comorbidity rates for PTSD and CPTSD with MDD, GAD, AUD, suicidality, and chronic illness were evaluated using the Pearson chi-square tests. There was very little missing data present (0.19%) and the default listwise deletion method in SPSS was used in all analyses. Yes), history of emigration (0 = No, 1 = Yes), relationship status (0 = Not in a committed relationship, 1 = In a committed relationship), number of children under the age of 16, employment status (0 = Employed, 1 = Unemployed), and attendance of college/university (0 = No, 1 = Yes). Five trauma-related variables were entered into the model at step 2 and these included: the time passed since trauma (1. < 6 months ago, 2. 6-12 months ago, 3. 1-5 years ago, 4. 5-10 years ago, 5. 10 to 20 years ago, 6. > 20 years ago), childhood non-interpersonal trauma, childhood interpersonal trauma, adulthood non-interpersonal trauma, and adulthood interpersonal trauma. The comorbidity rates for PTSD and CPTSD with MDD, GAD, AUD, suicidality, and chronic illness were evaluated using the Pearson chi-square tests. There was very little missing data present (0.19%) and the default listwise deletion method in SPSS was used in all analyses. 3.1 Life events and common mental health problems The most common traumatic event in childhood was physical assault (32.4%, n = 340) and the most common traumatic event in adulthood was the sudden and unexpected death of a loved one (47.4%, n = 498). The sudden and unexpected death of a loved one was most commonly reported as the ‘most distressing traumatic event’ (29.4%, n = 309). With respect to the time passed since the trauma, the most common response was ‘more than 20 years ago’ (40.1%, n = 421). The mean number of childhood non-interpersonal trauma types was .84 (SD = 1.31, range = 0-9) and the mean number of childhood interpersonal trauma types was also .84 (SD = 1.20, range = 0-7). The mean number of adulthood non- interpersonal trauma types was 1.43 (SD = 1.49, range = 0-9), and the mean number of adulthood interpersonal trauma types was .82 (SD = 1.27, range 0-7). Bivariate correlations between the different types of trauma were all statistically significant (p < .001) and ranged 9 9 from .26 (between childhood interpersonal trauma and adulthood non-interpersonal trauma) to .52 (between childhood interpersonal trauma and childhood non-interpersonal trauma). As reported in Cloitre et al. (2018), the PTSD diagnostic criteria were met by 56 respondents (5.3% [95% CI = 4.0% - 6.7%]) and the CPTSD diagnostic criteria were met by 136 respondents (12.9% [95% CI = 10.9% - 15.0%]). Furthermore, based on the recommended cut-off scores of > 10 for the PHQ-9 and GAD-7, respectively, 368 respondents (35% [95% CI = 32.1% - 37.9%]) had a probable MDD diagnosis and 300 (28.5% [95% CI = 25.8% - 31.3%]) had a probable GAD diagnosis. Additionally, 353 (33.6% [95% CI = 30.7% - 36.4%]) participants had a probable AUD diagnosis based on the recommended cut-off score of > 4 for the AUDIT-C, 329 (31.3% [95% CI = 28.5% - 34.1%]) reported a history of suicidality, and 322 (30.6% [95% CI = 27.8% - 33.4%]) reported having been diagnosed with a chronic illness. 3.2 Risk factors for PTSD and CPTSD The results of the two binary logistic regression analyses for PTSD and CPTSD are presented in Table 2. In the case of meeting diagnostic criteria for PTSD, step 1 of the model (which included the demographic variables) was statistically significant (χ2 (8) = 23.03, p = .003), and growing up in an urban area (OR = 2.02) and being in a committed relationship (OR = 2.30) were significantly associated with PTSD. The trauma variables were entered at step 2 and did not significantly improve the model (χ2 (5) = 5.68, p = .339). The full model was statistically significant (χ2 (13) = 28.71, p = .007) and, when adjusting for all demographic and trauma factors, four variables were significantly associated with a diagnosis of PTSD: being female (OR = 2.08), having grown up in an urban area (OR = 2.05), being in a committed relationship (OR = 2.30) and less time having passed since trauma (OR = .81). TABLE 2 HERE 10 In the case of meeting diagnostic criteria for CPTSD, step 1of the model was statistically significant (χ2 (8) = 65.52, p < .001), and age (OR = .96), growing up in an urban area (OR = 1.96), and having emigrated (OR = 1.62) were significantly associated with CPTSD. The inclusion of the trauma variables at step 2 significantly improved the model (χ2 (5) = 67.70, p < .001), and the model as a whole was significant: χ2 (13) = 133.25, p < .001). When adjusting for all demographic and trauma factors, four variables were significantly associated with a diagnosis of CPTSD: younger age (OR = .97), growing up in an urban area (OR = 1.72), childhood interpersonal trauma (OR = 1.52), and adulthood interpersonal trauma (OR = 1.20). 3.3 ICD-11 PTSD and CPTSD comorbidities The comorbidities for PTSD ranged from a low of 30.4% with GAD to a high of 60.0% with AUD (see Table 3). Meeting criteria for PTSD was significantly (all ps < .05) associated with an increased likelihood of having a probable MDD diagnosis (OR = 1.79), a chronic illness (OR = 1.90), an AUD (OR = 2.04), and a history of suicidality (OR = 3.13). The comorbidities for CPTSD ranged from a low of 45.6% with chronic illness to 89.0% with MDD. Meeting criteria for CPTSD significantly increased the likelihood of having every other condition (all ps < .01) including being approximately 25 times more likely to have a probable MDD diagnosis and being 22 times more likely to have a probable GAD diagnosis. The associations between CPTSD and AUD, suicidality, and chronic illness, respectively, were of a similar magnitude to those observed for PTSD. TABLE 3 HERE 4. Discussion 4.1 Main findings Our results showed that physical abuse and the unexpected death of a loved one were the most common traumatic events during childhood and adulthood, respectively. We also 11 11 found that PTSD was independently associated with being female, being in a committed relationship, and less time having passed since traumatic exposure. In contrast, CPTSD was independently associated with younger age, having experienced a higher number of different childhood interpersonal traumas, and having experienced a higher number of different adulthood interpersonal traumas. A shared risk factor associated with PTSD and CPTSD was growing up in an urban environment. High rates of comorbidity were observed for PTSD and CPTSD however in the cases of MDD and GAD, CPTSD was substantially more strongly associated with these disorders than was PTSD. Presence of PTSD and CPTSD increased the likelihood of suicidality by more than three times, and an AUD and a chronic illness by approximately two times. 4.2 Comparison with other studies In this trauma-exposed community sample of the UK population, we found that CPTSD was a more common diagnosis than PTSD. This is in contrast to findings from a nationally representative sample of the Israeli population that reported higher rates of PTSD (9.0%) than CPTSD (2.6%) (Ben-Ezra et al., 2018), and findings from a nationally representative sample of the United States population that reported similar rates of PTSD (3.4%) and CPTSD (3.8%) (Cloitre et al., in press). It is probable that the trauma-exposed In this trauma-exposed community sample of the UK population, we found that CPTSD was a more common diagnosis than PTSD. This is in contrast to findings from a nationally representative sample of the Israeli population that reported higher rates of PTSD (9.0%) than CPTSD (2.6%) (Ben-Ezra et al., 2018), and findings from a nationally representative sample of the United States population that reported similar rates of PTSD (3.4%) and CPTSD (3.8%) (Cloitre et al., in press). It is probable that the trauma-exposed inclusion criteria used in this study selected for more distressed members of the general population. Consequently, while prior studies suggest that PTSD may be as or more common than CPTSD in the general population, the current findings suggest that CPTSD may be the more common diagnosis among members of the general population that are trauma-exposed. This is an important discovery given the 70% of the world’s population has experienced at least one traumatic life event (Benjet et al., 2016). inclusion criteria used in this study selected for more distressed members of the general population. Consequently, while prior studies suggest that PTSD may be as or more common than CPTSD in the general population, the current findings suggest that CPTSD may be the more common diagnosis among members of the general population that are trauma-exposed. This is an important discovery given the 70% of the world’s population has experienced at least one traumatic life event (Benjet et al., 2016). 12 Our findings regarding the risk factors for PTSD and CPTSD are generally consistent with much of the theoretical and empirical psychotrauma literature. In the current study, females were twice as likely as males to experience PTSD and this aligns with a substantial body of evidence relating to DSM-based models of PTSD (Tolin & Foa 2006). Such differences have been predominantly attributed to biological factors (e.g., hormonal factors; Kajantie & Phillips, 2006). Possible explanations for this effect include differences in social support, stressful life events, and familial liability (Peen & Decker, 2004). 4.3 Strengths and limitations of the study A notable strength of the current study is that it is the first to empirically assess the risk factors and comorbidities associated with the ICD-11 diagnoses of PTSD and CPTSD in a community sample of trauma-exposed adults. Nonetheless, there were several limitations. The community sample was drawn from a nationally representative panel, however, it cannot be ensured that the trauma-exposed participant sample itself was nationally representative, especially considering the high levels of comorbidities reported. Second, diagnoses were based on self-report measures rather than clinician-administered interviews and should therefore be interpreted cautiously. Third, the current study did not include children or adolescents, therefore, it is unknown if the current findings are relevant for younger persons exposed to trauma. However, it is important to highlight that many reported exposure to trauma as a younger person. Finally, the sample in the present study was the same as in Cloitre et al. (2018) although in the study by Cloitre et al. (2018), a more extended version of the ITQ was used. 4.2 Comparison with other studies The same applies for the observed association between less time having passed since traumatic exposure and the increased risk for PTSD. This association has been explained in terms of increased amygdala activity in the aftermath of traumatic exposure and thus an increased risk for fear-based responses (Ganzel, Casey, Glover , Voss & Temple, 2007). Consistent with theoretical models of CPTSD (Cloitre, Garvert, Brewin, Bryant & Maercker, 2013), and findings from clinical (Karatzias et al., 2017, Hyland et al., 2018) and general population (Cloitre et al., in press) samples, we showed that interpersonal trauma is an independent risk-factor for CPTSD. Interpersonal trauma exposure, particularly in childhood, tends to be more toxic with regard to its effects on health and well-being predominantly because it compromises a child’s ability to successfully master certain developmental tasks (e.g., affect regulation, secure attachments; Cichetti & Toth, 1995). Consistent with theoretical models of CPTSD (Cloitre, Garvert, Brewin, Bryant & Maercker, 2013), and findings from clinical (Karatzias et al., 2017, Hyland et al., 2018) and general population (Cloitre et al., in press) samples, we showed that interpersonal trauma is an independent risk-factor for CPTSD. Interpersonal trauma exposure, particularly in childhood, tends to be more toxic with regard to its effects on health and well-being predominantly because it compromises a child’s ability to successfully master certain developmental tasks (e.g., affect regulation, secure attachments; Cichetti & Toth, 1995). Interpersonal trauma also compromises the development of neurobiological systems involved in regulating arousal, emotion, stress responses, and reward processing (McLaughlin, Fox, Zeanah, & Nelson, 2011), which are all related to the development of traumatic stress. Current findings are also in-line with the broader scientific literature, which indicates that urban dwelling increases risk of multiple forms of psychiatric diagnoses, particularly those characterised by anxiety and mood problems (Peen, Schoevers, Beekam & Dekker, 2010). Possible explanations for this effect include differences in social support, stressful life events, and familial liability (Peen & Decker 2004) Current findings are also in-line with the broader scientific literature, which indicates that urban dwelling increases risk of multiple forms of psychiatric diagnoses, particularly those characterised by anxiety and mood problems (Peen, Schoevers, Beekam & Dekker, 2010). Possible explanations for this effect include differences in social support, stressful life events, and familial liability (Peen & Decker, 2004). 13 13 4.4 Implications for clinicians and future research Clinicians should be acutely aware of the symptom profiles of ICD-11 PTSD and CPTSD, and consider a diagnosis of CPTSD to be especially pertinent in cases where there is a history of interpersonal trauma (in childhood or in adulthood). It is critical that clinicians are cognizant of, and accurately diagnose, CPTSD given that it is (a) so highly comorbid with a range of life-threatening conditions, and (b) may require enhanced interventions distinct from those currently offered for PTSD (Karatzias et al., 2018b). Work is ongoing to test the efficacy and acceptability of specific clinical interventions for CPTSD (Karatzias et al., 2018b) however, accurate recognition of CPTSD in general practice, and referral for appropriate interventions, will facilitate recovery from the deleterious effects of CPTSD. 14 Declaration of Interest Marylène Cloitre participated as member of the World Health Organization Working Group on the Classification of Disorders Specifically Associated with Stress, reporting to the International Advisory Group for the Revision of ICD-10 Mental and Behavioural Disorders. However, the views expressed reflect the opinions of the authors and not necessarily the Working Group or Advisory Group and the content of this article does not represent WHO policy. Author Contribution TK and PH conceptualized the manuscript. MS, PH, AB conducted the statistical analysis and wrote the first draft of the manuscript. All authors contributed to and have approved the final manuscript. Data Sharing The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. 15 15 References American Psychiatric Association. (2013). Diagnostic and Statistical Manual of Mental Disorders (5th ed). Washington, DC: American Psychiatric Press. Benjet, C., Bromet, E., Karam, E. G., Kessler, R. C., McLaughlin, K. A., Ruscio, A. M….Koenen, K. C. (2016). The epidemiology of traumatic event exposure worldwide: results from the World Mental Health Survey Consortium. 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(1993). https://doi.org/10.1016/j.genhosppsych.2014.09.009 McLaughlin, K. A., Fox, N. A., Zeanah, C. H., & Nelson, C. A. (2011). Adverse rearing environments and neural development in children: The development of frontal electroencephalogram asymmetry. Biological Psychiatry, 70(11), 1008–1015. https://doi.org/10.1016/j.biopsych.2011.08.006 McLaughlin, K. A., Fox, N. A., Zeanah, C. H., & Nelson, C. A. (2011). Adverse rearing environments and neural development in children: The development of frontal electroencephalogram asymmetry. Biological Psychiatry, 70(11), 1008–1015. https://doi.org/10.1016/j.biopsych.2011.08.006 Nayak, M. B., Bond, J. C., Cherpitel, C., Patel, V., & Greenfield, T. K. (2009). Detecting alcohol-related problems in developing countries: a comparison of 2 screening measures in India. Alcoholism, Clinical and Experimental Research, 33(12), 2057– 2066. Peen, J., & Dekker, J. (2004). Is urbanicity an environmental risk-factor for psychiatric disorders? Lancet, 363(9426), 2012–2013. https://doi.org/10.1016/S0140- 6736(04)16486-6 Peen J, Schoevers RA, Beekam AT, Dekker, J. (2010). The current status of urban-rural differences in psychiatric disorders. Acta Psychiatrica Scandinavica , 121, 84–93. https://doi.org/10.1111/j.1600-0447.2009.01438.x Saunders, J. B., Aasland, O. G., Babor, T. F., & de la Fuente, J. R. (1993). Development of the Alcohol Use Disorders Identification Test (AUDIT): WHO collaborative project Saunders, J. B., Aasland, O. G., Babor, T. F., & de la Fuente, J. R. (1993). Development of the Alcohol Use Disorders Identification Test (AUDIT): WHO collaborative project 20 on early detection of persons with harmful alcohol consumption: II. Addiction, 88(6), 791–804. https://doi.org/10.1111/j.1360-0443.1993.tb02093.x Shevlin, M., Hyland, P., Vallieres, F., Bisson, J., Makhashvili, N., Javakhishvili, Kharchenk, N., Shpiker, M., & Roberts, B. (2018). A comparison of DSM-5 and ICD-11 Posttraumatic Stress Disorder rates and comorbidity: An analysis of the Ukrainian Internally Displaced Person’s Mental Health Survey. Acta Psychiatrica Scandinavica, 137, 138-147. doi: 10.1111/acps.12840 (IF = 4.984) Spitzer R, Kroenke K, Williams J, L. B. (2006). A brief measure for assessing generalized anxiety disorder—the GAD-7. Archives of Internal Medicine, 166(10), 1092–1097. https://doi.org/10.1001/archinte.166.10.1092 Tolin, D. F., & Foa, E. B. (2006). Sex differences in trauma and posttraumatic stress disorder: a quantitative review of 25 years of research. Psychological Bulletin, 132(6):959-92. DOI: 10.1037/0033-2909.132.6.95 Weathers, F., Blake, D. D., Schnurr, P. P., Kaloupek, D. G., Marx, B. P., & Keane, T. M. (2013). The Life Events Checklist for DSM-5 (LEC-5). National Center for PTSD, 5(October), Instrument available from the National Center for PTSD at www.ptsd.va.gov Weathers, F., Blake, D. D., Schnurr, P. P., Kaloupek, D. G., Marx, B. P., & Keane, T. M. (2013). The Life Events Checklist for DSM-5 (LEC-5). https://doi.org/10.1016/j.genhosppsych.2014.09.009 National Center for PTSD, 5(October), Instrument available from the National Center for PTSD at www.ptsd.va.gov World Health Organization. (2018). International statistical classification of diseases and related health problems (11th Revision). Retrieved February 11, 2019 from https://icd.who.int/browse11/l-m/en 21 Table 1. Table 1. Symptom and diagnostic profile for ICD-11 PTSD and CPTSD. Symptom and diagnostic profile for ICD-11 PTSD and CPTSD. Symptom and diagnostic profile for ICD-11 PTSD and CPTSD. PTSD CPTSD Traumatic exposure (required) All PTSD diagnostic requirements necessary and disturbances in self organisation (DSO) Re-experiencing in the here and now (1 of 2 required) Affective dysregulation (1 of 2 required) 1. Upsetting dreams 7. Emotional reactivity 2. Flashbacks 8. Emotional numbing Avoidance of traumatic reminders (1 of 2 required) Negative self-concept (1 of 2 required) 3. Internal reminders 9. Failure 4. External reminders 10. Worthless Sense of threat (1 of 2 required) Disturbances in relationships (1 of 2 required) 5. Vigilance 11. Cut-off from people 6. Hyperarousal 12. Hard to stay close to others Functional impairment associated with these symptoms (required) Functional impairment associated with these symptoms (required) y p g p PTSD CPTSD Traumatic exposure (required) All PTSD diagnostic requirements necessary and disturbances in self organisation (DSO) Re-experiencing in the here and now (1 of 2 required) Affective dysregulation (1 of 2 required) 1. Upsetting dreams 7. Emotional reactivity 2. Flashbacks 8. Emotional numbing Avoidance of traumatic reminders (1 of 2 required) Negative self-concept (1 of 2 required) 3. Internal reminders 9. Failure 4. External reminders 10. Worthless Sense of threat (1 of 2 required) Disturbances in relationships (1 of 2 required) 5. Vigilance 11. Cut-off from people 6. Hyperarousal 12. Hard to stay close to others Functional impairment associated with these symptoms (required) Functional impairment associated with these symptoms (required) 22 Table 2. Table 2. Hierarchical binary logistic regression models for PTSD and CPTSD diagnosis. ICD-11 PTSD ICD-11 CPTSD OR (95% CI) OR (95% CI) Step 1 Sex (female) 2.02 (.97, 4.20) .74 (.49, 1.14) Age .99 (.97, 1.01) .96 (.94, .97)*** Grew up in an urban area 2.02 (1.15, 3.58)* 1.96 (1.34, 2.88)** History of emigration 1.22 (.61, 2.45) 1.62 (1.03, 2.54)* In a relationship 2.30 (1.05, 5.02)* .92 (.60, 1.42) Number of children 1.14 (.87. 1.49) 1.07 (.88. 1.29) Unemployed .98 (.55, 1.74) 1.37 (.93, 2.02) University education 1.17 (.64, 2.15) .79 (.53, 1.17) Step 2 Sex (female) 2.08 (.99, 4.36)* .88 (.55, 1.40) Age 1.00 (.97, 1.02) .97 (.95, .98)*** Grew up in an urban area 2.05 (1.15, 3.64)* 1.72 (1.15, 2.58)** History of emigration 1.16 (.56, 2.40) 1.02 (.61, 1.70) In a relationship 2.30 (1.05, 5.03)* .93 (.60, 1.45) Number of children 1.12 (.85. 1.48) .92 (.74. 1.14) Unemployed .98 (.55, 1.77) 1.39 (.92, 2.10) University education 1.21 (.65, 2.23) .94 (.61, 1.44) Time since trauma .81 (.66, 1.00)* .92 (.79, 1.07) Childhood non-interpersonal trauma .88 (.67, 1.15) 1.03 (.86, 1.22) Childhood interpersonal trauma 1.04 (.78, 1.39) 1.52 (1.27, 1.83)*** Adult non-interpersonal trauma 1.05 (.83, 1.32) 1.08 (.92, 1.26) Adult interpersonal trauma 1.07 (.82, 1.39) 1.20 (1.01, 1.43)* Note: N = 1049; OR (95% CI) = Odds Ratio with 95% confidence intervals; * p < .05, p < .01, * p < .001. Hierarchical binary logistic regression models for PTSD and CPTSD diagnosis. Note: N = 1049; OR (95% CI) = Odds Ratio with 95% confidence intervals; * p < .05, p < .01, * p < .001. 23 Table 3. Table 3. Proportion of those with PTSD and CPTSD meeting probable diagnosis for other disorders. Note: PTSD = Posttraumatic stress disorder; CPTSD = Complex PTSD; MDD = Major depressive disorder; GAD = Generalized anxiety disorder; AUD = Alcohol use disorder; OR (95% CI) = Odds ratio (95% confidence intervals); statistical significance = * p < .05, p < .01, * p < .001. Table 2. % with MDD % with GAD % with AUD % with Suicidality % with Chronic Illness OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI) PTSD 48.2% 30.4% 60.0% 57.1% 44.6% 1.79 (1.04 – 3.07)* 1.10 (0.61 – 1.97) 2.04 (1.02 – 4.08)* 3.13 (1.81 – 5.41)*** 1.90 (1.10 – 3.27)* CPTSD 89.0% 86.0% 58.8% 56.6% 45.6% 21.82 (12.51 – 38.04)* 24.63 (14.77 – 41.07)* 2.07 (1.36 – 3.15) 3.43 (2.37 – 4.70)* 2.11 (1.46 – 3.05)*** Note: PTSD = Posttraumatic stress disorder; CPTSD = Complex PTSD; MDD = Major depressive disorder; GAD = Generalized anxiety disorder; AUD = Alcohol use disorder; OR (95% CI) = Odds ratio (95% confidence intervals); statistical significance = * p < .05, p < .01, * p < .001. 24
https://openalex.org/W2193541516	https://europepmc.org/articles/pmc4703187?pdf=render	English	null	Ultrasound of Sternal Fracture	The western journal of emergency medicine/Western journal of emergency medicine	2,015	cc-by	754	Images in Emergency Medicine Images in Emergency Medicine PRESENTATION A 61-year-old female was brought in by ambulance after being the restrained driver of a head-on motor vehicle collision at 40MPH. There was positive airbag deployment and intrusion from the other vehicle. During workup, the patient complained of midline chest pain, and left chest wall pain. The patient was not in acute respiratory distress, and had the following vital signs: temperature 37°C, heart rate 84, blood pressure of 150/64, respiratory rate 18, and oxygen saturation of 97% on two liters of oxygen. On physical exam, breath sounds were heard bilaterally, with no acute cardiopulmonary issues identified. A bruise was identified on the lower abdomen, which was thought to be a potential seatbelt sign. A focused assessment with sonography for trauma was negative, and an ultrasound of additional chest and mediastinal structures was performed for the chest tenderness (Figure 1). Figure 1. Ultrasound image of the sternum, with the red labeling the two ends of a displaced sternal fracture. University of California, Irvine, Department of Emergency Medicine, Irvine, California University of California, Irvine, Department of Emergency Medicine, Irvine, California Section Editor: Sean O. Henderson, MD Submission history: Submitted September 11, 2015; Accepted September 24, 2015 Electronically published December 8, 2015 Full text available through open access at http://escholarship.org/uc/uciem_westjem DOI: 10.5811/westjem.2015.9.28645 j [West J Emerg Med. 2015;16(7):1057-1058.] [West J Emerg Med. 2015;16(7):1057-1058.] [West J Emerg Med. 2015;16(7):1057-1058.] Recent studies have compared the sensitivity and specificity of chest radiographs and ultrasound in determining the presence of sternal fracture. The sensitivity and specificity of chest radiograph were 70.8% and 75.0%, respectively with ultrasound showing a sensitivity and specificity as high as 100%.4 Ultrasound has the advantage of increased sensitivity and specificity for diagnosing sternal fractures in comparison to chest radiographs, and avoids the excess radiation and time commitment of mobilizing patients to perform a chest CT.4 Ultrasound is not accurate in identifying the degree of displacement of sternal fractures, but can accurately identify related hematomas and pleural effusions.4 Ultrasound of Sternal Fracture Shadi Lahham, MD, MS Jonathan Patane, MD Nathaniel Lane, MD DIAGNOSIS Sternal fracture has been observed in approximately 10% of patients with blunt chest trauma, with the most common mechanism of injury being motor vehicle accidents.1 Isolated sternal fractures most often have a benign course, but can rarely cause secondary cardiac injury.2 Patients with chest trauma typically undergo radiograph imaging in the emergency department to help rule out acute life-threatening cardiopulmonary injuries such as aortic dissection, tension pneumothorax, and cardiac tamponade, among other pathologies. Typically, these imaging techniques involve a portable chest radiograph, followed by a computed tomography (CT) of the chest if applicable.3 Standard AP chest radiographs have a low sensitivity for diagnosing sternal fractures, with the majority of fractures being identified by lateral chest radiograph or CT (Figure 2). Because lateral chest radiographs are typically not performed in the acute trauma workup, many sternal fractures are not diagnosed until later in the trauma evaluation.1,4 Figure 1. Ultrasound image of the sternum, with the red labeling the two ends of a displaced sternal fracture. Volume XVI, no. 7 : December 2015 Western Journal of Emergency Medicine 1057 Lahham et al. Ultrasound of Sternal Fracture Figure 2. Computed tomography identifying the same displaced sternal fracture. Address for Correspondence: Shadi Lahham MD, MS, 333 The City Boulevard West, Suite 640, Rt 128-01, Orange, CA 92868. Email: slahham@uci.edu. Conflicts of Interest: By the WestJEM article submission agreement, all authors are required to disclose all affiliations, funding sources and financial or management relationships that could be perceived as potential sources of bias. The authors disclosed none. Copyright: © 2015 Lahham et al. This is an open access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) License. See: http://creativecommons.org/ licenses/by/4.0/ Western Journal of Emergency Medicine Volume XVI, no. 7 : December 2015 REFERENCES 1. Engin G, Yekeler E, Güloğlu R, et al. US versus conventional radiology in the diagnosis of sternal fractures. Acta Radiol. 2000;41(3):296-299. 2. Hills MW, Delprado AM, Deane SA. Sternal fractures. Associated injuries and management. J Trauma. 1993;35(1):55-60. 3. Rodriguez RM, Anglin D, Landorf MI, et al. NEXUS chest: validation of a decision instrument for selective chest imaging in blunt trauma. JAMA Surg. 2013;148(10):940-6. 4. You JS, Chung YE, Kim D, et al. Role of somography in the emergency room to diagnose sternal fractures. J Clin Ultrasound. 2010;38(3):135-7. Figure 2. Computed tomography identifying the same displaced sternal fracture. Volume XVI, no. 7 : December 2015 Western Journal of Emergency Medicine 1058
https://openalex.org/W4298442789	https://zenodo.org/record/5740731/files/source.pdf	English	null	Catalogue of type specimens of true bugs (Hemiptera: Heteroptera) deposited in the National Museum, Prague, Czech Republic	Zenodo (CERN European Organization for Nuclear Research)	2,013	cc-by	52,603	ACTA ENTOMOLOGICA MUSEI NATIONALIS PRAGAE Published 15.xi.2013 Volume 53(2), pp. 821–890 ISSN 0374-1036 http://zoobank.org/urn:lsid:zoobank.org:pub:146A1A16-2E72-4ACA-B9F7-D2D1ED48574B ACTA ENTOMOLOGICA MUSEI NATIONALIS PRAGAE Published 15.xi.2013 Volume 53(2), pp. 821–890 ISSN 0374-1036 http://zoobank.org/urn:lsid:zoobank.org:pub:146A1A16-2E72-4ACA-B9F7-D2D1ED48574B Introduction The number of species-group type specimens of insects in the Department of Entomology of the National Museum, Prague (NMP; NMPC when referring to the collection) is estimated to several tens of thousands (majority of them belonging to Coleoptera), and the presence of some of them in the collection is still largely unknown (see BEZDĚK & HÁJEK 2009). As the International Code of Zoological Nomenclature encourages institutions to catalogue and make accessible the type material in their care (ICZN 1999: Recommendation 72F), the curators of the NMP have started cataloguing the types in the NMPC to improve the knowledge about the collections and provide information to the entomological community. This effort has already resulted in seven parts of the catalogue devoted to selected families of Coleoptera (see BEZDĚK & HÁJEK 2009, 2010a,b, 2011, 2012, 2013; HÁJEK & ŠVIHLA 2012). This part represents a start of another series dedicated to Hemiptera. Along with the paper, photos of the types and a copy of their original description are available upon request. The Department of Entomology was founded by Dr. Jan Obenberger as an entomological unit of the Department of Zoology in 1920 and became entirely independent in 1952. The history of insect collections deposited in the NMP is, however, much longer. The oldest specimens originate from the end of the 18th century (BEZDĚK & HÁJEK 2009). Unlike the more popular Coleoptera and Lepidoptera, the beginnings of the Heteroptera collection were more modest. The ﬁ rst specialized Hemiptera collection acquired by the museum was that of the secondary school teacher Ladislav Duda (1854–1895) donated by his sister in 1896 (KOLEŠKA 1980). The Duda collection included valuable voucher specimens of Heteroptera, Auchenorrhyncha and Psylloidea on which his pioneer papers on Hemiptera of Bohemia (DUDA 1884, 1885–1886, 1892a,b) were based, but contained types of only one infraspeciﬁ c taxon of Heteroptera described by him, Ischnorrhynchus (= Kleidoceyrs) resedae var. ﬂ avicornis Duda, 1885. The second founding collection of Heteroptera in the NMP was part of the important Nickerls collection, i.e. the joint collection of František Antonín Nickerl (1813–1871), Otakar Nickerl Sr. (1838–1920) and Otakar Nickerl Jr. (1873–1904). The collection of the father, son and grandson Nickerl came to the museum as a legacy in 1920 (VÁVRA 1923). It represents one of the NMP’s oldest collections and includes predominantly Coleoptera, Lepidoptera and Heteroptera, which were incorporated into the general collection. Enicocephalomorpha, Dipsocoromorpha, Nepomorpha, Gerromorpha, and Leptopodomorpha Petr KMENT & Zdislava KOLÍNOVÁ Department of Entomology, National Museum, Kunratice 1, CZ-148 00 Praha 4, Czech Republic; e-mail: sigara@post.cz, Zakova.slavka@seznam.cz Abstract. Type specimens from the collection of true bugs (Hemiptera: Hetero- ptera) deposited in the Department of Entomology, National Museum, Prague, are currently being catalogued. In this part of the catalogue dealing with the ﬁ ve basal infraorders, Enicocephalomorpha, Dipsocoromorpha, Nepomorpha, Gerro- morpha, and Leptopodomorpha, we present precise information about the types of 166 taxa belonging to the families Enicocephalidae (2 taxa), Schizo pteridae (1 taxon), Nepidae (4 taxa), Gelastocoridae (1 taxon), Corixidae (4 taxa), Micro- nectidae (7 taxa), Naucoridae (4 taxa), Potamocoridae (1 taxon), Aphelocheiridae (4 taxa), Notonectidae (2 taxa), Helotrephidae (6 taxa), Mesoveliidae (1 taxon), Hebridae (7 taxa), Hydrometridae (5 taxa), Macroveliidae (1 taxon), Veliidae (66 taxa), Gerridae (28 taxa), Saldidae (20 taxa), and Leptopodidae (2 taxa), including holotypes (or dissected parts of holotypes) of 42 taxa, and neotype of one taxon. The correct spelling of Brachymetra anduze Drake & Harris, 1942 and Naboandelus bergevini pygmaeus Linnavuori, 1971 (both Gerridae) is ﬁ xed. Key words. Catalogue, type specimens, nomenclature, National Museum, Hemiptera, Heteroptera, Enicocephalomorpha, Dipsocoromorpha, Nepomorpha, Gerromorpha, Leptopodomorpha * Catalogue of type specimens in NMPC, part 8 (Heteroptera I) 822 KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. Introduction The Heteroptera section of the Nickerls collection contains mostly specimens collected by the ophthalmolog and heteropterist O. Nickerl Jr. in Bohemia (see NICKERL 1905), including also types of the single infraspeciﬁ c taxon he described before his untimely death, Adomerus biguttatus var. concolor Nickerl, 1892 (NICKERL 1892). However, the Nicklerls collection includes also material of exotic Heteroptera and a few type specimens from the 19th century obtained by the Nicklerls’ active communication, exchange and purchase of material from many entomologists of their era, e.g., types of Anton Dohrn (1840–1909), whose main collection deposited in the Museum für Naturkunde Stettin (currently Szczecin, Poland) was destroyed during World War II.; the depository of these types was largely unknown (KOLEŠKA 1988; BEZDĚK & HÁJEK 2009, 2010b). After the World War I, the entomological collection of the NMP started to grow thanks to collecting effort of the staff in the Mediterranean (J. Obenberger, J. Mařan) and especially through acquisition of material from the voluntary Czech collectors working abroad, e.g. Vlasta Kálalová-di Lotti (Iraq), Jaro Mráz (Brazil), Jiří Baum (mostly tropical Africa and Asia), or 823 Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 Stanislav Škulina (tropical Africa), as well as thanks to purchase of additional exotic material (Lamberton’s material from Madagascar) which became part of the general collection. ( g ) p g An important part of the history of the NMP’s heteropterological collection is connected with the name of its ﬁ rst curator-specialist, Ludvík Hoberlandt (1918–2005). Hoberlandt ﬁ rst started to work in the NMP as a volunteer in 1939 and was its employee from January 1, 1945 until his retirment in 1985 but he continued to work as a scientiﬁ c associate as long as his health allowed. During his 60 years long engagement in the NMPC, L. Hoberlandt participated in several collecting trips (Bulgaria, Montenegro, Turkey, Iran, India) yielding numerous specimens of Heteroptera, as well as in identiﬁ cation and description of new taxa based on material of other expeditions (e.g. Afghanistan, Pakistan, Angola, Madagascar, Mongolia), keeping the multiple specimens in the NMPC. Ludvík Hoberlandt also maintained contacts with many of his contemporaries who participated in identiﬁ cation and description of new taxa from the NMPC or exchanged specimens and paratypes with him (e.g. N. M. Andersen, R. H. Cobben, C. J. Drake, H. B. Hungerford, A. Jansson, M. Josifov, I. M. Kerzhner, J. D. Lattin, P. Lindskog, R. Linnavuori, J. Introduction Péricart, L. R. Rolston, T. R. Schuh, G. G. E. Scudder, G. Seidenstücker, J. A. Slater, J. L. Stehlík, J. M. Štusák, L. Tamanini, A. Villiers, E. Wagner, L.-Y. Zheng) (see ŠTYS 1979, JEŽEK 1987, STEHLÍK 1999, HEISS 2005). g) ( ) In 1985, Vladimír Švihla (1952), a renowned specialist in Coleoptera: Cantharidae, Oede- meridae, replaced L. Hoberlandt as a curator of Heteroptera, partly assisting L. Hoberlandt with his work (HOBERLANDT & ŠVIHLA 1990a,b) but the scientiﬁ c processing of the collection slowed down due to fewer visits and loans by foreign specialists (except for e.g. J. A. Lis). In 2005, Petr Kment (1977) was put in charge of the Hemiptera collection (joining the former Heteroptera and Homoptera collections). Since 2005, the NMPC has suceeded in acquiring new interesting materials of Heteroptera (United Arab Emirates, Socotra, Laos, China, and smaller collections from other countries), and new or renewed co-operations have beend started with many specialists all around the world (e.g. H. Brailovsky, A. Carapezza, P.-P. Chen, D. Chłond, J. Czaja, J. A. M. Fernandes, D. Gapon, E. Heiss, E. V. Kanyukova, A. Kocorek, E. Kondorosy, F. Konstantinov, R. Linnavuori, Ph. Magnien, A. Matocq, P. Moulet, N. Nieser, D. Rédei, J. L. Stehlík, J.-F. Tsai, N. N. Vinokurov, A. Wolski, H. Zettel) already resulting in several descriptions of new taxa based in the NMPC. Material and methods The system used to arrange the taxa mostly follows AUKEMA & RIEGER (1995); in addition it accepts erection of the superfamily Aphelocheiroidea (HEBSGAARD et al. 2004) and the family status of Micronectidae (NIESER 2002, NIESER & CHEN 2006). Within each subfamily/tribe, the genera and species are arranged alphabetically. Each entry includes: – the name of the taxon in original combination. – the name of the taxon in original combination. – the name of the taxon in original combination and spelling, with the author and year of description. Pagination, ﬁ gures and plates are also given. – the name of the taxon in original combination and spelling, with the author and year of description. Pagination, ﬁ gures and plates are also given. – the type status, number of specimens (including their sex if known) and exact label data. Our remarks are found in square brackets: [p] – preceding data are printed, [hw] – preced- ing data are handwritten. Separate labels are indicated by a double slash ‘//’ and lines within – the type status, number of specimens (including their sex if known) and exact label data. Our remarks are found in square brackets: [p] – preceding data are printed, [hw] – preced- ing data are handwritten. Separate labels are indicated by a double slash ‘//’ and lines within 824 KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. Figs 1−4. Type labels of Ludvík Hoberlandt. 1−2 – Velia rhadamantha Hoberlandt, 1941, holotypus, characteristic style of labels in the 1940s; 3−4 – Sigara samani Hoberlandt, 1952, characteristic labels for the 1950s and later period. (Photos: L. Macháčková). PARATYPE (): ‘Andohahelo / 1800m R P. [p] // PARATYPE [p, red label] // [p] // Desystellores / pauliani / n g n. sp. [hw] / A Villiers det 195[p]7 [hw]’. Figs 1−4. Type labels of Ludvík Hoberlandt. 1−2 – Velia rhadamantha Hoberlandt, 1941, holotypus, characteristic style of labels in the 1940s; 3−4 – Sigara samani Hoberlandt, 1952, characteristic labels for the 1950s and later period. (Photos: L. Macháčková). each label are separated by a slash ‘/’. Unless otherwise indicated, it means black ink and white label. To mark the wing development of the specimens, the following abbreviations are used: apter. – apterous, brach. – brachypterous, micr. – micropterous, macr. – macropterous, submacr. – submacropterous). When appropriate, the sex of the specimen was identiﬁ ed and the corresponding label attached to the specimen. In case the original labels did not contain precise information on the current status of the type specimen, one additional printed red label was attached by the curator. For samples of typical type labels by Ludvík Hoberlandt see Figs 1−4. g – the type condition is mentioned for considerably damaged specimens (given in parentheses following the particular specimen). Regular mounts between a slide and a cover glass, de- posited separately from the specimen are referred to as slides; mounts between two cover slides attached to the same pin as the specimen are referred to as microslides. – the current taxonomic status. As subgeneric division of Ranatra Fabricius, 1790 and Rhagovelia Mayr, 1865 is still controversial, we refrain from using subgenera within those taxa (see e.g. POLHEMUS 1995a, AUKEMA et al. 2013). – the current taxonomic status. As subgeneric division of Ranatra Fabricius, 1790 and Rhagovelia Mayr, 1865 is still controversial, we refrain from using subgenera within those taxa (see e.g. POLHEMUS 1995a, AUKEMA et al. 2013). – any taxonomic problems and incosistencies are mentioned under Remarks. Full reference to each publication can be found in References section. Two paratypes are deposited in NMPC: PARATYPES ( macr., brach.): ‘(Kyushu) / Korasan / (Chikugo) [p] / 6. iii. [hw] 195[p]5 [hw] / S. Miyamoto [p] // [p] // [p] // PARATYPUS / Kokeshia / esakii Miy. / n. g. et n. sp. 1960 [hw, pale green label]’. (Both specimens are glued to pointed pieces of card and placed on the same pin). Current status. Valid species (see KERZHNER 1995, RÉDEI 2008). Family NEPIDAE Latreille, 1802 Subfamily Nepinae Latreille, 1802 Tribe Nepini Latreille, 1802 Family NEPIDAE Latreille, 1802 Subfamily Nepinae Latreille, 1802 Tribe Nepini Latreille, 1802 Tribe Nepini Latreille, 1802 Laccotrephes elongatus Montandon, 1907 Laccotrephes elongatus Montandon, 1907: 330−331 (original description). One syntype is deposited in NMPC: SYNTYPE (): ‘Bombay. [hw] // [p] // Laccotrephes / elongatus Montand [hw, black ink] / co-type 1903 [hw, red ink].’ Current status. Valid species: Laccotrephes (Laccotrephes) elongatus Montandon, 1907 (see THIRUMALAI 2007). Subfamily Ranatrinae Douglas & Scott, 1865 Tribe Ranatrini Douglas & Scott, 1865 Ranatra aethiopica Montandon, 1903 Ranatra aethiopica Montandon, 1903a: 20−21 (original description). One syntype is deposited in NMPC: SYNTYPE (): ‘Scioa / Falle 8. 2. 85 / Ragazzi [p, black frame submarginally] // R. Gestro [hw] // [p] // Ranatra / Embolorrhinus ambrinus Villiers, 1969 PARATYPE (): ‘Madagascar Nord / Montagne d’Ambre / Les Roussettes 1100m / II-59 / Pierre Soga [p] // PARATYPE [p, red label] // [p] // Embolorrhinus / ambrinus / n. sp. [hw] / A Villiers det 19[p]68 [hw]’. Current status. Valid species: Embolorrhinus (Ceratotrachelus) ambrinus Villiers, 1969 (see ŠTYS 1969, 2002). Catalogue Family ENICOCEPHALIDAE Stål, 1860 Subfamily Enicocephalinae Stål, 1860 Tribe Enicocephalini Stål, 1860 Family ENICOCEPHALIDAE Stål, 1860 Subfamily Enicocephalinae Stål, 1860 Tribe Enicocephalini Stål, 1860 Desystellores pauliani Villiers, 1958 Desystellores pauliani Villiers, 1958: 34–35, Figs 38–41 (original description). esystellores pauliani Villiers, 1958: 34–35, Figs 38–41 (original description). One paratype is deposited in NMPC: One paratype is deposited in NMPC: One paratype is deposited in NMPC: PARATYPE (): ‘Andohahelo / 1800m R P. [p] // PARATYPE [p, red label] // [p] // Desystellores / pauliani / n g n. sp. [hw] / A Villiers det 195[p]7 [hw]’. 825 Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 Current status. Valid species: Euchelichir (Desystellores) pauliani (Villiers, 1958) (see VILLIERS 1969, ŠTYS 2002). b l h b Current status. Valid species: Euchelichir (Desystellores) pauliani (Villiers, 1958) (see VILLIERS 1969, ŠTYS 2002). Current status. Valid species: Euchelichir (Desystellores) pauliani (Villiers, 1958) (see VILLIERS 1969, ŠTYS 2002). Kokeshia esakii Myiamoto, 1960 Kokeshia esakii Myiamoto, 1960: 163–169, Text: Figs 1A–F, Pl. 18: Figs A–B, Pl. 19: Figs A–P (original descrip- tion). Kokeshia esakii Myiamoto, 1960: 163–169, Text: Figs 1A–F, Pl. 18: Figs A–B, Pl. 19: Figs A–P (original descrip- tion) Two paratypes are deposited in NMPC: Family GELASTOCORIDAE Kirkaldy, 1897 Subfamily Nerthrinae Kirkaldy, 1906 Family GELASTOCORIDAE Kirkaldy, 1897 Subfamily Nerthrinae Kirkaldy, 1906 Ranatra dispar Montandon, 1903 Ranatra dispar Montandon, 1903 Ranatra dispar Montandon, 1903b: 104−105 (original description). Ranatra dispar: LANSBURY (1972): 323 (lectotype designation). p , Ranatra dispar Montandon, 1903b: 104−105 (original description). Ranatra dispar: LANSBURY (1972): 323 (lectotype designation). One paralectotype is deposited in NMPC: PARALECTOTYPE (): ‘VICTORIA / Alexandra / F.L.Billinghurst. [p] // R. dispar Montd / [hw, black ink] co-type [hw, red ink] // PARALECTOTYPUS / RANATRA / DISPAR / Montandon, 1903 / labelled: P. KMENT 2013 [p, red label]’. Current status. Valid species: Ranatra dispar Montandon, 1903 (see LANSBURY 1972, ANDERSEN & WEIR 2004). Laccotrephes elongatus Montandon, 1907 Laccotrephes elongatus Montandon, 1907: 330−331 (original description). One syntype is deposited in NMPC: SYNTYPE (): ‘Bombay. [hw] // [p] // Laccotrephes / elongatus Montand [hw, black ink] / co-type 1903 [hw, red ink].’ Current status. Valid species: Laccotrephes (Laccotrephes) elongatus Montandon, 1907 One syntype is deposited in NMPC: SYNTYPE (): ‘Bombay. [hw] // [p] // Laccotrephes / elongatus Montand [hw, black ink] / co-type 1903 [hw, red ink].’ SYNTYPE (): Bombay. [hw] // [p] // Laccotrephes / elongatus Montand [hw, black ink] / co-type 1903 [hw, red ink].’ Current status. Valid species: Laccotrephes (Laccotrephes) elongatus Montandon, 1907 (see THIRUMALAI 2007). Current status. Valid species: Laccotrephes (Laccotrephes) elongatus Montandon, 1907 (see THIRUMALAI 2007). Subfamily Ranatrinae Douglas & Scott, 1865 Tribe Ranatrini Douglas & Scott, 1865 Ranatra aethiopica Montandon, 1903 Ranatra aethiopica Montandon, 1903a: 20−21 (original description). One syntype is deposited in NMPC: SYNTYPE (): ‘Scioa / Falle 8. 2. 85 / Ragazzi [p, black frame submarginally] // R. Gestro [hw] // [p] // Ranatra / Subfamily Ranatrinae Douglas & Scott, 1865 Tribe Ranatrini Douglas & Scott, 1865 826 KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. Æthiopica Montand [hw, black ink] / co-type. 1903. [hw, red ink]’. Æthiopica Montand [hw, black ink] / co-type. 1903. [hw, red ink]’. Æthiopica Montand [hw, black ink] / co type. 1903. [hw, red ink] . Current status. Valid species: Ranatra aethiopica Montandon, 1903 (see POISSON 1965a). Ranatra bottegoi Montandon, 1903 Ranatra bottegoi Montandon, 1903a: 22−23 (original description). One syntype is deposited in NMPC: SYNTYPE (): ‘SOMALI / BASSO GANANA / VII-VIII. 93 / V. BOTTEGO [p, black frame submarginally] // [orange square with black stripe medially] // Mus Civ Genoa / R. Gestro [hw] // Ranatra / Bottegoi Montd / [hw, black ink] co-type [hw, red ink]’. Current status. Valid species: Ranatra bottegoi Montandon, 1903 (see POISSON 1965a, LIN- NAVUORI 1981, KEFFER 2004). Ranatra bottegoi Montandon, 1903 Ranatra bottegoi Montandon, 1903a: 22−23 (original description). One syntype is deposited in NMPC: Nerthra williamsi Todd, 1955 Nerthra williamsi Todd, 1955: 348 (key), 383–384, 469: Fig. 112 (original description). The holotype and 6 paratypes (including the allotype) are deposited in NMPC: HOLOTYPE (): ‘SAÕ [sic!] PAULO / BRAS. MRÁZ LGT. / MUS. PRAGENSE [p, black frame submarginally] // Holotype [p] / NERTHRA / WILLIAMSI [hw] / E. L. Todd [p, red label] // Nerthra / williamsi / N. sp. / Det. E. L. Todd [hw, black frame submarginally] // [p]’. (Genitalia dissected, placed dry in a glass microvial attached to the same pin.) PARATYPE (): ‘SAÕ [sic!] PAULO / BRAS. MRÁZ LGT. / MUS. PRAGENSE [p, black frame submarginally] // Allotype [p] / NERTHRA / WILLIAMSI [hw] / E. L. Todd [p, red label] // [p]’. PARATYPE (): ‘SAÕ [sic!] PAULO / BRAS. MRÁZ LGT. / MUS. PRAGENSE [p, black frame submarginally] // Allotype [p] / NERTHRA / WILLIAMSI [hw] / E. L. Todd [p, red label] // [p]’. PARATYPES (2 ): ‘SAÕ [sic!] PAULO / BRAS. MRÁZ LGT. / MUS. PRAGENSE [p, black frame submargin- ally] // Paratype [p] / NERTHRA / WILLIAMSI [hw] / E. L. Todd’ [p, blue label] // [p]’. (One paratype with dissected genitalia placed in a glass microvial attached to the same pin.) Á PARATYPES (2 ): ‘SAÕ [sic!] PAULO / BRAS. MRÁZ LGT. / MUS. PRAGENSE [p, black frame submargin- ally] // Paratype [p] / NERTHRA / WILLIAMSI [hw] / E. L. Todd’ [p, blue label] // [p]’. (One paratype with dissected genitalia placed in a glass microvial attached to the same pin.) PARATYPES (2 ): ‘SAÕ [sic!] PAULO / BRAS. MRÁZ LGT. / MUS. PRAGENSE [p, black line submarginally] // Paratype [p] / NERTHRA / WILLIAMSI [hw] / E. L. Todd’ [p, blue label] // [p]’. (One paratype seriously damaged, only torso of thorax and hemelytra left.) PARATYPES (2 ): ‘SAÕ [sic!] PAULO / BRAS. MRÁZ LGT. / MUS. PRAGENSE [p, black line submarginally] // Paratype [p] / NERTHRA / WILLIAMSI [hw] / E. L. Todd’ [p, blue label] // [p]’. (One paratype seriously damaged, only torso of thorax and hemelytra left.) PARATYPE (): ‘Sao PAULO; Mráz / legit. Brasilia / mus. R. BOH, [p] // Paratype [p] / NERTHRA / WILLIAMSI [hw] / E. L. Todd’ [p, blue label] // [p]’. PARATYPE (): ‘Sao PAULO; Mráz / legit. Brasilia / mus. R. The holotype and two paratypes are deposited in NMPC: (Male genitalia dissected, preserved dry in a glass microvial attached to the same pin). PARATYPE (): ‘Ambanja / Madagascar / Mus Praha [p] // Paratypus [p, red label with black margins] // I. i936. / F. Lamberton [hw] // Agraptocorixa / stepaneki Hob. / Paratypus - i942 [hw] // Mus. Nat. Pragae / Inv. [p] 11.123 [hw, orange label]’. PARATYPE (): ‘Ambanja / Madagascar / Mus Praha [p] // Paratypus [p, red label with black margins] // I. i936. / F. Lamberton [hw] // Agraptocorixa / stepaneki Hob. / Paratypus - i942 [hw] // Mus. Nat. Pragae / Inv. [p] 11.123 [hw, orange label]’. Current status. Valid species (see POISSON 1951, 1963). Current status. Valid species (see POISSON 1951, 1963). The neotype is deposited in NMPC: The neotype is deposited in NMPC: NEOTYPE (): ‘Jezero / Plöckenstein- / ské. Dr.Štolc [hw] // Neotype [p] [hw] / Corisa propinqua / Fieber / det. A. Jansson 1985 [hw, red label]’. NEOTYPE (): ‘Jezero / Plöckenstein- / ské. Dr.Štolc [hw] // Neotype [p] [hw] / Corisa propinqua / Fieber / det. A. Jansson 1985 [hw, red label]’. Current status. Valid species: Glaenocorisa propinqua (Fieber, 1860) (see JANSSON 1986a, 1995, 2000; KANYUKOVA 2006; AUKEMA et al. 2013). Agraptocorixa stepaneki Hoberlandt, 1942 Agraptocorixa štěpáneki Hoberlandt, 1942a: 47–50 , Figs 1–11 (original description). Agraptocorixa štěpáneki Hoberlandt, 1942a: 47–50 , Figs 1–11 (original description). Nerthra williamsi Todd, 1955 BOH, [p] // Paratype [p] / NERTHRA / WILLIAMSI [hw] / E. L. Todd’ [p, blue label] // [p]’. Current status. Valid species (see HECKMAN 2011, MOREIRA et al. 2011). 827 Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 Corisa propinqua Fieber, 1860 Corisa propinqua Fieber, 1860: 99 (original description). Corisa propinqua Fieber, 1860: 99 (original description). Corisa propinqua: JANSSON (1986a): 26 (neotype designation). Corisa propinqua: JANSSON (1986a): 26 (neotype designation). Corisa propinqua: JANSSON (1986a): 26 (neotype designation). The neotype is deposited in NMPC: NEOTYPE (): ‘Jezero / Plöckenstein- / ské. Dr.Štolc [hw] // Neotype [p] [hw] / Corisa propinqua / Fieber / det. The neotype is deposited in NMPC: The holotype and two paratypes are deposited in NMPC: HOLOTYPE (): ‘Ambanja / Madagascar / Mus Praha [p] // Holotypus [p, red label with black margins] // I-i936. / F. Lamberton [hw] // Agraptocorixa / štěpáneki n. sp. [hw, red ink] / L. Hoberlandt det. [p] // Mus. Nat. Pragae / Inv. [p] 11.121 [hw, orange label]’. (Only head + pronotum and rest of thorax without wings and legs + base of abdomen are glued separately to a piece of card, remaining body parts mounted in a series of 7 slides stored in Slide Box 1). HOLOTYPE (): ‘Ambanja / Madagascar / Mus Praha [p] // Holotypus [p, red label with black margins] // I-i936. / F. Lamberton [hw] // Agraptocorixa / štěpáneki n. sp. [hw, red ink] / L. Hoberlandt det. [p] // Mus. Nat. Pragae / Inv. [p] 11.121 [hw, orange label]’. (Only head + pronotum and rest of thorax without wings and legs + base of abdomen are glued separately to a piece of card, remaining body parts mounted in a series of 7 slides stored in Slide Box 1). PARATYPE (): ‘Ambanja / Madagascar / Mus Praha // I. i936. / F. Lamberton [hw] // Paratypus [p, red label with black margins] // Mus. Nat. Pragae / Inv. [p] 11.122 [hw, orange label] // Paratypus [hw, red ink] / Agraptocorixa [hw, black ink] / 19[p]42 štěpáneki sp. n. [hw, black ink] / [hw, black ink] / L. Hoberlandt det. [p]’. (Male genitalia dissected, preserved dry in a glass microvial attached to the same pin). PARATYPE (): ‘Ambanja / Madagascar / Mus Praha // I. i936. / F. Lamberton [hw] // Paratypus [p, red label with black margins] // Mus. Nat. Pragae / Inv. [p] 11.122 [hw, orange label] // Paratypus [hw, red ink] / Agraptocorixa [hw, black ink] / 19[p]42 štěpáneki sp. n. [hw, black ink] / [hw, black ink] / L. Hoberlandt det. [p]’. (Male genitalia dissected, preserved dry in a glass microvial attached to the same pin). PARATYPE (): ‘Ambanja / Madagascar / Mus Praha // I. i936. / F. Lamberton [hw] // Paratypus [p, red label with black margins] // Mus. Nat. Pragae / Inv. [p] 11.122 [hw, orange label] // Paratypus [hw, red ink] / Agraptocorixa [hw, black ink] / 19[p]42 štěpáneki sp. n. [hw, black ink] / [hw, black ink] / L. Hoberlandt det. [p]’. Pseudoglaenocorisa linnavuorii Jansson, 1986 Pseudoglaenocorisa linnavuorii Jansson, 1986b: 102–104, Figs 1, 3, 5, 7, 9, 11, 13 (original description). One paratype is deposited in NMPC: PARATYPE (): ‘Ethiopia / Mai Chew 1. VI. 63 / Linnavuori [p] // Paratype [p] [hw] / Pseudoglaeno- / corisa linnavuorii / Jansson [hw, red label]’. PARATYPE (): ‘Ethiopia / Mai Chew 1. VI. 63 / Linnavuori [p] // Paratype [p] [hw] / Pseudoglaeno- / corisa linnavuorii / Jansson [hw, red label]’. Current status. Valid species. Current status. Valid species. Sigara (Subsigara) samani Hoberlandt, 1952 Sigara (Subsigara) samani Hoberlandt, 1952: 15–17, 20, Pl. III: Figs 27–30, Pl. IV: Figs 23–30, P. IV: Figs 31–35, Pl. V: Figs 36–39, Pl. IX: Fig. 71 (original description, key to species). The holotype and one paratype (allotype) are deposited in NMPC: KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. 828 HOLOTYPE (): ‘Mersin [hw] / Anat. [p] 25. VIII. 47 [hw] / Exp. N. Mus. ČSR [p] // Holotypus [p, red label] // Holotypus [p] / Sigara / (Subsigara) / samani sp. n. [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 1015 [hw] / Mus. Nat. Pragae [p, orange label]’ (see Figs 3−4). (Left fore, middle and hind leg, left hemelytron, and apical part of abdomen are removed from the specimen and mounted on 4 slides stored in Slide Box 1). HOLOTYPE (): ‘Mersin [hw] / Anat. [p] 25. VIII. 47 [hw] / Exp. N. Mus. ČSR [p] // Holotypus [p, red label] // Holotypus [p] / Sigara / (Subsigara) / samani sp. n. [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 1015 [hw] / Mus. Nat. Pragae [p, orange label]’ (see Figs 3−4). (Left fore, middle and hind leg, left hemelytron, and apical part of abdomen are removed from the specimen and mounted on 4 slides stored in Slide Box 1). PARATYPE (): ’Mersin [hw] / Anat. [p] 25. VIII. 47 [hw] / Exp. N. Mus. ČSR [p] // Allotypus [p, red label] // Al- lotypus [p] / Sigara / (Subsigara) / samani sp. n. / [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // Inv. [p] 1016 [hw] / Mus. Nat. Pragae [p, orange label]’. PARATYPE (): ’Mersin [hw] / Anat. [p] 25. VIII. 47 [hw] / Exp. N. Mus. Micronecta crinita Chen, Nieser & Wattanachaiyingcharoen, 2002 Micronecta crinita Chen, Nieser & Wattanachaiyingcharoen, 2002: 194–197, 199, Figs 1, 3–9, 20 (original descrip- tion). Two paratypes are deposited in NMPC (exchange with Natural History Museum, V PARATYPES (2 brach.): ‘Thailand:Khon Kaen Prov. / Phu Phan Kham NP, Bon Noon / Hua Chang, Huai Sam Caen / 21.11.1995,leg.H.Zettel(20a) [p] // [p] // Paratype / Micronecta / crinita / Chen & Nieser [p, red label]’. Current status. Junior synonym of Micronecta punctinotum L.-C. Chen, 1960 (see NIESER et al. 2004); not assigned to a particular subgenus. Pseudoglaenocorisa linnavuorii Jansson, 1986 ČSR [p] // Allotypus [p, red label] // Al- lotypus [p] / Sigara / (Subsigara) / samani sp. n. / [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // Inv. [p] 1016 [hw] / Mus. Nat. Pragae [p, orange label]’. Current status. Valid species: Sigara (Subsigara) samani samani Hoberlandt, 1952 (see JANSSON 1986a,c, 1995; AUKEMA et al. 2013). Current status. Valid species: Sigara (Subsigara) samani samani Hoberlandt, 1952 (see JANSSON 1986a,c, 1995; AUKEMA et al. 2013). Family MICRONECTIDAE Jaczewski, 1924 Subfamily Micronectinae Jaczewski, 1924 Micronecta christiniana Lansbury, 1954 Micronecta christiniana Lansbury, 1954: 140–142, Figs 1A–G (original description). Micronecta christiniana Lansbury, 1954: 140–142, Figs 1A–G (original description). Micronecta christiniana Lansbury, 1954: 140–142, Figs 1A–G (original description). Four paratypes are deposited in NMPC: PARATYPES (2 macr.): ‘Para- / type [p, white circle with yellow margin] // At light [p] // A.E.SUDAN: / Wau. x.1952. / E.T.M.Reid. / B.M.1952-535 [p] // Micronecta / christiniana / Lansbury / PARATYPE. [p]’. PARATYPES (2 macr.): ‘Para- / type [p, white circle with yellow margin] // At light [p] // A.E.SUDAN: / Wau. x.1952. / E.T.M.Reid. / B.M.1952-535 [p] // Micronecta / christiniana / Lansbury / PARATYPE. [p]’. PARATYPES (2 macr.): ‘Para- / type [p, white circle with yellow margin] // At light [p] // A.E.SUDAN: / Wau. x.1952. / E.T.M.Reid. / B.M.1952-535 [p] // Micronecta / christiniana / Lansbury / PARATYPE. [p]’. PARATYPES (2 macr.): ‘Para- / type [p, white circle with yellow margin] // At light [p] // A.E.SUDAN: / Wau. x.1952. / E.T.M.Reid. / B.M.1952-535 [p] // Micronecta / christiniana / Lansbury / PARATYPE. [p]’. PARATYPES (2 macr.): ‘Para- / type [p, white circle with yellow margin] // At light [p] // A.E.SUDAN: / Wau. x.1952. / E.T.M.Reid. / B.M.1952-535 [p] // Micronecta / christiniana / Lansbury / PARATYPE. [p]’. x.1952. / E.T.M.Reid. / B.M.1952-535 [p] // Micronecta / christiniana / Lansbury / PARATYPE. [p]’. PARATYPES (2 macr.): ‘Para- / type [p, white circle with yellow margin] // At light [p] // A.E.SUDAN: / Wau. x.1952. / E.T.M.Reid. / B.M.1952-535 [p] // Micronecta / christiniana / Lansbury / PARATYPE. [p]’. PARATYPES (2 macr.): ‘Para- / type [p, white circle with yellow margin] // At light [p] // A.E.SUDAN: / Wau. x.1952. / E.T.M.Reid. / B.M.1952-535 [p] // Micronecta / christiniana / Lansbury / PARATYPE. [p]’. Current status. Valid species, not assigned to any of the subgenera (see LINNAVUORI 1971, 1981). Micronecta crinita Chen, Nieser & Wattanachaiyingcharoen, 2002 Micronecta drepani Nieser, 2000 icronecta drepani Nieser, 2000: 278–281, 287, Figs 1–10 (original description, key). Two paratypes are deposited in NMPC (exchange with Natural History Museum, Vienna): PARATYPE ( macr.): ‘Thailand:Mae Hong Son Prov. / Phe Bong, 12km S Mae Hong / Son, 12.11.1995 / leg. H. Zettel (13a) [p] // [p] // P a r a t y p u s / Micronecta / drepani sp.n. / des. Nico Nieser [p, red label]’. PARATYPE ( macr.): ‘Thailand:Phetchabun Prov. / Nam Nao NP,Huai Phrom / Laeng, 24.11.1995 / leg. H. Zettel (22) [p] // [p] // P a r a t y p u s / Micronecta / drepani sp.n. / des. Nico Nieser [p, red label]’. PARATYPE ( macr.): ‘Thailand:Mae Hong Son Prov. / Phe Bong, 12km S Mae Hong / Son, 12.11.1995 / leg. H. Zettel (13a) [p] // [p] // P a r a t y p u s / Micronecta / drepani sp.n. / des. Nico Nieser [p, red label]’. PARATYPE ( macr.): ‘Thailand:Phetchabun Prov. / Nam Nao NP,Huai Phrom / Laeng, 24.11.1995 / leg. H. Zettel (22) [p] // [p] // P a r a t y p u s / Micronecta / drepani sp.n. / des. Nico Nieser [p, red label]’. Current status. Valid species: Micronecta (Micronecta) drepani Nieser, 2000 (see NIESER et al. 2005, AUKEMA et al. 2013). 829 Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 Micronecta macrothoracica Jordan, 1943 Micronecta macrothoracica Jordan, 1943: 237–240, Figs 1, 3–4, 8–9 (original description). Micronecta macrothoracica: JANSSON (1986a): 18 (lectotype designation). Four paratypes are deposited in NMPC: PARALECTOTYPE ( macr.): ‘ [p] // Paratypus [p, red label] // Dr. Jordan [p, vertical line] / 14. 6. 43 / Cune - / walde [hw] / Oberlausitz [p] // Micronecta / macrothoracica / n. sp. [hw] / det.K.H.C.Jordan, Bautzen [p, black frame submarginally] // [p] // Mus. Nat. Pragae / Inv. [p] 1207 [hw, orange label] // PARALECTOTYPUS / MICRO- NECTA / MACROTHORACICA / Jordan, 1943 / labelled: P. KMENT 2013 [p, red label]’. PARALECTOTYPES (3 macr.): ‘ [p] // Paratypus [p, red label] // Dr. Jordan [p, vertical line] / 14. 6. 43 / Cune - / walde [hw] / Oberlausitz [p] // Mus. Nat. Pragae / Inv. [p] 1205, 1206, 1208 [respectively; hw, orange label] // PARALEC- TOTYPUS / MICRONECTA / MACROTHORACICA / Jordan, 1943 / labelled: P. KMENT 2013 [p, red label]’. Current status. Junior synonym of Micronecta (Micronecta) poweri poweri (Douglas & Scott, 1869) (see WRÓBLEWSKI 1958; JANSSON 1986a, 1988, 1995). Micronecta pacheta Nieser & Chen, 2003 Micronecta pacheta Nieser & Chen, 2003: 133, 140–142, Figs 2, 38–48 (original description). Two paratypes are deposited in NMPC (exchange with Natural History Museum, Vienna): PARATYPE ( macr.): ‘Philippinen: N. Samar / San Joaquin, Lologayan / Felis, 27.1.2000 / leg. H. Zettel (219a) [p] // [p] // Paratype / Micronecta / pacheta / Nieser & Chen [p, red label]’. PARATYPE ( macr.): ‘Philippinen: N. Samar / San Joaquin, Lologayan / Felis, 27.1.2000 / leg. H. Zettel (219a) [p] // [p] // Paratype / Micronecta / pacheta / Nieser & Chen [p, red label]’. Current status. Valid species, not assigned to any subgenus (see CHEN et al. 2005). Micronecta (Micronecta) wui alkani Hoberlandt, 1952 Micronecta (Micronecta) wui alkani Hoberlandt, 1952: 7–10, Pl. I: Figs 1–12, Pl. IX: Figs 65–68, Pl. X: Fig. 74 (original description, key to subspecies). The holotype and 21 paratypes (including the allotype) are deposited in NMPC; however, only the 13 paratypes from Alacakilise and Armutlu represent the genuine M. w. alkani (see WRÓBLEWSKI 1962a,b): The holotype and 21 paratypes (including the allotype) are deposited in NMPC; however, only the 13 paratypes from Alacakilise and Armutlu represent the genuine M. w. alkani (see WRÓBLEWSKI 1962a,b): HOLOTYPE ( brach.): ‘Alacakilise [hw] / Anat. [p] 21.VIII.47 [hw] / Exp. N. Mus. ČSR [p] // Holotypus [p, red label] // Holotypus [p] / Micronecta (M.) / wui alkani / ssp. n. [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // Micronecta (M.) / wui alkani [hw] / 19[p]48 / ssp. n. [hw] / L. Hoberlandt det. [p] // [p] // Mus. Nat. Pragae / Inv. [p] 1190 [hw, orange label]’. (The specimen is glued to a piece of card, head missing). PARATYPE ( brach.): ‘Alacakilise [hw] / Anat. [p] 21.VIII.47 [hw] / Exp. N. Mus. ČSR [p] // Allotypus [p, red label] // Allotypus [p] / Micronecta / (M.) wui alkani / ssp. n. [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // Micronecta (M.) [hw] / 19[p]48 wui alkani [hw] / ssp. n. [hw] / L. Hoberlandt det. [p] [hw] // [p] // Mus. Nat. Pragae / Inv. [p] 1197 [hw, orange label]’. PARATYPES (2 brach.): ‘Alacakilise [hw] / Anat. [p] 21.VIII.47 [hw] / Exp. N. Mus. ČSR [p] // Paratypus [p, red label] // Paratypus [p] / Micronecta / wui alkani / ssp. n. [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // Micronecta (M.) [hw] / 19[p]48 wui alkani / ssp. n. [hw] / L. Hoberlandt det. [p] // Mus. Nat. Pragae / Inv. [p] 1181, 1182 [respectively; hw, orange label]’. PARATYPES (2 brach.): ‘Alacakilise [hw] / Anat. [p] 21.VIII.47 [hw] / Exp. N. Mus. ČSR [p] // Paratypus [p, red label] // Paratypus [p] / Micronecta / wui alkani / ssp. n. [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // Micronecta (M.) [hw] / 19[p]48 wui alkani / ssp. n. [hw] / L. Hoberlandt det. [p] // Mus. Nat. Pragae / Inv. [p] 1181, 1182 [respectively; hw, orange label]’. Micronecta (Micronecta) wui alkani Hoberlandt, 1952 [ p y g ] PARATYPES (9 brach.): ‘Alacakilise [hw] / Anat. [p] 21.VIII.47 [hw] / Exp. N. Mus. ČSR [p] // Paratypus [p, red label] // Paratypus [p] / Micronecta (M.) / wui alkani / ssp. n. [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 1192, 1193, 1194, 1195, 1196, 1198, 1199, 1200, 1201 [respectively; hw, orange label]’. 830 KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. PARATYPE ( brach.): ‘Armutlu [hw] / Anat. [p] 7.VII.44 [hw] / C. Kosswig lgt. [p] // Paratypus / Micronecta / wui alkani / ssp. n. [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // Paratypus [p, red label] // Mus. Nat. Pragae / Inv. [p] 1191 [hw, orange label] // M. wui alkani / Hoberl. / A. Wróblewski det / prep / nm p Č..4. 1961 [hw]’. (Specimen slide-mounted, stored in Slide Box 1). Eight paratypes from Suluhan were misidentiﬁ ed and in fact belong to Micronecta (Micro- necta) anatolica anatolica Lindberg, 1922 (see WRÓBLEWSKI 1962a,b): Eight paratypes from Suluhan were misidentiﬁ ed and in fact belong to Micronecta (Micro- necta) anatolica anatolica Lindberg, 1922 (see WRÓBLEWSKI 1962a,b): PARATYPES (2 brach.): ‘Suluhan, Toros / Anat. 11. VIII. 47 / Exp. N.Mus. ČSR. [p] // Paratypus [p, red label] // Paratypus / Micronecta / wui alkani / ssp. n. [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // Micro- necta (M.) [hw] / 19[p]48 wui alkani [hw] / ssp. n. [hw] / L. Hoberlandt det. [p] // Mus. Nat. Pragae / Inv. [p] 1178, 1179 [respectively; hw, orange label] // MICRONECTA / ANATOLICA / Lindberg, 1922, det. A. WRÓBLEWSKI [p]’. PARATYPE ( brach.): ‘Suluhan, Toros / Anat. 11. VIII. 47 / Exp. N.Mus. ČSR. [p] // Paratypus / Micronecta / wui alkani / ssp. n. [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // Paratypus [p, red label] // Mus. Nat. Pragae / Inv. [p] 1180 [hw, orange label] // Micronecta (M.) [hw] / 19[p]48 wui alkani [hw] / ssp. n. [hw] / L. Hoberlandt det. [p] // M. anatolica Lindb. / A. Wróblewski det / prep / nmp Č. 2. 1961 [hw]’. (Specimen slide-mounted, stored in Slide Box 1). PARATYPE ( brach.): ‘Suluhan, Toros / Anat. 11. VIII. 47 / Exp. N.Mus. ČSR. Micronecta (Micronecta) wui alkani Hoberlandt, 1952 [p] // Paratypus [p, red label] // Paratypus [p] / Micronecta / wui alkani / ssp. n. [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 1185 [hw, orange label] MICRONECTA / ANATOLICA / Lindberg, 1922, det. A. WRÓBLEWSKI [p]’. PARATYPES (2 brach.): ‘Suluhan, Toros / Anat. 11. VIII. 47 / Exp. N.Mus. ČSR. [p] // Paratypus [p, red label] // Paratypus [p] / Micronecta / wui alkani / ssp. n. [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // Micronecta (M.) [hw] / 19[p]48 wui alkani / ssp. n. [hw] / L. Hoberlandt det. [p] // Mus. Nat. Pragae / Inv. [p] 1186, 1189 [re- spectively; hw, orange label] MICRONECTA / ANATOLICA / Lindberg, 1922, det. A. WRÓBLEWSKI [p]’. PARATYPE ( brach.): ‘Suluhan, Toros / Anat. 11. VIII. 47 / Exp. N.Mus. ČSR. [p] // Paratypus / Micronecta / wui alkani / ssp. n. [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // Paratypus [p, red label] // Mus. Nat. Pragae / Inv. [p] 1187 [hw, orange label] // Micronecta (M.) [hw] / 19[p]48 wui alkani [hw] / ssp. n. [hw] / L. Hoberlandt det. [p] // M. anatolica Lindb. / A. Wróblewski det / prep / nmp Č. 3. 1961 [hw]’. (Specimen slide-mounted, stored in Slide Box 1). PARATYPE ( brach.): ‘Suluhan, Toros / Anat. 11. VIII. 47 / Exp. N.Mus. ČSR. [p] // Paratypus / Micronecta / wui alkani / ssp. n. [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // Paratypus [p, red label] // Mus. Nat. Pragae / Inv. [p] 1188 [hw, orange label] // Micronecta (M.) [hw] / 19[p]48 wui alkani [hw] / ssp. n. [hw] / L. Hoberlandt det. [p] // M. anatolica Lindb. / A. Wróblewski det / prep / nmp Č. 5. 1961 [hw]’. (Specimen slide-mounted, stored in Slide Box 1). Current status. Valid subspecies: Micronecta (Micronecta) wui alkani Hoberlandt, 1952 (see WRÓBLEWSKI 1962a, JANSSON 1995, FENT et al. 2011). Subfamily Cryphocricinae Montandon, 1897 Subfamily Cryphocricinae Montandon, 1897 Micronecta (Micronecta) wui kosswigi Hoberlandt, 1952 Micronecta (Micronecta) wui kosswigi Hoberlandt, 1952a: 9–10, Pl. I: Figs 13–14, Pl. II: Figs 15–19, Pl. IX: Fig. 69 (original description, key to subspecies). The holotype and 15 paratypes (including the allotype) are deposited in NMPC: HOLOTYPE ( brach.): ‘Afrin near / Musabeyli [hw] / Anat. [p] 20.VIII.47 [hw] / Exp. N. Mus. ČSR. [p] // Holotypus [p, red label] // Holotypus [p] / Micronecta / wui kosswigi / ssp. n. [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // [p] // Mus. Nat. Pragae / Inv. [p] 1163 [hw, orange label]’. PARATYPES (3 brach.): ‘Afrin near / Musabeyli [hw] / Anat. [p] 20.VIII.47 [hw] / Exp. N. Mus. ČSR. [p] // Pa- ratypus [p, red label] // Paratypus [p] / Micronecta / wui kosswigi / ssp. n. [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 1160, 1161, 1162 [respectively; hw, orange label]’. PARATYPE ( brach.): ‘Afrin near / Musabeyli [hw] / Anat. [p] 20.VIII.47 [hw] / Exp. N. Mus. ČSR. [p] // Allotypus [p, red label] // Allotypus [p] / Micronecta / wui kosswigi / ssp. n. [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // [p] // Mus. Nat. Pragae / Inv. [p] 1166 [hw, orange label]’. 831 Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 PARATYPES (8 brach.): ‘Afrin near / Musabeyli [hw] / Anat. [p] 20.VIII.47 [hw] / Exp. N. Mus. ČSR. [p] // Paratypus [p, red label] // Paratypus [p] / Micronecta (M.) / wui kosswigi / ssp. n. [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 1167, 1168, 1169, 1170, 1171, 1173, 1174, 1176 [respec- tively; hw, orange label]’. PARATYPES (2 brach.): ‘Abacĭlar [hw] / Anat. [p] 7.VIII.47 [hw] / Exp. N. Mus. ČSR. [p] // Paratypus [p, red label] // Paratypus [p] / Micronecta (M.) / wui kosswigi / ssp. n. [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 1172, 1175 [respectively; hw, orange label]. Current status. Junior synonym of Micronecta (Micronecta) wui alkani Hoberlandt, 1952 (see WRÓBLEWSKI 1962a, JANSSON 1995, FENT et al. 2011). aratypes are deposited in NMPC (exchange with Natural History Museum, Vienna) Two paratypes are deposited in NMPC (exchange with Natural History Museum, Vienna): PARATYPE (): ‘PHILIPPINEN: Mindanao / Agusan N., stream at / Kicharao, 27.III.1993 / leg. N. Nieser N9326 [p] // Paratype / Austhenocoris [sic!] / australis / Zettel, Nieser & / D. Polhemus [p, red label] // [p]’. PARATYPE (): ‘PHILIPPINEN: Mindanao / Agusan N., stream at / Kicharao, 27.III.1993 / leg. N. Nieser N9326 [p] // Paratypus / Asthenocoris / australis sp.n. Zettel, / Nieser & D. Polhemus / des H. Zettel 1998/99 [p, red label] // [p]’. Current status. Valid species (see CHEN et al. 2005). Family NAUCORIDAE Leach, 1815 Subfamily Cheirochelinae Montandon, 1897 Tribe Sagocorini La Rivers, 1971 Tribe Sagocorini La Rivers, 1971 Asthenocoris australis Zettel, Nieser & D. A. Polhemus, 1999 Cryphocricos granulosus De Carlo, 1967 Cryphocricos granulosus De Carlo, 1967 Cryphocricos granulosus De Carlo, 1967: 190−191, Lámina 1: Figs 4−5, 8, Lámina 2: Fig. 18 (original descrip- tion). Two paratypes are deposited in NMPC: PARATYPES (2 ): ‘Brasilien Cipo / Rio Grande do Sul / 10.9.-8.12.1960 / C. Ribeiro leg. [p] // [p] // Cryphocricos / granulosus / de Carlo / PARATYPUS [hw]’. Current status. Valid species (see HECKMAN 2011, MOREIRA et al. 2011). Subfamily Laccocorinae Stål, 1876 Laccocoris spurcus congoensis Poisson, 1949 Laccocoris spurcus congoensis Poisson, 1949a: 67, Figs 69, 70B, 71A–B, 72A–B, 73A–F (original description). One syntype is deposited in NMPC: SYNTYPE (): ‘256. [hw, vertical line] / Sake (Kivu) (1560) / 19-22-II-1934 / G. F. de Witte / PARC NAT. ALBERT [p] // R. POISSON det., 1945 [p] / Laccocoris spurcus / f. congoensis nov. [hw] // Ma [hw, pink pencil] // SYNTYPUS / LACCOCORIS / SPURCUS CONGOENSIS / Poisson, 1949 / labelled: P. KMENT 2013 [p, red label]’. Current status. Valid subspecies (see LINNAVUORI 1987). Cryphocricos granulosus De Carlo, 1967: 190−191, Lámina 1: Figs 4−5, 8, Lámina 2: Fig. 18 (original descrip- tion). Cryphocricos granulosus De Carlo, 1967: 190−191, Lámina 1: Figs 4−5, 8, Lámina 2: Fig. 18 (original descrip- tion). PARATYPES (2 ): ‘Brasilien Cipo / Rio Grande do Sul / 10.9.-8.12.1960 / C. Ribeiro leg. [p] // [p] // Cryphocricos / granulosus / de Carlo / PARATYPUS [hw]’. Current status. Valid species (see HECKMAN 2011, MOREIRA et al. 2011). Asthenocoris australis Zettel, Nieser & D. A. Polhemus, 1999 Asthenocoris australis Zettel, Nieser & D. A. Polhemus, 1999: 45, 81–83, 95, Figs 4, 74, 86–89, 100–102, 136–148, 151 (original description, key). Subfamily Naucorinae Leach, 1815 urrent status. Valid species (see SITES & MBOGHO 2012). Current status. Valid species. Current status. Valid species. Laccocoris spurcus congoensis Poisson, 1949 y yp p SYNTYPE (): ‘256. [hw, vertical line] / Sake (Kivu) (1560) / 19-22-II-1934 / G. F. de Witte / PARC NAT. ALBERT [p] // R. POISSON det., 1945 [p] / Laccocoris spurcus / f. congoensis nov. [hw] // Ma [hw, pink pencil] // SYNTYPUS / LACCOCORIS / SPURCUS CONGOENSIS / Poisson, 1949 / labelled: P. KMENT 2013 [p, red label]’. Current status. Valid subspecies (see LINNAVUORI 1987). KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. 832 Family APHELOCHEIRIDAE Fieber, 1851 Aphelocheirus (Aphelocheirus) goellnerae Zettel, 2012 Aphelocheirus (Aphelocheirus) gusenleitneri Zettel, 2009 Aphelocheirus (s.str.) gusenleitneri Zettel, 2009: 1072–1074, 1076–1077, Figs 1–13 (original description). One paratype is deposited in NMPC (exchange with Natural History Museum, Vienna): PARATYPE ( micr.): ‘MYANMAR: Sagaing Div. / Alaungdaw Katthapa NP / Wakya Stream, 400 m / 22° 19.173′ N 94°29.654′ E / 8.5.2003, lg. Boukal & al. (115) [p] // PARATYPUS / Aphelocheirus (s.str.) / gusenleitneri sp.n. / des. H. Zettel 2009 [p, red label] // [p].’ One paratype is deposited in NMPC (exchange with Natural History Museum, Vienna): PARATYPE ( micr.): ‘MYANMAR: Sagaing Div. / Alaungdaw Katthapa NP / Wakya Stream, 400 m / 22° 19.173′ N One paratype is deposited in NMPC (exchange with Natural History Museum, Vienna): PARATYPE ( micr.): ‘MYANMAR: Sagaing Div. / Alaungdaw Katthapa NP / Wakya Stream, 400 m / 22° 19.173′ N 94°29.654′ E / 8.5.2003, lg. Boukal & al. (115) [p] // PARATYPUS / Aphelocheirus (s.str.) / gusenleitneri sp.n. / des. H. Zettel 2009 [p, red label] // [p].’ Current status. Valid species. Aphelocheirus pygmaeus La Rivers, 1971 Aphelocheirus pygmaeus La Rivers, 1971: 69−70, Fig. 1 (original description). One paratype is deposited in NMPC: PARATYPE ( macr.): ‘ASSAM / Kohara, / Kaziranga / 110m X-7-61 // Collectors: / E. S. Ross / D. Q. Cavagnaro [p] // [p] // APHELOCHEIRUS / PYGMAEUS N. SP / PARATYPE [p, blue label]’. (Left fore leg glued separately to a triangular piece of card.) Current status. Valid species: Aphelocheirus (Micraphelocheirus) pygmaeus La Rivers, 1971 (see HOBERLANDT & ŠTYS 1979, POLHEMUS & POLHEMUS 1989, ZETTEL & PAPÁČEK 2006). Current status. Valid species. Current status. Valid species. Aphelocheirus (Aphelocheirus) goellnerae Zettel, 2012 Aphelocheirus (s.str.) goellnerae Zettel, 2012a: 111–113, Figs 1–11 (original description). The holotype and four paratypes are deposited in NMPC: The holotype and four paratypes are deposited in NMPC: HOLOTYPE ( micr.): ‘Vohémar / Madagascar [p] // COLLECTIO / NATIONAL MUSEUM / Praha, Czech Repub- lic [p] // HOLOTYPUS / Aphelocheirus (s.str.) / goellnerae sp.n. / des. H. Zettel 2012 [p, red label] // [p].’ (Dissected male genitalia mounted in microslides attached to the same pin.) PARATYPE ( micr.): ‘Vohémar / Madagascar [p] // COLLECTIO / NATIONAL MUSEUM / Praha, Czech Republic [p] // [p] // PARATYPUS / Aphelocheirus (s.str.) / goellnerae sp.n. / des. H. Zettel 2012 [p, red label]’ PARATYPE ( micr.): ‘Madagascar / Inv. č. [p] Vohémar [hw] // COLLECTIO / NATIONAL MUSEUM / Praha, Czech Republic [p] // [p] // PARATYPUS / Aphelocheirus (s.str.) / goellnerae sp.n. / des. H. Zettel 2012 [p, red label]’ PARATYPES (2 micr.): ‘Madagascar / Inv. č. [p] // COLLECTIO / NATIONAL MUSEUM / Praha, Czech Republic [p] // [p] // PARATYPUS / Aphelocheirus (s.str.) / goellnerae sp.n. / des. H. Zettel 2012 [p, red label]’ Aphelocheirus pygmaeus La Rivers, 1971 Aphelocheirus pygmaeus La Rivers, 1971: 69−70, Fig. 1 (original description). Aphelocheirus pygmaeus La Rivers, 1971: 69−70, Fig. 1 (original description). One paratype is deposited in NMPC: PARATYPE ( macr.): ‘ASSAM / Kohara, / Kaziranga / 110m X-7-61 // Collectors: / E. S. Ross / D. Q. Cavagnaro [p] // [p] // APHELOCHEIRUS / PYGMAEUS N. SP / PARATYPE [p, blue label]’. (Left fore leg glued separately to a triangular piece of card.) One paratype is deposited in NMPC: PARATYPE ( macr.): ‘ASSAM / Kohara, / Kaziranga / 110m X-7-61 // Collectors: / E. S. Ross / D. Q. Cavagnaro [p] // [p] // APHELOCHEIRUS / PYGMAEUS N. SP / PARATYPE [p, blue label]’. (Left fore leg glued separately to a triangular piece of card.) Current status. Valid species: Aphelocheirus (Micraphelocheirus) pygmaeus La Rivers, 1971 (see HOBERLANDT & ŠTYS 1979, POLHEMUS & POLHEMUS 1989, ZETTEL & PAPÁČEK 2006). 833 Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 Four paratypes (including the allotype) are deposited in NMPC: PARATYPE ( macr.): ‘VIETNAM, Lao-cai, 300 m / fôret trop. second. / à la lumière [p] // 21. IX. 1963 / T. PÓCS [p] // [p] // Allotypus [p] / Tamopocoris / asiaticus sp.n. / et P. Štys [hw] / Det. L. Hoberlandt, 19[p]76 [hw, red label]’. PARATYPE ( macr.): ‘VIETNAM, Lao-cai, 300 m / fôret trop. second. / à la lumière [p] // 21. IX. 1963 / T. PÓCS [p] // [p] // Paratypus [p] / Tamopocoris / asiaticus sp.n. / et P. Štys [hw] / Det. L. Hoberlandt, 19[p]76 [hw, red label]’. PARATYPES (2 macr.): ‘VIETNAM, Lao-cai, 300 m / fôret trop. second. / à la lumière [p] // 21. IX. 1963 / T. PÓCS [p] // [p] // Paratypus [p] / Tamopocoris / asiaticus sp.n. / et P. Štys [hw] / Det. L. Hoberlandt, 19[p]76 [hw, red label]’. Current status. Valid species: Aphelocheirus (Micraphelocheirus) asiaticus (Hoberlandt & Štys, 1979) (see POLHEMUS & POLHEMUS 1989, ZETTEL & PAPÁČEK 2006). Anisops persica Kaiser, 1940 & Anisops persica Lindberg, 1941 Anisops persica Kaiser, 1940: 139–157 (morphology, biology, ecology). Anisops persica Lindberg, 1941: 17–18, Figs 1–4 (original description). Family NOTONECTIDAE Latreille, 1802 Family NOTONECTIDAE Latreille, 1802 Subfamily Anisopinae Hutchinson, 1929 Subfamily Anisopinae Hutchinson, 1929 Potamocoris parvus Hungerford, 1941 Potamocoris parvus Hungerford, 1941: 1–4, Pl. I: Figs 1–8 (original description). Potamocoris parvus Hungerford, 1941: 1–4, Pl. I: Figs 1–8 (original description). Two paratypes are deposited in NMPC: PARATYPES (2 ): ‘Horqueta / Paraguay / 45 miles E. // Paraguay Riv. [p] / XII. 7 [hw] 1934 / Alberto Schulze [p] // PARATYPE / Potamocaris [sic!] / parvus / H. B. Hungerford [p, blue label] // [p]’. Current status. Valid species (see POLHEMUS & POLHEMUS 1983, VAN DOESBURG 1984, HECK- MAN 2011). p y , Tampocoris asiaticus Hoberlandt & Štys, 1979: 2, 4–9, Figs 3, 10–13 (original description, key). p y Tampocoris asiaticus Hoberlandt & Štys, 1979: 2, 4–9, Figs 3, 10–13 (original description, ke Four paratypes (including the allotype) are deposited in NMPC: Tampocoris asiaticus Hoberlandt & Štys, 1979 p y , Tampocoris asiaticus Hoberlandt & Štys, 1979: 2, 4–9, Figs 3, 10–13 (original description, key). Helotrephes recurvatus Zettel, 2000 p Helotrephes recurvatus Zettel, 2000a: 4–5, 19, 21, Figs 6–8, 15, 18, 22 (original description, key). One paratype is deposited in NMPC (exchange with Natural History Museum, Vienna): PARATYPE (): ‘Malaysia, Sabah, Crocker / Range, Sunsuron, 10.-11.VI. / 1996, 8a, Sunsuron riv. Flowing / th- rough deforested area [p] // [p] // P a r a t y p u s / Helotrephes / recurvatus sp.n. / des. H. Zettel 1998 [p, red label]’. Current status. Valid species (see PAPÁČEK & ZETTEL 2004, CHEN et al. 2005). Two paratypes are deposited in NMPC: Two paratypes are deposited in NMPC: PARATYPE (): ‘[Formosa] / Ankô, near / Taihoku / 25.V.1941 / S. Miyamoto [hw] // Paratypus / Helotrephes / for- mosanus [p] / [hw] / Esaki et Miyamoto, / 1948 [p, blue label]’. PARATYPE (): ‘[Formosa] / Ankô / 25.V.1941 / S. Miyamoto [hw] // Paratypus / Helotrephes / formosanus [p] / [hw] / Esaki et Miyamoto, / 1948 [p, blue label]’. Current status. Valid subspecies: Helotrephes semiglobosus formosanus Esaki & Miyamoto, 1943 (see POLHEMUS 1995c, ZETTEL & POLHEMUS 1998, PAPÁČEK & ZETTEL 2004, AUKEMA et al. 2013). Enithares unguistigris Zettel, 2012 Enithares unguistigris Zettel, 2012b: 24–26, Figs 1–9, 13 (original description). The holotype is deposited in NMPC (donation of P. Šrámek): HOLOTYPE (): ‘N 27° 08´ 22“ E 076° 20´ 38“ / INDIA BOR. OCC., Rajasthan st. / Alwar distr., Naranimata env., / shadowy pool / 29. 7. 2002, lgt. P. Šrámek [p] // COLLECTIO / Petr ŠRÁMEK / (Jilemnice, Czech Rep.) [p] // [p] // H O L O T Y P U S / Enithares / unguistigris sp. n. / des. H. Zettel 2011 [p, red label]’. Current status. Valid species. Family HELOTREPHIDAE Esaki & China, 1927 Subfamily Helotrephinae Esaki & China, 1927 Tribe Helotrephini Esaki & China, 1927 Helotrephes formosanus Esaki & Miyamoto, 1943 Helotrephes formosanus Esaki & Miyamoto, 1943: 485–494, Figs 1–6 (original description). Helotrephes formosanus Esaki & Miyamoto, 1943: 485–494, Figs 1–6 (original description). Helotrephes formosanus Esaki & Miyamoto, 1943: 485–494, Figs 1–6 (original description). Two syntypes are deposited in NMPC: SYNTYPE (): ‘Bushir / Iran 19.III.37 / E. W. Kaiser lgt. [hw] // Paratypus [p, red label] // Anisops / persica Lindb. / Det. H. Lindberg. [hw] // SYNTYPUS / ANISOPS / PERSICUS / Kaiser, 1940 + Lindberg, 1941 / labelled: P. KMENT 2013 [p, red label]’. SYNTYPE (): ‘Bushir / Iran 19.III.37 / E. W. Kaiser lgt [hw] // Paratypus [p, red label] // Anisops / persica Lindb. SYNTYPE (): ‘Bushir / Iran 19.III.37 / E. W. Kaiser lgt [hw] // Paratypus [p, red label] // Anisops / persica Lindb. / Det. H. Lindberg. [hw] // SYNTYPUS / ANISOPS / PERSICUS / Kaiser, 1940 + Lindberg, 1941 / labelled: P. KMENT 2013 [p, red label]’. SYNTYPE (): ‘Bushir / Iran 19.III.37 / E. W. Kaiser lgt [hw] // Paratypus [p, red label] // Anisops / persica Lindb. / Det. H. Lindberg. [hw] // SYNTYPUS / ANISOPS / PERSICUS / Kaiser, 1940 + Lindberg, 1941 / labelled: P. KMENT 2013 [p, red label]’. SYNTYPE (): ‘Bushir / Iran 19.III.37 / E. W. Kaiser lgt [hw] // Paratypus [p, red label] // Anisops / persica Lindb. / Det. H. Lindberg. [hw] // SYNTYPUS / ANISOPS / PERSICUS / Kaiser, 1940 + Lindberg, 1941 / labelled: P. KMENT 2013 [p, red label]’. Current status. Anisops persica Lindberg, 1941 is a junior homonym and a junior objective synonym of Anisops persica Kaiser, 1940. Both names are junior synonyms of Anisops debilis perplexus Poisson, 1929 (see LANSBURY 1964, POLHEMUS 1995b). Neither KAISER (1940) nor LINDBERG (1941) did designate a holotype in the original publication so all the type specimens must be regarded as syntypes. 834 KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. Subfamily Notonectinae Latreille, 1802 Tribe Notonectini Latreille, 1802 Enithares unguistigris Zettel, 2012 Enithares unguistigris Zettel, 2012b: 24–26, Figs 1–9, 13 (original description). The holotype is deposited in NMPC (donation of P. Šrámek): HOLOTYPE (): ‘N 27° 08´ 22“ E 076° 20´ 38“ / INDIA BOR. OCC., Rajasthan st. / Alwar distr., Naranimata env., / shadowy pool / 29. 7. 2002, lgt. P. Šrámek [p] // COLLECTIO / Petr ŠRÁMEK / (Jilemnice, Czech Rep.) [p] // [p] // H O L O T Y P U S / Enithares / unguistigris sp. n. / des. H. Zettel 2011 [p, red label]’. aratypes are deposited in NMPC (exchange with Natural History Museum, Vienna) PARATYPES (1 1 ): ‘PHILIPPINEN: Mindanao / Bukidnon, 4km NE Lantapan / Kastuan, Kutasihan Riv., 850m / 9.11.1996, leg.H.Zettel (93) [p] // P a r a t y p u s / Hydrotrephes / stereos sp.n. / des. Nieser & Chen 1997 [p, red label]’. Current status. Valid species (see ZETTEL 2003, PAPÁČEK & ZETTEL 2004, CHEN et al. 2 Hydrotrephes bicolanus seyferti Zettel, 2003 Hydrotrephes bicolanus seyferti Zettel, 2003: 54–55, 77, 79, 85–86, 95, Figs 79–84, 95, 102, 127 (original descrip- tion, key). Two paratypes are deposited in NMPC (exchange with Natural History Museum, Vienna): PARATYPES (1 1 ): ‘Philippinen: LZ, Albay / Malinao, Palali Falls / 200 m, 14.3.1999 / leg. H. Zettel (201) [p] // P a r a t y p u s / Hydrotrephes / bicolanus / seyferti ssp. n. / des. H. Zettel 2002 [p, red label]. Current status. Valid subspecies (see PAPÁČEK & ZETTEL 2004, CHEN et al. 2005). 835 Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 Tribe Limnotrephini J. T. Polhemus, 1990 Tribe Limnotrephini J. T. Polhemus, 1990 Tribe Limnotrephini J. T. Polhemus, 1990 Family MESOVELIIDAE Douglas & Scott, 1867 Subfamily Mesoveliinae Douglas & Scott, 1867 Family MESOVELIIDAE Douglas & Scott, 1867 Subfamily Mesoveliinae Douglas & Scott, 1867 Mixotrephes (Pictotrephes) pictus Papáček & Zettel, 2011 Mixotrephes (Pictotrephes) pictus Papáček & Zettel, 2011 Mixotrephes (Pictotrephes) pictus Papáček & Zettel, 2011: 399, 402–404, Figs 1–6, 9, 11–16 (original descripti- on). The holotype and three paratypes are deposited in NMPC: HOLOTYPE ( brach.): ‘LAOS, CHAMPASAK prov. / Bolavens Plateau / ca. 1 km S Ban Lak 40 [vill.] / TAD YUEANG waterfall, 900-970 m / 15°10.8’N, 106°08.3’E / Jiří Hájek leg. 28.iv.2010’ [p] // HOLOTYPUS / Mixotrephes / (Pictotrephes subgen.n.) / pictus sp.n. des. M. / Papáček & H. Zettel 2011 [p, red label]’. (Genital capsula, aedeagus and both parameres dissected and glued to the same piece of card as the specimen). PARATYPES (3 brach.): ‘LAOS, CHAMPASAK prov. / Bolavens Plateau / ca. 1 km S Ban Lak 40 [vill.] / TAD YUEANG waterfall, 900-970 m / 15°10.8’N, 106°08.3’E / Jiří Hájek leg. 28.iv.2010’ [p] // PARATYPUS / Mixotrephes / (Pictotrephes subgen.n.) / pictus sp.n. des. M. / Papáček & H. Zettel 2011 [p, red label]’. (In two females terminalia and subgenital plate dissected and glued to the same piece of card as the specimens). Current status. Valid species. Family MESOVELIIDAE Douglas & Scott, 1867 Subfamily Mesoveliinae Douglas & Scott, 1867 Mesovelia dentiventris Linnavuori, 1971 Mesovelia dentiventris Linnavuori, 1971: 363–364, Figs 20m–p (original description). One paratype is deposited in NMPC: PARATYPE ( apter.): ‘Sudan. Equatoria / 30 km N of Terakeka / 4–5. III. 63 / Linnavuori [p] // [p] // Paratypus [p, red label] // Mesovelia / dentiventris / Lv. [hw, blue ink]’. Current status. Valid species: Mesovelia dentiventris dentiventris Linnavuori, 1971 (see LINNAVUORI 1975, DAMGAARD et al. 2012). Current status. Valid species. Current status. Valid species. Hydrotrephes visayesensis Zettel, 2003 Hydrotrephes visayesensis Zettel, 2003: 53, 55, 69–72, 94, Figs 38–40, 49, 54, 59, 126 (original description, keys to males and females). One paratype is deposited in NMPC (exchange with Natural History Museum, Vi PARATYPE (): ‘PHILIPPINEN: Panay, Ilo-Ilo / 10 km NE Igbaras, Nadsadan / Falls. 500m, 22.3.1994 / leg. H. Zettel (47) [p] // P a r a t y p u s / Hydrotrephes / visayasensis sp. n. / des. H. Zettel 2002 [p, red label]’. Current status. Valid species (see PAPÁČEK & ZETTEL 2004, CHEN et al. 2005). Hydrotrephes stereos Nieser & Chen, 1999 Hydrotrephes stereos Nieser & Chen, 1999: 98–99, 101, 104–107, 119, 123, Figs 84, 89, 104, 106, 110–112 (original description). Two paratypes are deposited in NMPC (exchange with Natural History Museum, Vienna): Mesovelia dentiventris Linnavuori, 1971 Mesovelia dentiventris Linnavuori, 1971: 363–364, Figs 20m–p (original description). Mesovelia dentiventris Linnavuori, 1971: 363–364, Figs 20m–p (original description). Mesovelia dentiventris Linnavuori, 1971: 363–364, Figs 20m–p (original description). One paratype is deposited in NMPC: PARATYPE ( apter.): ‘Sudan. Equatoria / 30 km N of Terakeka / 4–5. III. 63 / Linnavuori [p] // [p] // Paratypus [p, red label] // Mesovelia / dentiventris / Lv. [hw, blue ink]’. One paratype is deposited in NMPC: PARATYPE ( apter.): ‘Sudan. Equatoria / 30 km N of Terakeka / 4–5. III. 63 / Linnavuori [p] // [p] // Paratypus [p, red label] // Mesovelia / dentiventris / Lv. [hw, blue ink]’. Current status. Valid species: Mesovelia dentiventris dentiventris Linnavuori, 1971 (see LINNAVUORI 1975, DAMGAARD et al. 2012). 836 KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. Parts of the holotype and two paratypes are deposited in NMPC: Parts of the holotype and two paratypes are deposited in NMPC: HOLOTYPE ( macr.): Two slides (Slide Box 2), one including antenna, fore, middle and hind leg, and hemelytron, and the other one including male genitalia, labelled: ‘N. Mus. Praha / č. k. [p] / Heteroptera / Zpr. M.Z. / (1430-17) 1950 [hw] / Kassai 1. V. 1949 / lgt. A. Machado [hw, white label with black frame submarginally] // Holotypus [p] / Hebrus (s. str.) / angolensis / sp.n. / f. macr. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. (Body of the holotype returned to the Dundo Museum, Angola). HOLOTYPE ( macr.): Two slides (Slide Box 2), one including antenna, fore, middle and hind leg, and hemelytron, and the other one including male genitalia, labelled: ‘N. Mus. Praha / č. k. [p] / Heteroptera / Zpr. M.Z. / (1430-17) 1950 [hw] / Kassai 1. V. 1949 / lgt. A. Machado [hw, white label with black frame submarginally] // Holotypus [p] / Hebrus (s. str.) / angolensis / sp.n. / f. macr. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. (Body of the holotype returned to the Dundo Museum, Angola). PARATYPE ( macr.): ‘Dundo, Lunda / Angola [p] VI. 49 [hw] / A. B. Machado lgt. [p] // Ang. [p] No 1525. 12 [hw] / coll.A.B.Machado [p] // Paratypus [p, red label] // Paratypus [p] / Hebrus (s. str.) / angolensis / sp. n. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // \|\|\| [p]’. PARATYPE ( macr.): Dundo, Lunda / Angola [p] VI. 49 [hw] / A. B. Machado lgt. [p] // Ang. [p] No 1525. 12 [hw] / coll.A.B.Machado [p] // Paratypus [p, red label] // Paratypus [p] / Hebrus (s. str.) / angolensis / sp. n. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // \|\|\| [p]’. Current status. Valid species. Hebrus (Hebrusella) machadoi Hoberlandt, 1951 Hebrus (Hebrusella) machadoi Hoberlandt, 1951a: 40–44, Figs 129–136 (original description, key) PARATYPE ( macr.): ‘VI-VII.48 Tshikapa [hw] / Dundo, Angola / A. B. Machado lg. [p] // Ang. [p] No 880. 4 / coll. A.B.Machado [p] // Paratypus [p, red label] // Paratypus [p] / Hebrus / (Hebrusella) / machadoi / sp. n. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // \|\|\| [p]’. Note. Collection also includes two slides with legs, antennae and hemelytron of additional paratype specimens (Slide Box 2). Current status. Valid species: Hebrus (Hebrusella) machadoi machadoi (see POISSON 1955). Hebrus (Hebrus) mizae Hoberlandt, 1951 Hebrus mizae Hoberlandt, 1951a: 47–48, Figs 157–164 (original description). Hebrus mizae Hoberlandt, 1951a: 47–48, Figs 157–164 (original description). Four paratypes are deposited in NMPC: Hebrus (Hebrus) angolensis Hoberlandt, 1951 ebrus (Hebrus) angolensis Hoberlandt, 1951a: 45–46, Figs 150–156 (original description). Four paratypes are deposited in NMPC: PARATYPE ( macr.): ‘VI-VII. 48 Tshikapa [hw] / Dundo, Angola / A B Machado lgt. [p] // Ang. [p] No 880. 4 [hw] / coll.A.B.Machado [p] // Paratypus [p, red label] // Paratypus [p] / Hebrus (s. str.) / mizae. / sp. n. f. macr. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] / \|\|\| [p]’. PARATYPE ( macr.): ‘VI-VII. 48 Tshikapa [hw] / Dundo, Angola / A B Machado lgt. [p] // Ang. [p] No 880. 4 [hw] / coll.A.B.Machado [p] // Paratypus [p, red label] // Paratypus [p] / Hebrus (s. str.) / mizae. / sp. n. f. macr. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. PARATYPE ( macr.): ‘VI-VII. 48 Tshikapa [hw] / Dundo, Angola / A B Machado lgt. [p] // Ang. [p] No 880. 4 [hw] / coll.A.B.Machado [p] // Paratypus [p, red label] // Paratypus [p] / Hebrus (s. str.) / mizae. / sp. n. f. macr. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] / \|\|\| [p]’. PARATYPE ( macr.): ‘VI-VII. 48 Tshikapa [hw] / Dundo, Angola / A B Machado lgt. [p] // Ang. [p] No 880. 4 [hw] / coll.A.B.Machado [p] // Paratypus [p, red label] // Paratypus [p] / Hebrus (s. str.) / mizae. / sp. n. f. macr. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. 837 Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 Note. Collection also includes two slides with legs, antennae and hemelytron of additional paratype specimens (Slide Box 2). Note. Collection also includes two slides with legs, antennae and hemelytron of additional paratype specimens (Slide Box 2). Current status. Valid species. Current status. Valid species. Hebrus pusillus canariensis Poisson, 1954 Hebrus pusillus canariensis Poisson, 1954: 1–2, Figs 1A–E (original description). Hebrus pusillus canariensis Poisson, 1954: 1–2, Figs 1A–E (original description). A syntype is deposited in NMPC: A syntype is deposited in NMPC: A syntype is deposited in NMPC: SYNTYPE ( macr.): ‘Gran Canaria / Aldea S. Nicolas / 1. 3. 49 Lindberg [p] // [p] // Paratypus [p] / Hebr. pusillus / ssp. canarien- / sis / Poiss. [hw, red label] // SYNTYPUS / HEBRUS / PUSILLUS CANARIENSIS / Poisson, 1954 / labelled: P. KMENT 2013 [p, red label]’. SYNTYPE ( macr.): ‘Gran Canaria / Aldea S. Nicolas / 1. 3. 49 Lindberg [p] // [p] // Paratypus [p] / Hebr. pusillus / ssp. canarien- / sis / Poiss. [hw, red label] // SYNTYPUS / HEBRUS / PUSILLUS CANARIENSIS / Poisson, 1954 / labelled: P. KMENT 2013 [p, red label]’. Current status. Junior synonym of Hebrus (Hebrus) pusillus pusillus (see BAENA 1996, AUKEMA et al. 2013). Remarks. POISSON (1954) described the species based on 2 and 4 without designating a holotype, so all the specimens must be regarded as syntypes (see ANDERSEN 1995). Current status. Junior synonym of Hebrus (Hebrus) pusillus pusillus (see BAENA 1996, AUKEMA et al. 2013). ) Remarks. POISSON (1954) described the species based on 2 and 4 without designating a holotype, so all the specimens must be regarded as syntypes (see ANDERSEN 1995). Hebrus sondani campestris Linnavuori, 1971 Hebrus sondani Ps. ssp. campestris Linnavuori, 1971: 365, Figs 21j–m (original description). Two paratypes are deposited in NMPC: Two paratypes are deposited in NMPC: PARATYPES (2 macr.): ‘Sudan / Equatoria. Juba- / Terakeka 2–6 III. 63 / Linnavuori [p] // [p] // Paratypus [p, red label] // Hebrus / sondani ssp. / campestris / Lv. [hw, blue ink]’. (Both specimens on a single piece of card). Current status. Valid species: Hebrus (Hebrus) campestris Linnavuori, 1971 (upgraded to species rank by LINNAVUORI 1981). Hebrus (Hebrusella) wygodzinskyi Hoberlandt, 1951 Hebrus (Hebrusella) wygodzinskyi Hoberlandt, 1951 Hebrus (Hebrusella) wygodzinskyi Hoberlandt, 1951a: 42–44, Figs 137–145 (original description, key). Hebrus (Hebrusella) wygodzinskyi Hoberlandt, 1951a: 42–44, Figs 137–145 (original description, key). A paratype is deposited in NMPC: PARATYPE ( macr.): ‘Dundo, Lunda / Angola [p] 11.VII.48 [hw] / A. B. Machado lgt. [p] // Paratypus [p, red label] // Paratypus [p] / Hebrus / (Hebrusella) / wygodzinskyi / sp. n. f. macr. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // \|\|\| [p]’. Note. Collection also includes two slides with legs, antennae and hemelytron of additional paratype specimens (Slide Box 2). Current status. Valid species (see POISSON 1955). The holotype is deposited in NMPC: The holotype is deposited in NMPC: HOLOTYPE ( macr.): ‘Dundo - ANGOLA [p] / Forest gallery of the river / Coachino, detritus on / soil / 10– [hw]194[p]6[hw] A.B.Machado [p] // [p] // Heterocleptes / hoberlandti / China, Usinger, / Villiers [hw, white label with black frame submarginally] // HOLOTYPE [p] / Heterocleptes / hoberlandti / China, Usinger, Villiers [hw, red label]’. (Specimen card-mounted, both detached fore legs glued to the same card; male genitalia mounted on a microslide attached to a card and placed on the same pin.) HOLOTYPE ( macr.): ‘Dundo - ANGOLA [p] / Forest gallery of the river / Coachino, detritus on / soil / 10– [hw]194[p]6[hw] A.B.Machado [p] // [p] // Heterocleptes / hoberlandti / China, Usinger, / Villiers [hw, white label with black frame submarginally] // HOLOTYPE [p] / Heterocleptes / hoberlandti / China, Usinger, Villiers [hw, red label]’. (Specimen card-mounted, both detached fore legs glued to the same card; male genitalia mounted on a microslide attached to a card and placed on the same pin.) Current status. Valid species (see ANDERSEN 1982). Merragata lindbergi Poisson, 1954 A paratype is deposited in NMPC: A paratype is deposited in NMPC: SYNTYPE ( macr.): ‘Tenerife / Puerto de S. Juan [p] / 18. 1. [hw] 49 Lindberg // [p] // Paratypus [p] / Merragata / Lindbergi / Poiss. [hw, red label] // SYNTYPUS / MERRAGATA / LINDBERGI / Poisson, 1954 / labelled: P. KMENT 2013 [p, red label]’. Current status. Junior synonym of Merragata hebroides White, 1877 (see DRAKE & CHAPMAN 1958, DRAKE & COBBEN 1960, ANDERSEN 1995, MOREIRA et al. 2011). 838 KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. Remarks. POISSON (1954) described the species based on 2 and 4 without designating a holotype, so all the specimens must be regarded as syntypes (see ANDERSEN 1995). Remarks. POISSON (1954) described the species based on 2 and 4 without designating a holotype, so all the specimens must be regarded as syntypes (see ANDERSEN 1995). Heterocleptes hoberlandti China, Usinger & Villiers, 1950 Heterocleptes hoberlandti China, Usinger & Villiers, 1950: 338–340, Figs 1–3 (original description, differential diagnosis). eptes hoberlandti China, Usinger & Villiers, 1950: 338–340, Figs 1–3 (original description, differentia osis). Two paratypes are deposited in NMPC. PARATYPE ( macr.): ‘Peru S A / Recardo Palma / Nov. 27-29 1934 / F. Woytkowski [p] // 1250 m above sea / 44 km E of Lima / (see letter) [p] // [p] // P A R A T Y P E / Bacillometra / woytkowskii / H. B. Hungerford [p, blue label]’. PARATYPE (): ‘Peru S A / Recardo Palma / Nov. 27-29 1934 / F. Woytkowski [p] // 1250 m above sea / 44 km E of Lima / (see letter) [p] // [p] // P A R A T Y P E / Bacillometra / woytkowskii / H. B. Hungerford [p, blue label]’. Current status. Valid species: Bacillometroides woytkowskii (Hungerford, 1935) (see POLHE- MUS & POLHEMUS 2010, HECKMAN 2011). Bacillometra woytkowskii Hungerford, 1935 Bacillometra woytkowskii Hungerford, 1935: 119−123, Figs 1 male, a, b; 2 female, a,b (original description, key). Hydrometra greeni Kirkaldy, 1898 Hydrometra greeni Kirkaldy, 1898: 2 (original description). One syntype is deposited in NMPC: y yp p SYNTYPE ( macr.): ‘Surface of / stagnant Pond [hw] / Punduloya. / Ceylon. / E. K. Green [p] / May 90 [hw] // Geo W. Kirkaldy [p] // Hydrometra / greeni / - Kirk. [hw] // Hydrometra / greeni / Kirk. / SYNTYPE. [hw] // [p]’. Current status. Valid species (see ANDERSEN 1995, POLHEMUS & POLHEMUS 1995a, CHEN et al. 2005, YANG & ZETTEL 2005). Hydrometra marani Hoberlandt, 1942 The holotype and three paratypes are deposited in NMPC: 839 Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 HOLOTYPE ( macr.): ‘Chartum / Sudan / coll. Baum [p] / reverse: V. 1931 [hw] // Holotypus [p, red label with black margins] // Hydrometra / mařani n. sp. [hw, red ink] / L. Hoberlandt det. [p]’. (Holotype mounted to a piece of card; right fore leg, mesotibia, mesotarsus and metatarsus, and left middle and hind leg missing). HOLOTYPE ( macr.): ‘Chartum / Sudan / coll. Baum [p] / reverse: V. 1931 [hw] // Holotypus [p, red label with black margins] // Hydrometra / mařani n. sp. [hw, red ink] / L. Hoberlandt det. [p]’. (Holotype mounted to a piece of card; right fore leg, mesotibia, mesotarsus and metatarsus, and left middle and hind leg missing). PARATYPES (3 macr.): ‘Chartum / Sudan / coll. Baum [p] / reverse: V. 1931 [hw] // Paratypus [p, red label, black margins] // Hydrometra mařani n. sp. [hw, red ink] / L. Hoberlandt det. [p]’. (Paratypes badly damaged; one of them missing head and all apendages, another one missing head and four legs). PARATYPES (3 macr.): ‘Chartum / Sudan / coll. Baum [p] / reverse: V. 1931 [hw] // Paratypus [p, red label, black margins] // Hydrometra mařani n. sp. [hw, red ink] / L. Hoberlandt det. [p]’. (Paratypes badly damaged; one of them missing head and all apendages, another one missing head and four legs). Current status. Valid species (see POISSON 1949b). It was omitted in the monograph on water bugs of Sudan by LINNAVUORI (1971). Current status. Valid species (see POISSON 1949b). It was omitted in the monograph on water bugs of Sudan by LINNAVUORI (1971). Three paratypes are deposited in NMPC: Three paratypes are deposited in NMPC: PARATYPE ( submacr.): ‘Canal Zone / Pan.2-10-39 / C. J. Drake [p] // Paratype [p] / Hydrometra / zeteki / Drake [hw, red label] // [p]’. PARATYPE ( submacr.): Canal Zone / Pan.2-10-39 / C. J. Drake [p] // Paratype [p] / Hydrometra / zeteki / Drake [hw, red label] // [p]’. PARATYPES (2 submacr.): ‘Canal Zone / Pan.2-10-39 / C. J. Drake [p] // Paratype [p] / Hydrometra / zeteki / Drake [hw, red label] // C J Drake / Coll. 1956 [p] // [p]’. Current status. Valid species (see FROESCHNER 1999, HECKMAN 2011). Family VELIIDAE Brullé, 1836 Subfamily Microveliinae China & Usinger, 1949 (1860) Hydrometra zeteki Drake, 1952: 2–3 (original description). Hydrometra zeteki Drake, 1952: 2–3 (original description). Hydrometra zeteki Drake, 1952: 2–3 (original description). Three paratypes are deposited in NMPC: Oravelia pege Drake & Chapm Oravelia pege Drake & Chapman, 1963: 227−233, Figs 1, 3a−c (original description, key). Oravelia pege Drake & Chapman, 1963: 227−233, Figs 1, 3a−c (original description, key). Two paratypes are deposited in NMPC: PARATYPE ( apter.): ‘Tollhouse / Fresno co. / Calif. 16– X.62 [hw] / reverse: Fresno / Calif [hw] // [p] // Oravelia / pege / D & C [hw] / PARATYPE [p, red label]’. PARATYPE ( apter.): Tollhouse / Fresno co. / Calif 16.X.62 // [p] // Oravelia / pege / D. & C [hw] / PARATYPE [p, red label] // PARATYPE [p in blue] / Oravelia / pege / D & C [hw, white label with blue margins]’. Current status. Valid species (see FROESCHNER 1988). Microvelia (Microvelia) angolensis Hoberlandt, 1951 a) angolensis Hoberlandt, 1951a: 24–27, Figs 48–57 (original description). Microvelia (Microvelia) angolensis Hoberlandt, 1951a: 24–27, Figs 48–57 (original description). One paratype is deposited in NMPC: One paratype is deposited in NMPC: PARATYPE ( apter.): ‘Cranberry Lake / N. Y. 7. 19-19 [p] // C. J. Drake / Collector [p] // Bean / Pond [hw] // Paratype [p] / Microvelia / buenoi / Drake [hw, red label] // \|\| [p]’. p yp p PARATYPE ( apter.): ‘Cranberry Lake / N. Y. 7. 19-19 [p] // C. J. Drake / Collector [p] // Bean / Pond [hw] // Paratype [p] / Microvelia / buenoi / Drake [hw, red label] // \|\| [p]’. Current status. Valid species: Microvelia (Microvelia) buenoi Drake, 1920 (see SMITH 1988a, ANDERSEN 1995, KANYUKOVA 2006, AUKEMA et al. 2013). Three paratypes are deposited in NMPC: Three paratypes are deposited in NMPC: PARATYPES (2 apter.): ‘Linha Facao / St. Catarina / Bras. [p] V [hw] 57 / F. Plaumann [p] // C. J. Drake / Coll. 1956 [p] // [p] // PARATYPE / By C. J. Drake [p] / Microvelia / arca [hw, red label]’. PARATYPES (2 apter.): ‘Linha Facao / St. Catarina / Bras. [p] V [hw] 57 / F. Plaumann [p] // C. J. Drake / Coll. 1956 [p] // [p] // PARATYPE / By C. J. Drake [p] / Microvelia / arca [hw, red label]’. PARATYPE (1 apter.): ‘Linha Facao / St. Catarina / Bras. [p] V [hw] 57 / F. Plaumann [p] // C. J. Drake / Coll. 1956 [p] // [p] // PARATYPE / By C. J. Drake [p] / Microvelia / arca [hw, red label]’. PARATYPE (1 apter.): ‘Linha Facao / St. Catarina / Bras. [p] V [hw] 57 / F. Plaumann [p] // C. J. Drake / Coll. 1956 [p] // [p] // PARATYPE / By C. J. Drake [p] / Microvelia / arca [hw, red label]’. Current status. Valid species, not attributed to any subgenus (see HECKMAN 2011, MOREIRA et al. 2011). Current status. Valid species, not attributed to any subgenus (see HECKMAN 2011, MOREIRA et al. 2011). Current status. Valid species. Current status. Valid species. Current status. Valid species. Five paratypes are deposited in NMPC: Five paratypes are deposited in NMPC: PARATYPE ( apter.): ‘Dundo, Lunda / Angola [p] III. 48 [hw] / A. B. Machado lgt. [p] // Ang. [p] No. 608.3 [hw] / coll. A. B. Machado [p] // Paratypus [p, red label] // Paratypus [p] / Microvelia / angolensis / n. sp. f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // \|\|\| [p] // Mus. Nat. Pragae / Inv. [p] 1209 [hw, orange label]’. PARATYPES (2 apter.): ‘Dundo, Lunda / Angola [p] III. 48 [hw] / A. B. Machado lgt. [p] // Ang. [p] No. 608.3 [hw] / coll. A.B. Machado [p] // Paratypus [p, red label] // Paratypus [p] / Microvelia / angolensis / n. sp. f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 1210, 1211 [respectively; hw, orange label]’. KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. 840 PARATYPE ( apter.): ‘N. Mus. Praha / č. k. [p] / Heteroptera / Zpr. M. Z. / (608.3 ) 1950 [hw] / Dundo Lunda III. 1948 / lgt. A. Machado [hw, white label with black frame submarginally] // Paratypus [p] / Microvelia (s. str.) / angolensis / apt. sp.n. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. (The specimen is mounted on a slide, male genitalia and all legs except the right hind leg are removed; male genitalia, and one fore and both middle legs mounted on two separate slides. All the slides deposited in Slide Box 2). PARATYPE ( apter.): ‘Dundo, Lunda / Angola [p] III. 48 [hw] / A B Machado lgt. [p] // Ang. [p] No. 608.3 [hw] / coll. A.B. Machado [p] // Paratypus [p, red label] // Paratypus [p] / Microvelia / angolensis / n. sp. f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // \|\|\| [p] // Mus. Nat. Pragae / Inv. [p] 1212 [hw, orange label]’. Microvelia arca Drake, 1958 Microvelia arca Drake, 1958: 59−60, Fig. 1 (original description). Three paratypes are deposited in NMPC: Microvelia buenoi Drake, 1920 Microvelia buenoi Drake, 1920: 20–21 (original description). One paratype is deposited in NMPC: Microvelia (Pseudovelia) crinita Hoberlandt, 1951 Microvelia (Pseudovelia) crinita Hoberlandt, 1951a: 36–38, Figs 103–113 (original description). One paratype is deposited in NMPC: One paratype is deposited in NMPC: PARATYPE ( apter.): ‘Habana / II-24.43 Cuba / S. C. Bruner [hw] // PARATYPE / By C. J. Drake [p] / Microvelia / cubana [hw, red label] // [p]’. p yp p PARATYPE ( apter.): ‘Habana / II-24.43 Cuba / S. C. Bruner [hw] // PARATYPE / By C. J. Drake [p] / Microvelia / cubana [hw, red label] // [p]’. Current status. Valid species: Microvelia (Microvelia) cubana Drake, 1951 (see SMITH 1988a). Current status. Valid species: Microvelia (Microvelia) cubana Drake, 1951 (see SMITH 1988a). Six paratypes are deposited in NMPC: Six paratypes are deposited in NMPC: PARATYPE ( macr.): ‘Cameroun - 1949-50 [p] / 26[hw]/[p]II[hw] – [p] 85 [hw] / Lok. [p] 174 [hw] / J. B.-S. J. D. [p] // n. sp. [hw] // Paratypus [p] / Microvelia / (Pseudovelia) / dahli n. sp. [hw] / Det. L. Hoberlandt, 19[p]58 [hw, red label]’. PARATYPE ( macr.): ‘Cameroun 1949-50 / 13/12 - 222 / Lok. 174 / J.B.-S. J.D. [hw] // Paratypus [p] / Microvelia / (Pseudovelia) / dahli n. sp. [hw] / Det. L. Hoberlandt, 19[p]58 [hw, red label]’. (Male genitalia dissected and stored in a set of two microslides attached to pieces of cards and placed on the same pin). PARATYPE ( macr.): ‘Cameroun 1949-50 / 26/II. loc. 124/85 / J. B.-S. J. D. [hw] // Paratypus [p] / Microvelia / (Pseudovelia) / dahli n. sp. [hw] / Det. L. Hoberlandt, 19[p]58 [hw, red label]’. (Left antenna, fore and hind leg, hemelytron, and male genitalia dissected and stored in a set of four microslides attached to pieces of cards and placed on the same pin). p p ) PARATYPE ( macr.): ‘Cameroun - 1949-50 / 27/II – 84 / Lok. 174 / J. B. – S. J. D. [hw] // Paratypus [p] / Micro- velia / (Pseudovelia) / dahli n. sp. [hw] / Det. L. Hoberlandt, 19[p]58 [hw, red label]’. PARATYPE ( macr.): ‘Cameroun 1949-50 / 26 /II – 85 / Lok 174 / J. B. – S. J. D. [hw] // Paratypus [p] / Microvelia / (Pseudovelia) / dahli n. sp. [hw] / Det. L. Hoberlandt, 19[p]58 [hw, red label]’. PARATYPE ( macr.): ‘Cameroun - 1949-50 / 20/12 – 264 / Lok. 174 / J. B.–S. J. D. [hw] // Paratypus [p] / Mi- crovelia / (Pseudovelia) / dahli n. sp. [hw] / Det. L. Hoberlandt, 19[p]58 [hw, red label]’. Current status. Valid species: Pseudovelia (Pseudovelia) dahli dahli (Hoberlandt, 1958) (see LINNAVUORI 1977, ANDERSEN 1983). Microvelia (Pseudovelia) crinita Hoberlandt, 1951a: 36–38, Figs 103–113 (original description). Parts of the holotype and one paratype are deposited in NMPC: HOLOTYPE (): Two slides (Slide Box 2), one including antenna, fore, middle and hind leg, and the other one includ- ing male genitalia, labelled: ‘N. Mus. Praha / č. k. [p] / Heteroptera / Zpr. M.Z. / (880.4) 1950 [hw] / Tshikapa Dundo, Lunda / VI.-VII. 48 lgt. A. Machado [hw, white label with black frame submarginally] // Holotypus [p] / Microvelia / (Pseudovelia subgen. n.) / crinita sp.n. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. (Body of the holotype returned to the Dundo Museum, Angola). PARATYPE ( apter.): ‘VI.-VII.48 Tshikapa [hw] / Dundo, Angola / A. B. Machado lgt. [p] // Ang. [p] No 880.4 [hw] / coll A.B.Machado [p] // Paratypus [p, red label] // Paratypus [p] / Microvelia (Pseudovelia subg. n.) / crinita / sp. n. f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // \|\|\| [p]’. Current status. Valid species: Pseudovelia (Pseudovelia) crinita (Hoberlandt, 1951) (see LINNAVUORI 1977, ANDERSEN 1983). Current status. Valid species: Pseudovelia (Pseudovelia) crinita (Hoberlandt, 1951) (see L 1977 A 1983) 841 Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 , Microvelia cubana Drake, 1951a: 41–42 (original description). One paratype is deposited in NMPC: Microvelia (Pseudovelia) dahli Hoberlandt, 1958 Microvelia (Pseudovelia) dahli Hoberlandt, 1958: 1354–1356, Figs 1–5 (original description). Microvelia hozari Hoberlandt, 1952 Microvelia hozari Hoberlandt, 1952: 29–32, Pl. VII: Figs 50–55, Pl. X: Fig. 71 (original description, differential diagnosis). The holotype and 79 paratypes (including the allotype) are deposited in NMPC: HOLOTYPE (): ‘Kilis [hw] / Anat. [p] 20.VIII.47 [hw] / Exp. N. Mus. ČSR [p] // Holotypus [p, red label] // Holotypus [p] / Microvelia / hozari sp. n. / apt. [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // [p] // Mus. Nat. Pragae / Inv. [p] 1077 [hw, orange label]’. PARATYPES (15 apter.): ‘Kilis [hw] / Anat. [p] 20.VIII.47 [hw] / Exp. N. Mus. ČSR [p] // Paratypus [p, red label] // Paratypus [p] / Microvelia / hozari sp. n. / apt. [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1088, 1090, 1091, 1092, 1093, 1094 [respectively; hw, orange label]’. PARATYPE ( apter.): ‘Kilis [hw] / Anat. [p] 20.VIII.47 [hw] / Exp. N. Mus. ČSR [p] // Allotypus [p, red label] // Allotypus [p] / Microvelia / hozari sp. n. / apt. [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // [p] // Mus. Nat. Pragae / Inv. [p] 1086 [hw, orange label]’. 842 KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. PARATYPES (19 apter.): ‘Kilis [hw] / Anat. [p] 20.VIII.47 [hw] / Exp. N. Mus. ČSR [p] // Paratypus [p, red label] // Paratypus [p] / Microvelia / hozari sp. n. / apt. [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 1097, 1098, 1099, 1100, 1101, 1103, 1104, 1105, 1106, 1107, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1118 [respectively; hw, orange label]’. Č PARATYPES (3 apter.): ‘Suluhan, Toros / Anat. 1[p]0.[hw] VIII. 47 / Exp. N. Mus. ČSR [p] // Paratypus [p, red label] // Paratypus [p] / Microvelia / hozari sp. n. / apt. [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 1119, 1120, 1121 [respectively; hw, orange label]’. PARATYPE ( macr.): ‘Afrin near / Musabeyli [hw] / Anat. [p] 20.VIII.47 [hw] / Exp. N. Mus, ČSR [p] // Paratypus [p, red label] // Paratypus [p] / Microvelia / hozari sp. n. / f. macr. [hw] / Det. L. Microvelia hozari Hoberlandt, 1952 Hoberlandt, 19[p]48 [hw, red label] // [p] // Mus. Nat. Pragae / Inv. [p] 1122 [hw, orange label]’. PARATYPES (14 macr.): ‘Kilis [hw] / Anat. [p] 20.VIII.47 [hw] / Exp. N. Mus. ČSR [p] // Paratypus [p, red label] // Paratypus [p] / Microvelia / hozari sp. n. / f. macr. [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1137 [respectively; hw, orange label]’. PARATYPES (21 macr.): ‘Kilis [hw] / Anat. [p] 20.VIII.47 [hw] / Exp. N. Mus. ČSR [p] // Paratypus [p, red label] // Paratypus [p] / Microvelia / hozari sp. n. / f. macr. [hw] / Det. L. Hoberlandt, 19[p]48 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 1138, 1139, 1140, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159 [respectively; hw, orange label]’. PARATYPE ( apter.): ‘Paratypus [hw, red pencil] / Microvelia / hozari sp. n. / f. apt. / Kilis, Anatolia / 20.VIII.47 / Exp. N. Mus. [hw, white label with black frame submarginally] // Caput, antenna / thorax / abdomen / L. Hober- landt [hw, white label with black frame submarginally]’. (Specimen mounted on a slide; Slide Box 2). PARATYPES ( apter., apter.): ‘Paratypus [hw, red pencil] / Microvelia / hozari sp. n. / f. apt. + / Kilis, Anatolia / 20.VIII.47 / Exp. N. Mus. [hw, white label with black frame submarginally] // Corpora / L. Hoberlandt [hw, white label with black frame submarginally]’. (Specimens mounted on a single slide; Slide Box 2). Note. Collection also contains 10 slides with various body parts of paratype specimens (Slide Box 2). ) Current status. Valid species: Microvelia (Picaultia) hozari Hoberlandt, 1952 (see ANDERSEN 1995, ANDERSEN & WEIR 2003, AUKEMA et al. 2013). Microvelia inannana Drake & Hottes, 1952 Microvelia inannana Drake & Hottes, 1952: 67 (original description). ratype is deposited in NMPC: One paratype is deposited in NMPC: PARATYPE ( apter.): ‘Tigre, B. Aires / Arg. Dec. 10, 1938 / Carl J. Drake [p] // Paratype [p] / Microvelia / inannana / D & H [hw, red label] // [p]’. Current status. Valid species, not attributed to any subgenus (see HECKMAN 2011, MOREIRA et al. 2011). Microvelia japonica Esaki & Miyamoto, 1955 Microvelia japonica Esaki & Miyamoto, 1955: 179, 187–190, Figs 7A–M, Pl. 25: Fig. A, Pl. 29: Figs E–F (original description, key). Two paratypes are deposited in NMPC: Two paratypes are deposited in NMPC: PARATYPE ( apter.): ‘(Kyushu) / Kurume / Chikugo / 2. IX. 1952 / S. Miyamoto [hw] // [p] // PARATYPUS / Microvelia / kyushuensis / Esaki et Miyamoto / reverse: [Kyushu] / Kurume / Chikugo / 2. IX. 1952 / S. Miya- moto [hw] // PARATYPUS / MICROVELIA / KYUSHUENSIS / Esaki & Miyamoto, 1955 / labelled: P. KMENT 2013 [p, red label]’. PARATYPE ( apter.): ‘(Kyushu) / Kurume / Chikugo / 2. IX. 1952 / S. Miyamoto [hw] // [p] // PARATYPUS / MICROVELIA / KYUSHUENSIS / Esaki & Miyamoto, 1955 / labelled: P. KMENT 2013 [p, red label]’. Current status. Valid species: Microvelia (Paciﬁ covelia) kyushuensis Esaki & Miyamoto, 1955 (see ANDERSEN 1995, ANDERSEN & WEIR 2003, AUKEMA et al. 2013). Two paratypes are deposited in NMPC: Two paratypes are deposited in NMPC: PARATYPE ( apter.): ‘(Kyushu) / Wakasugi-yama / Chikuzen / 28. VII. 1948 / S. Miyamoto [hw] // [p] // PARA- TYPUS / Microvelia / japonica / Esaki et Miyamoto / reverse: [Kyushu] / Wakasugi-yama / Chikuzen / 28. VII. 1948 / S. Miyamoto [hw] // PARATYPUS / MICROVELIA / JAPONICA / Esaki & Miyamoto, 1955 / labelled: P. KMENT 2013 [p, red label]’. 843 Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 PARATYPE ( apter.): ‘(Kyushu) / Wakasugi-yama / Chikuzen / 28. VII. 1948 / S. Miyamoto [hw] // [p] // PARA- TYPUS / MICROVELIA / JAPONICA / Esaki & Miyamoto, 1955 / labelled: P. KMENT 2013 [p, red label]’. PARATYPE ( apter.): ‘(Kyushu) / Wakasugi-yama / Chikuzen / 28. VII. 1948 / S. Miyamoto [hw] // [p] // PARA- TYPUS / MICROVELIA / JAPONICA / Esaki & Miyamoto, 1955 / labelled: P. KMENT 2013 [p, red label]’. PARATYPE ( apter.): ‘(Kyushu) / Wakasugi-yama / Chikuzen / 28. VII. 1948 / S. Miyamoto [hw] // [p] // PARA- TYPUS / MICROVELIA / JAPONICA / Esaki & Miyamoto, 1955 / labelled: P. KMENT 2013 [p, red label]’. Current status. Valid species: Microvelia (Picaultia) japonica Esaki & Miyamoto, 1955 (see ANDERSEN 1995, ANDERSEN & WEIR 2003, AUKEMA et al. 2013). Current status. Valid species: Microvelia (Picaultia) japonica Esaki & Miyamoto, 1955 (see ANDERSEN 1995, ANDERSEN & WEIR 2003, AUKEMA et al. 2013). Current status. Valid species. Current status. Valid species. Microvelia kyushuensis Esaki & Miyamoto, 1955 Microvelia kyushuensis Esaki & Miyamoto, 1955 Microvelia kyushuensis Esaki & Miyamoto, 1955: 172, 178, 181–185, Figs 2a, 6A–I, Pl. 24, Pl. 27: Figs E–F (original description). Microvelia (Microvelia) kamassanguensis Hoberlandt, 1951 Microvelia (Microvelia) kamassanguensis Hoberlandt, 1951: 28–30, Figs 69–72 (original description). Microvelia (Microvelia) kamassanguensis Hoberlandt, 1951: 28–30, Figs 69–72 (original description) The holotype and one paratype (allotype) are deposited in NMPC: HOLOTYPE ( apter.): ‘16.I.48 Kamassangu [hw] / Dundo, Angola / A. B. Machado lg. [p] // Ang. [p] No. 333.3 [hw] / coll.A.B.Machado [p] // Holotypus [p, red label] // Holotypus [p] / Microvelia / kamassangu- / ensis sp. n. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // \|\| [p]’. (The specimen is mounted on a piece of card; left hind leg, right antenna and all legs on the right side dissected, mounted on two slides deposited in Slide Box 2). g, g g g , p ) PARATYPE ( apter.): ‘16.I.48 Kamassangu [hw] / Dundo, Angola / A B Machado lgt. [p] // Ang. [p] No. 333.3 [hw] / coll.A.B Machado [p] // Allotypus [p, red label] // Allotypus [p] / Microvelia / kamassangu- / ensis sp. n. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // \|\|\| [p]’. Ten paratypes are deposited in NMPC: PARATYPE ( apter.): GBG. / 239. F [hw, vertical lines] / CAPE PROVINCE / Great Berg River [p] / 26. xi. 1951 [hw] / A. D. Harrison / C.S.I.R. Stream Survey [p] // Paratypus [p] / Microvelia / (Stenovelia) / lansburyi sbg. sp. n. [hw] / Det. L. Hoberlandt, 19[p]54 [hw, red label]’. PARATYPE ( apter.): GBG. / 239. F [hw, vertical lines] / CAPE PROVINCE / Great Berg River [p] / 26. xi. 1951 [hw] / A. D. Harrison / C.S.I.R. Stream Survey [p] // Paratypus [p] / Microvelia / (Stenovelia) / lansburyi sbg. sp. n. [hw] / Det. L. Hoberlandt, 19[p]54 [hw, red label]’. PARATYPE ( apter.): ‘GBG. / 339.C [hw, vertical lines] / CAPE PROVINCE / Great Berg River [p] / 29. viii. 1951 [hw] / A. D. Harrison / C.S.I.R. Stream Survey [p] // Paratypus [p] / Microvelia / (Stenovelia) / lansburyi sbg. sp. n. [hw] / Det. L. Hoberlandt, 19[p]54 [hw, red label]’. (Left antenna, right fore and hind leg, and male genitalia dissected and stored in a set of three microslides attached to pieces of card and placed on the same pin). PARATYPE ( apter.): ‘GBG. / 339.C [hw, vertical lines] / CAPE PROVINCE / Great Berg River [p] / 29. viii. 1951 [hw] / A. D. Harrison / C.S.I.R. Stream Survey [p] // Paratypus [p] / Microvelia / (Stenovelia) / lansburyi sbg. sp. n. [hw] / Det. L. Hoberlandt, 19[p]54 [hw, red label]’. (Left antenna, right fore and hind leg, and male genitalia dissected and stored in a set of three microslides attached to pieces of card and placed on the same pin). PARATYPE ( apter.): ‘GBG. / 566.C [hw, vertical lines] / CAPE PROVINCE / Great Berg River [p] / 28. v. 1952 [hw] / A. D. Harrison / C.S.I.R. Stream Survey [p] // Paratypus [p] / Microvelia / (Stenovelia) / lansburyi sbg. sp. n. [hw] / Det. L. Hoberlandt, 19[p]54 [hw, red label]’. PARATYPE ( apter.): ‘GBG. / 585.A [hw, vertical lines] / CAPE PROVINCE / Great Berg River [p] / 29. v. 1952 [hw] / A. D. Harrison / C.S.I.R. Stream Survey [p] // Paratypus [p] / Microvelia / (Stenovelia) / lansburyi sbg. sp. n. [hw] / Det. L. Hoberlandt, 19[p]54 [hw, red label]’. Microvelia lacunana Drake & Plaumann, 1953 Microvelia lacunana Drake & Plaumann, 1953: 415−416 (original decription). One paratype (allotype) is deposited in NMPC: One paratype (allotype) is deposited in NMPC: PARATYPE ( macr.): ‘1953 [p, vertical line] / Brasilien / Rio Caragualà / 21° 48´ B. 52° 27´ L. / Fritz Plaumann [p, horizontal lines] / 400 m [p, vertical line; white label with light blue vertical lines and black frame submarginally] // Allotype [p] / Microvelia / lacunana / D & P. [hw, red label] // [p]’. Current status. Valid species: Xiphovelia lacunana (Drake & Plaumann, 1953) (see POLHEMUS 1977, HECKMAN 2011, MOREIRA et al. 2011). However, ZETTEL (2012c: 100) expressed serious doubts concerning placement of this Nearctic species in Xiphovelia; further revision seems necessary to elucidate its generic placement. KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. 844 Ten paratypes are deposited in NMPC: (Antenna and all legs on the left side and male genitalia dissected and stored in a set of three microslides attached to pieces of card and placed on the same pin). PARATYPE ( apter.): GBG. / 269. B [hw, vertical lines] / CAPE PROVINCE / Great Berg River [p] / 29. v. 1951 [hw] / A. D. Harrison / C.S.I.R. Stream Survey [p] // Paratypus [p] / Microvelia / (Stenovelia) / lansburyi sbg. sp. n. [hw] / Det. L. Hoberlandt, 19[p]54 [hw, red label]’. PARATYPE ( apter.): ‘GBG. / 339. C. [hw, vertical lines] / CAPE PROVINCE / Great Berg River [p] / 29. viii. 1951 [hw] / A. D. Harrison / C.S.I.R. Stream Survey [p] // Paratypus [p] / Microvelia / (Stenovelia) / lansburyi sbg. sp. n. [hw] / Det. L. Hoberlandt, 19[p]54 [hw, red label]’. PARATYPE ( apter.): ‘GBG. / 443.F. [hw, vertical lines] / CAPE PROVINCE / Great Berg River [p] / 1. xii. 1951 [hw] / A. D. Harrison / C.S.I.R. Stream Survey [p] // Paratypus [p] / Microvelia / (Stenovelia sbg. n.) / lansburyi sp. n. [hw] / Det. L. Hoberlandt, 19[p]54 [hw, red label]’. PARATYPE ( apter.): ‘GBG. / 478. E. [hw, vertical lines] / CAPE PROVINCE / Great Berg River [p] / 28. xii. 1951 [hw] / A. D. Harrison / C.S.I.R. Stream Survey [p] // Paratypus [p] / Microvelia / (Stenovelia) / lansburyi sbg. sp. n. [hw] / Det. L. Hoberlandt, 19[p]54 [hw, red label]’. PARATYPE ( apter.): ‘GBG. / 585. A [hw, vertical lines] / CAPE PROVINCE / Great Berg River [p] / 29. v. 1951 [hw] / A. D. Harrison / C.S.I.R. Stream Survey [p] // Paratypus [p] / Microvelia / (Stenovelia) / lansburyi sbg. sp. n. [hw] / Det. L. Hoberlandt, 19[p]54 [hw, red label]’. p p PARATYPE ( apter.): ‘GBG. / 607. G. [hw, vertical lines] / CAPE PROVINCE / Great Berg River [p] / 9. ix. 1952 [hw] / A. D. Harrison / C.S.I.R. Stream Survey [p] // Paratypus [p] / Microvelia / (Stenovelia) / lansburyi sbg. sp. n. [hw] / Det. L. Hoberlandt, 19[p]54 [hw, red label]’. Current status. Valid species: Pseudovelia (Pseudovelia) lansburyi (Hoberlandt, 1956) (see ANDERSEN 1983). Current status. Valid species: Pseudovelia (Pseudovelia) lansburyi (Hoberlandt, 1956) (see A S 1983) ANDERSEN 1983). Microvelia (Pseudovelia) lansburyi Hoberlandt, 1956 Microvelia (Pseudovelia) lansburyi Hoberlandt, 1956: 181–184, Figs 1–9 (original description). Ten paratypes are deposited in NMPC: Ten paratypes are deposited in NMPC: Microvelia limaiana Drake, 1951 Microvelia limaiana Drake, 1951b: 79−80 (original description). Eight paratypes are deposited in NMPC: Eight paratypes are deposited in NMPC: Microvelia (Trichovelia) machadoi Hoberlandt, 1951 Microvelia (Trichovelia) machadoi Hoberlandt, 1951a: 38–41, Figs 114–125 (original description). Parts of the holotype and one paratype are deposited in NMPC: HOLOTYPE ( apter.): Two slides (Slide Box 2), one including antenna, fore, middle and hind leg, and the other one including male genitalia, labelled: ‘N. Mus. Praha / č. k. [p] / Heteroptera / Zpr. M.Z. / (880.4) 1950 [hw] / Tshikapa Dundo, Lunda / VI.-VII. 48 lgt. A. Machado [hw, white label with black frame submarginally] // Holotypus [p] / Microvelia / (Trichovelia subgen. n.) / machadoi sp.n. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. (Body of the holotype returned to the Dundo Museum, Angola). PARATYPE ( apter.): ‘VI-VII.48 Tshikapa [hw] / Dundo, Angola / A. B. Machado lgt. [p] // Ang. [p] No 880.4 [hw] / coll.A.B Machado [p] // Paratypus [p, red label] // Paratypus [p] / Microvelia / (Trichovelia subg. n.) / machadoi / sp. n. f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // \|\| [p]’. Current status. Valid species: Pseudovelia (Trichovelia) machadoi (Hoberlandt, 1951) (see LINNAVUORI 1975, 1977; ANDERSEN 1983). Microvelia (Pseudovelia) lundaensis Hoberlandt, 1951 Microvelia (Pseudovelia) lundaensis Hoberlandt, 1951a: 33–35, Figs 92–102 (original description). PARATYPE ( apter.): ‘VI.-VII.48 Tshikapa [hw] / Dundo, Angola / A. B. Machado lgt [p] // Ang. [p] No 880.4 [hw] / coll.A.B.Machado [p] // Paratypus [p, red label] // Paratypus [p] / Microvelia (Pseudovelia subg. n) / lundaensis / sp. n. f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // \|\|\| [p]’. Note. Collection also includes one slide with hemelytron of an additional paratype speci- men. Current status. Valid species: Pseudovelia (Pseudovelia) lundaensis (Hoberlandt, 1951) (see ANDERSEN 1983). Microvelia (Trichovelia) machadoi Hoberlandt, 1951 Microvelia (Trichovelia) machadoi Hoberlandt, 1951a: 38–41, Figs 114–125 (original descript Microvelia (Pseudovelia) lundaensis Hoberlandt, 1951 Microvelia (Pseudovelia) lundaensis Hoberlandt, 1951a: 33–35, Figs 92–102 (original description). Microvelia (Pseudovelia) lundaensis Hoberlandt, 1951 Microvelia (Pseudovelia) lundaensis Hoberlandt, 1951a: 33–35, Figs 92–102 (original description) Parts of the holotype and two paratypes are deposited in NMPC: Parts of the holotype and two paratypes are deposited in NMPC: HOLOTYPE ( apter.): Two slides (Slide Box 2), one including antenna, fore, middle and hind leg, and the other one including male genitalia, labelled: ‘N. Mus. Praha / č. k. [p] / Heteroptera / Zpr. M.Z. / (880.4) 1950 [hw] / Tshikapa Dundo, Lunda / VI.-VII. 48 lgt. A. Machado [hw, white label with black frame submarginally] // Holotypus [p] / Microvelia / (Pseudovelia subgen. n.) / lundaensis sp.n. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. (Body of the holotype returned to the Dundo Museum, Angola). HOLOTYPE ( apter.): Two slides (Slide Box 2), one including antenna, fore, middle and hind leg, and the other one including male genitalia, labelled: ‘N. Mus. Praha / č. k. [p] / Heteroptera / Zpr. M.Z. / (880.4) 1950 [hw] / Tshikapa Dundo, Lunda / VI.-VII. 48 lgt. A. Machado [hw, white label with black frame submarginally] // Holotypus [p] / Microvelia / (Pseudovelia subgen. n.) / lundaensis sp.n. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. (Body of the holotype returned to the Dundo Museum, Angola). ] ( y yp g ) PARATYPE ( macr.): ‘VI.-VII.48 Tshikapa [hw] / Dundo, Angola / A. B. Machado lgt. [p] // Ang. [p] No 880.4 [hw] / coll.A.B.Machado [p] // Paratypus [p, red label] // Paratypus [p] / Microvelia (Pseudovelia subg. n) / lundaensis / sp. n. f. macr. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // \|\|\| [p]’. PARATYPE ( macr.): ‘VI.-VII.48 Tshikapa [hw] / Dundo, Angola / A. B. Machado lgt. [p] // Ang. [p] No 880.4 [hw] / coll.A.B.Machado [p] // Paratypus [p, red label] // Paratypus [p] / Microvelia (Pseudovelia subg. n) / lundaensis / sp. n. f. macr. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // \|\|\| [p]’. p [ ] [p] [ ] \|\|\| [p] PARATYPE ( apter.): ‘VI.-VII.48 Tshikapa [hw] / Dundo, Angola / A. B. Machado lgt [p] // Ang. [p] No 880.4 [hw] / coll.A.B.Machado [p] // Paratypus [p, red label] // Paratypus [p] / Microvelia (Pseudovelia subg. n) / lundaensis / sp. n. f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // \|\|\| [p]’. Eight paratypes are deposited in NMPC: PARATYPE ( apter.): ‘Nova Teutonia / St. Catarina / Bras. [p] VI. 10 [hw] 1950 / Fritz Plaumann [p] / PARATYPE / By C. J. Drake [p] / Microvelia / limaiana [hw, red label] // Microvelia / limaiana / Drak. [hw, white label with black frame submarginally] // \|\| [p]’. PARATYPE ( apter.): ‘Nova Teutonia / St. Catarina / Bras. [p] VI. 10 [hw] 1950 / Fritz Plaumann [p] / PARATYPE / By C. J. Drake [p] / Microvelia / limaiana [hw, red label] // Microvelia / limaiana / Drak. [hw, white label with black frame submarginally] // \|\| [p]’. PARATYPES (5 apter.): ‘Nova Teutonia / St. Catarina / Bras. [p] VI. 10 [hw] 1950 / Fritz Plaumann [p] // PARA- TYPE / By C. J. Drake [p] / Microvelia / limaiana [hw, red label] // [p]’. PARATYPES (5 apter.): ‘Nova Teutonia / St. Catarina / Bras. [p] VI. 10 [hw] 1950 / Fritz Plaumann [p] // PARA- TYPE / By C. J. Drake [p] / Microvelia / limaiana [hw, red label] // [p]’. PARATYPES (5 apter.): ‘Nova Teutonia / St. Catarina / Bras. [p] VI. 10 [hw] 1950 / Fritz Plaumann [p] // PARA- TYPE / By C. J. Drake [p] / Microvelia / limaiana [hw, red label] // [p]’. PARATYPES (2 : 1 apter., 1 macr.): ‘Nova Teutonia / St. Catarina / Bras. [p] VI. 10 [hw] 1950 / Fritz Plaumann [p] // PARATYPE / By C. J. Drake [p] / Microvelia / limaiana [hw, red label] // \|\| [p]’. PARATYPES (2 : 1 apter., 1 macr.): ‘Nova Teutonia / St. Catarina / Bras. [p] VI. 10 [hw] 1950 / Fritz Plaumann [p] // PARATYPE / By C. J. Drake [p] / Microvelia / limaiana [hw, red label] // \|\| [p]’. PARATYPES (2 : 1 apter., 1 macr.): ‘Nova Teutonia / St. Catarina / Bras. [p] VI. 10 [hw] 1950 / Fritz Plaumann [p] // PARATYPE / By C. J. Drake [p] / Microvelia / limaiana [hw, red label] // \|\| [p]’. Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 845 Current status. Valid species, not attributed to any subgenus (see HECKMAN 2011, MOREIRA et al. 2011). Two paratypes are deposited in NMPC: Two paratypes are deposited in NMPC: Two paratypes are deposited in NMPC: PARATYPE ( macr.): ‘Mayaguez, Puerto / Rico, 4-4. 36 / H. D. Tate [p] // [p] // Paratype [p] / Microvelia / portoricensis / Drake. [hw, red label]’. PARATYPE ( apter.): ‘Mayaguez, Puerto / Rico, 4-4. 36 / H. D. Tate [p] // [p] // Paratype / By C. J. Drake [p] / Microvelia / portoricensis [hw, red label]’. PARATYPE ( macr.): ‘Mayaguez, Puerto / Rico, 4-4. 36 / H. D. Tate [p] // [p] // Paratype [p] / Microvelia / portoricensis / Drake. [hw, red label]’. PARATYPE ( apter.): ‘Mayaguez, Puerto / Rico, 4-4. 36 / H. D. Tate [p] // [p] // Paratype / By C. J. Drake [p] / Microvelia / portoricensis [hw, red label]’. Current status. Valid species, not attributed to any subgenus (see MALDONADO-CAPRILES & NAVARRO 1967). Microvelia (Microvelia) silvestris Hoberlandt, 1951a: 28, 30–32, Figs 73–82 (original description). Microvelia (Microvelia) silvestris Hoberlandt, 1951a: 28, 30–32, Figs 73–82 (original description). The holotype and one paratype are deposited in NMPC: The holotype and one paratype are deposited in NMPC: HOLOTYPE ( apter.): ‘Alex. [hw] / Egypt. [p] 12.9.33 [hw] / Dr. H. Priesner [p, white label with black frame submar- ginally] // Holotypus [p, red label] // Holotypus [p] / Microvelia (s. str.) / priesneri sp. n. / f. apt. [hw] / Det. L. Hoberlandt, 19[p]51 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 1213 [hw, orange label]’. (The specimen is card-mounted; antenna and all legs on the left side, and genitalia dissected, stored in a set of three microslides attached to three pieces of card placed on the same pin). PARATYPE ( apter.): ‘Alex. [hw] / Egypt. [p] 12.9.33 [hw] / Dr. H. Priesner [p, white label with black frame submar- ginally] // Paratypus [p, red label] // Paratypus [p] / Microvelia (s. str.) / priesneri sp. n. / f. apt. [hw] / Det. L. Hoberlandt, 19[p]51 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 1214 [hw, orange label]’. Current status. Junior synonym of Microvelia (Picaultia) popovi Brown, 1951 (see LIN- NAVUORI 1986, ANDERSEN 1995, ANDERSEN & WEIR 2003, AUKEMA et al. 2013). Current status. Valid species. Current status. Valid species. Microvelia (Trichovelia) troilos Linnavuori, 1975 Microvelia (Trichovelia) troilos Linnavuori, 1975: 50–53, Figs 19–25 (original description, key). PARATYPE ( apter.): ‘Côte d’Ivoire / Bouaké / IV-1964 / leg.R.H.Cobben [p] // Paratype / micr. (Trichovelia / troilos Linn. [sic!; hw, pale green label]’. Microvelia novana Drake & Plaumann, 1955 Microvelia novana Drake & Plaumann, 1955: 22−23 (original description). Microvelia novana Drake & Plaumann, 1955: 22−23 (original description). One paratype is deposited in NMPC: PARATYPE ( apter.): ‘Nova Teutonia / St. Catarina / Bras. [p] XI – [hw] 195[p]3 [hw] / Fritz Plaumann [p] // Paratype [p] / Microvelia / novana / D.& P. [hw, red label] // [p]’. One paratype is deposited in NMPC: Current status. Valid species, not attributed to any subgenus (see HECKMAN 2011, MOREIRA et al. 2011). KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. 846 The holotype is deposited in NMPC: The holotype is deposited in NMPC: HOLOTYPE ( macr.): ‘Dundo, Lunda / Angola [p] 10.XI.48 [hw] / A. B. Machado lgt. [p] // Ang. [p] No. 1209.2 [hw] / coll.A.B Machado // Holotypus [p, red label] // Holotypus [p] / Microvelia / silvestris / sp. n. f. macr. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // \|\|\| [p]’. (The specimen is card-mounted; left antenna, hemelytron, and all legs dissected, mounted on two separate slides deposited in Slide Box 2). Current status. Valid species. Microvelia (Microvelia) priesneri Hoberlandt, 1951 Microvelia (Microvelia) priesneri Hoberlandt, 1951b: 271–275, Figs 1–9 (original description, key). Microvelia tshingandana Linnavuori & Weber, 1974 One paratype is deposited in NMPC: PARATYPE ( apter.): ‘Zentral-Afrika [hw] // Tshinganda - Qache / 2450 m 5.4.72 / Statzner leg. [hw] // [p] // Paratypus / Microvelia / tshingandana / Lv. et Hb. [hw, red label]’. PARATYPE ( apter.): ‘Zentral-Afrika [hw] // Tshinganda - Qache / 2450 m 5.4.72 / Statzner leg. [hw] // [p] // Paratypus / Microvelia / tshingandana / Lv. et Hb. [hw, red label]’. Current status. Valid species, not attributed to any subgenus. Microvelia (Trichovelia) troilos Linnavuori, 1975 Microvelia (Trichovelia) troilos Linnavuori, 1975 Microvelia (Trichovelia) troilos Linnavuori, 1975: 50–53, Figs 19–25 (original description, key). PARATYPE ( apter.): ‘Côte d’Ivoire / Bouaké / IV-1964 / leg.R.H.Cobben [p] // Paratype / micr. (Trichovelia / troilos Microvelia (Trichovelia) troilos Linnavuori, 1975: 50–53, Figs 19–25 (original description, key). Microvelia (Trichovelia) troilos Linnavuori, 1975: 50–53, Figs 19–25 (original description, key). PARATYPE ( apter.): ‘Côte d’Ivoire / Bouaké / IV-1964 / leg.R.H.Cobben [p] // Paratype / micr. (Trichovelia / troilos Linn. [sic!; hw, pale green label]’. PARATYPE ( apter.): ‘Côte d’Ivoire / Bouaké / IV-1964 / leg.R.H.Cobben [p] // Paratype / micr. (Trichovelia / troilos Linn. [sic!; hw, pale green label]’. PARATYPE ( apter.): ‘Côte d’Ivoire / Bouaké / IV-1964 / leg.R.H.Cobben [p] // Paratype / micr. (Trichovelia / troilos Linn. [sic!; hw, pale green label]’. Current status. Valid species: Pseudovelia (Trichovelia) troilos (Linnavuori, 1975) (see LINNAVUORI 1977, ANDERSEN 1983). Current status. Valid species: Pseudovelia (Trichovelia) troilos (Linnavuori, 1975) (see LINNAVUORI 1977, ANDERSEN 1983). 847 Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 Microvelia (Microvelia) vilhenai Hoberlandt, 1951 Microvelia (Microvelia) vilhenai Hoberlandt, 1951a: 31–33, Figs 83–91 (original description). Seven paratypes are deposited in NMPC: Seven paratypes are deposited in NMPC: PARATYPES (4 apter.): ‘Dundo, Lunda / Angola [p] VI. 49 [hw] / A. B. Machado lgt. [p] // Ang. [p] No 1525.12 [hw] / coll.A.B.Machado [p] // Paratypus [p, red label] // Paratypus [p] / Microvelia / vilhenai / sp. n. [hw] / Det. L. Hoberlandt, 19[p]50 [hw. red label]’. PARATYPES (3 apter.): ‘Dundo, Lunda / Angola [p] VI. 49 [hw] / A. B. Machado lgt. [p] // Ang. [p] No 1525.12 [hw] / coll.A.B.Machado [p] // Paratypus [p, red label] // Paratypus [p] / Microvelia / vilhenai / sp. n. [hw] / Det. L. Hoberlandt, 19[p]50 [hw. red label]’. Note. Collection also includes two slides, one with antenna, fore, middle and hind leg, the other one with male genitalia, of an additional paratype specimen. Current status. Valid species (see POISSON 1957). Current status. Valid species (see POISSON 1957). Current status. Valid species (see POISSON 1957). Pseudovelia heissi Hecher, 2006 dovelia heissi Hecher, 2006: 436, 447–455, Figs 2, 12, 33–35, 46, 49, 58 (original description, key). Pseudovelia heissi Hecher, 2006: 436, 447–455, Figs 2, 12, 33–35, 46, 49, 58 (original description, key). Two paratypes are deposited in NMPC (exchange with Natural History Museum, Vienna): PARATYPE ( apter.): ‘Philippinen: Catanduanes / W Bato / Maribini Falls / 6.3.1999 / leg. H. Zettel (194) [p] // [p] // Paratypus / Pseudovelia / heissi sp.n. / des. Hecher 2006 [p, red label]’. PARATYPE ( apter.): ‘Philippinen: Camarines Sur / 20km E Naga, 3km E Carolina / Mainit Spring (Hydro) / 20.2.1998, leg.Zettel (142) [p] // [p] // Paratypus / Pseudovelia / heissi sp.n. / des. Hecher 2006 [p, red label]’. Current status. Valid species: Pseudovelia (Pseudovelia) heissi Hecher, 2006. Pseudovelia polhemi Hecher, 2006 Pseudovelia polhemi Hecher, 2006: 436, 438–440, 450–451, 453–454, Figs 11, 16–18, 43, 57–58 (original descrip- tion, key). Two paratypes are deposited in NMPC (exchange with Natural History Museum, Vienna): aratypes are deposited in NMPC (exchange with Natural History Museum, Vienna) PARATYPE ( apter.): ‘Philippinen: La Union / Bacnotan, Don Mariano / Marcos Mem. State Univ. / Casiaman Falls, 24.10. / 2002, leg.H.Zettel (312) [p] // [p] // Paratypus / Pseudovelia / polhemi sp. n. / des. Hecher 2006 [p, red label]’. ] PARATYPE ( apter.): ‘Philippinen: La Union / Bacnotan, Don Mariano / Marcos Mem. State Univ. / Casiaman Falls, 24.10. / 2002, leg.H.Zettel (312) [p] // [p] // Paratypus / Pseudovelia / polhemi sp. n. / des. Hecher 2006 [p, red label]’. Current status. Valid species: Pseudovelia (Pseudovelia) polhemi Hecher, 2006. KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. 848 Pseudovelia ullrichi Hecher, 2005 Pseudovelia ullrichi Hecher, 2005: 56–57, 59, 62–64, Figs 1, 13–19 (original description, key). Pseudovelia ullrichi Hecher, 2005: 56–57, 59, 62–64, Figs 1, 13–19 (original description, key). Two paratypes are deposited in NMPC (exchange with Natural History Museum, V PARATYPE ( apter.): ‘11 VIII 1998 Thaild. / Mae Hong Son / Mae Nam Cottage // N 019° 19′59.0″/ in Pai River / E 097° 57′ 13.0″[p] // among ﬂ oating / debris / Dr. W. Ullrich leg. [p] // Dr. Wolfgang G. / ULLRICH / collection [p] // [p] // Paratypus / Pseudovelia / ullrichi sp.n. / des. C. Hecher 2004 [p, red label]’. PARATYPE ( apter.): ‘11 VIII 1998 Thaild. / Mae Hong Son / Mae Nam Cottage // N 019° 19′59.0″/ in Pai River / E 097° 57′ 13.0″[p] // among ﬂ oating / debris / Dr. W. Ullrich leg. [p] // Dr. Wolfgang G. / ULLRICH / collection [p] // [p] // Paratypus / Pseudovelia / ullrichi sp.n. / des. C. Hecher 2004 [p, red label]’. Current status. Valid species: Pseudovelia (Pseudovelia) ullrichi Hecher, 2005. Pseudovelia schoenigi Hecher & Bongo, 2006 Pseudovelia schoenigi Hecher & Bongo, 2006: 92–98, Figs 1–9 (original description). Two paratypes are deposited in NMPC (exchange with Natural History Museum, Vienna): Two paratypes are deposited in NMPC (exchange with Natural History Museum, Vienna): PARATYPE ( apter.): ‘Philippinen: Masbate Isl. / 3.5 km SE Masbate, Tugbo / Tugbo River / 2.3.1998 / leg. H. Zettel (152) [p] // 1 [hw] // [p] // Paratypus / Pseudovelia / schoenigi sp.n., des. / Hecher & Bongo 2005 [p, red label]’ (The specimen is card-mounted, the detached male genitalia glued to the same piece of card). PARATYPE ( apter.): ‘Philippinen: Masbate Isl. / 3.5 km SE Masbate, Tugbo / Tugbo River / 2.3.1998 / leg. H. Zettel (152) [p] // 1 [hw] // [p] // Paratypus / Pseudovelia / schoenigi sp.n., des. / Hecher & Bongo 2005 [p, red label]’ (The specimen is card-mounted, the detached male genitalia glued to the same piece of card). ( p g g p ) PARATYPE ( apter.): ‘Philippinen: Masbate Isl. / 2 km S Baleno / stream, 4.3.1998 / leg. H. Zettel (154) [p] // [p] // Paratypus / Pseudovelia / schoenigi sp.n., des. / Hecher & Bongo 2005 [p, red label]’. (The specimen is card-mounted, glued on its dorsum). ( p g g p ) PARATYPE ( apter.): ‘Philippinen: Masbate Isl. / 2 km S Baleno / stream, 4.3.1998 / leg. H. Zettel (154) [p] // [p] // Paratypus / Pseudovelia / schoenigi sp.n., des. / Hecher & Bongo 2005 [p, red label]’. (The specimen is card-mounted, glued on its dorsum). Current status. Valid species: Pseudovelia (Pseudovelia) schoenigi Hecher & Bongo, 2005. Current status. Valid species: Pseudovelia (Pseudovelia) schoenigi Hecher & Bongo, 2005. Pseudovelia tibialis Esaki & Miyamoto, 1955 Pseudovelia tibialis Esaki & Miyamoto, 1955: 174–176, 179, 193–199, Figs 3A–D, 4D, 8A–E, 9A–J, Pl. 25: Fig. B, Pl. 29: Figs A–C (original description, key). Four paratypes are deposited in NMPC: Four paratypes are deposited in NMPC: PARATYPE ( apter.): ‘(Kyushu) / Mt. Kirishima / Hyuga / 28. IX. 1952 / R. Morimoto [hw] // [p] // PARATYPUS / PSEUDOVELIA / TIBIALIS / Esaki & Miyamoto, 1955 / labelled: P. KMENT 2013 [p, red label]’. PARATYPE ( apter.): ‘(Kyushu) / Mt. Kirishima / Hyuga / 28. IX. 1952 / R. Morimoto [hw] // [p] // PARATYPUS / Pseudovelia / tibialis / Esaki et Miyamoto / reverse: (Kyushu) / Mt. Kirishima / Hyuga / 28. IX. 1952 / R. Morimoto [hw] // PARATYPUS / PSEUDOVELIA / TIBIALIS / Esaki & Miyamoto, 1955 / labelled: P. KMENT 2013 [p, red label]’. PARATYPE ( macr.): ‘(Kyushu) / Mt. Kirishima / Hyuga / 28. IX. 1952 / R. Morimoto [hw] // [p] // PARATYPUS / Pseudovelia / tibialis / Esaki et Miyamoto / reverse: (Kyushu) / Mt. Kirishima / Hyuga / 28. IX. 1952 / R. Morimoto [hw] // PARATYPUS / PSEUDOVELIA / TIBIALIS / Esaki & Miyamoto, 1955 / labelled: P. KMENT 2013 [p, red label]’. PARATYPE ( macr.): ‘(Kyushu) / Mt. Kirishima / Hyuga / 28. IX. 1952 / R. Morimoto [hw] // [p] // PARATYPUS / PSEUDOVELIA / TIBIALIS / Esaki & Miyamoto, 1955 / labelled: P. KMENT 2013 [p, red label]’. (Detached antennal segment III and IV of both antennae glued on a separate piece of card). Current status. Valid species: Pseudovelia tibialis tibialis Esaki & Miyamoto, 1955 (see MIYAMOTO & LEE 1963; ANDERSEN 1983, 1995a; YE et al. 2013a). 849 Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 Thirteen paratypes are deposited in NMPC: [p] 411.7 [hw] / coll.A.B. Machado [p] // Paratypus [p, red label] // Paratypus [p] / Xiphoveloidea / chinai gen. et / sp. n.f. macr. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. PARATYPES (3 macr.): ‘Dundo, Lunda / Angola [p] III. 48 [hw] / A. B. Machado lgt. [p] // Ang. [p] 411.7 [hw] / coll.A.B. Machado [p] // Paratypus [p, red label] // Paratypus [p] / Xiphoveloidea / chinai gen. et / sp. n.f. macr. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. PARATYPES (2 macr.): ‘Dundo, Lunda / Angola II-III.48 [hw] / A B Machado lgt [p] // Ang. [p] No 411 [hw] / coll.A.B. Machado [p] // Paratypus [p, red label] // Paratypus [p] / Xiphoveloidea / chinai gen. et / sp. n.f. macr. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. PARATYPES (2 macr.): ‘Dundo, Lunda / Angola II-III.48 [hw] / A B Machado lgt [p] // Ang. [p] No 411 [hw] / coll.A.B. Machado [p] // Paratypus [p, red label] // Paratypus [p] / Xiphoveloidea / chinai gen. et / sp. n.f. macr. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. Note. Collection also includes six slides with various body parts of additional macropterous paratypes, and three slides with body parts of additional apterous paratypes (Slide Box 2). C t t t V lid i ( L 1977) Note. Collection also includes six slides with various body parts of additional macropterous paratypes, and three slides with body parts of additional apterous paratypes (Slide Box 2). Current status. Valid species (see LINNAVUORI 1977). paratypes, and three slides with body parts of additional apterous paratypes (Slide Box 2). Current status. Valid species (see LINNAVUORI 1977). p yp , y p p p yp ( ) Current status. Valid species (see LINNAVUORI 1977). Thirteen paratypes are deposited in NMPC: PARATYPES (2 apter.): ‘VI-VII.48 Tshikapa [hw] / Dundo, Angola / A. B. Machado lgt. [p] // Ang. [p] No 880.4 [hw] / coll.A.B. Machado [p] // Paratypus [p, red label] // Paratypus [p] / Xiphoveloidea / chinai gen. et / sp. n.f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. PARATYPES (2 apter.): ‘VI-VII.48 Tshikapa [hw] / Dundo, Angola / A. B. Machado lgt. [p] // Ang. [p] No 880.4 [hw] / coll.A.B. Machado [p] // Paratypus [p, red label] // Paratypus [p] / Xiphoveloidea / chinai gen. et / sp. n.f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. PARATYPES (4 apter.): ‘VI-VII.48 Tshikapa [hw] / Dundo, Angola / A. B. Machado lgt. [p] // Ang. [p] No 880.4 [hw] / coll.A.B.Machado [p] // Paratypus [p, red label] // Paratypus [p] / Xiphoveloidea / chinai gen. et / sp. n.f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. PARATYPES (4 apter.): ‘VI-VII.48 Tshikapa [hw] / Dundo, Angola / A. B. Machado lgt. [p] // Ang. [p] No 880.4 [hw] / coll.A.B.Machado [p] // Paratypus [p, red label] // Paratypus [p] / Xiphoveloidea / chinai gen. et / sp. n.f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. PARATYPE ( macr.): ‘Dundo, Lunda / Angola [p] III. 48 [hw] / A. B. Machado lgt. [p] // Ang. [p] 411.7 [hw] / coll. A.B. Machado [p] // Paratypus [p, red label] // Paratypus [p] / Xiphoveloidea / chinai gen. et / sp. n.f. macr. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. PARATYPE ( macr.): ‘Dundo, Lunda / Angola II-III.48 [hw] / A B Machado lgt [p] // Ang. [p] No 411 [hw] / coll. A.B. Machado [p] // Paratypus [p, red label] // Paratypus [p] / Xiphoveloidea / chinai gen. et / sp. n.f. macr. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. PARATYPE ( macr.): ‘Dundo, Lunda / Angola II-III.48 [hw] / A B Machado lgt [p] // Ang. [p] No 411 [hw] / coll. A.B. Machado [p] // Paratypus [p, red label] // Paratypus [p] / Xiphoveloidea / chinai gen. et / sp. n.f. macr. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. PARATYPES (3 macr.): ‘Dundo, Lunda / Angola [p] III. 48 [hw] / A. B. Machado lgt. [p] // Ang. Xiphoveloidea chinai Hoberlandt, 1951 Xiphoveloidea chinai Hoberlandt, 1951a: 12, 17–22, 25, Figs 17–38 (original description, differential diagnosis). Thirteen paratypes are deposited in NMPC: Parts of the holotype are deposited in NMPC: Parts of the holotype are deposited in NMPC: HOLOTYPE ( apter.): Four slides (Slide Box 2), one including antenna, one with fore, middle and hind leg, the two remaining slides including male genitalia, labelled: ‘N. Mus. Praha / č. k. [p] / Heteroptera / Zpr. M.Z. / (880.4) 1950 [hw] / Tshikapa Angola / VI.-VII. 48 lgt. A. Machado [hw, white label with black frame submarginally] // Holotypus [p] / Xiphoveloidea / pulchella / sp. n. / apter. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. (Body of the holotype returned to the Dundo Museum, Angola). Xiphoveloidea pulchella Hoberlandt, 1951 oveloidea pulchella Hoberlandt, 1951a: 22–25, Figs 39–47 (original description, differential diagnosis). Parts of the holotype are deposited in NMPC: HOLOTYPE ( apter.): Four slides (Slide Box 2), one including antenna, one with fore, middle and hind leg, the two remaining slides including male genitalia, labelled: ‘N. Mus. Praha / č. k. [p] / Heteroptera / Zpr. M.Z. / (880.4) 1950 [hw] / Tshikapa Angola / VI.-VII. 48 lgt. A. Machado [hw, white label with black frame submarginally] // Holotypus [p] / Xiphoveloidea / pulchella / sp. n. / apter. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. (Body of the holotype returned to the Dundo Museum, Angola). Current status. Valid species (see DRAKE & HUSSEY 1955). Subfamily Perittopinae China & Usinger, 1949 Perittopus asiaticus Zettel, 2001 Perittopus asiaticus Zettel, 2001: 110–113, 116, 118, Figs 2, 6, 14–15, 22, 25 (original description, key). Two paratypes are deposited in NMPC (exchange with Natural History Museum, Vienna): PARATYPES (2 : 1 apter., 1 macr.): ‘Malaysia: Penang Isl. / Penang Hill, Jelang / Waterfall,560 m, 10.1. / 1996,leg. Schwendinger [p] // [p] // P a r a t y p u s / Perittopus / asiaticus sp.n. / des. H. Zettel 2000 [p, red label]’. Current status. Valid species (see CHEN et al. 2005, AUKEMA et al. 2013, YE et al. 2013b). Perittopus asiaticus Zettel, 2001 Perittopus asiaticus Zettel, 2001 Perittopus asiaticus Zettel, 2001: 110–113, 116, 118, Figs 2, 6, 14–15, 22, 25 (original description, key). Two paratypes are deposited in NMPC (exchange with Natural History Museum, Vienna): Perittopus asiaticus Zettel, 2001: 110–113, 116, 118, Figs 2, 6, 14–15, 22, 25 (original description, key). ttopus asiaticus Zettel, 2001: 110–113, 116, 118, Figs 2, 6, 14–15, 22, 25 (original description, key). Perittopus asiaticus Zettel, 2001: 110–113, 116, 118, Figs 2, 6, 14–15, 22, 25 (original description, key). Two paratypes are deposited in NMPC (exchange with Natural History Museum, Vienna): PARATYPES (2 : 1 apter., 1 macr.): ‘Malaysia: Penang Isl. / Penang Hill, Jelang / Waterfall,560 m, 10.1. / 1996,leg. Schwendinger [p] // [p] // P a r a t y p u s / Perittopus / asiaticus sp.n. / des. H. Zettel 2000 [p, red label]’. Two paratypes are deposited in NMPC (exchange with Natural History Museum, Vienna): PARATYPES (2 : 1 apter., 1 macr.): ‘Malaysia: Penang Isl. / Penang Hill, Jelang / Waterfall,560 m, 10.1. / 1996,leg. Schwendinger [p] // [p] // P a r a t y p u s / Perittopus / asiaticus sp.n. / des. H. Zettel 2000 [p, red label]’. Current status. Valid species (see CHEN et al. 2005, AUKEMA et al. 2013, YE et al. 2013b). KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. 850 Current status. Valid species (see CHEN et al. 2005). Current status. Valid species (see CHEN et al. 2005). The holotype and 7 paratypes (including the allotype) are deposited in NMPC: The holotype and 7 paratypes (including the allotype) are deposited in NMPC The holotype and 7 paratypes (including the allotype) are deposited in NMPC: HOLOTYPE ( apter.): ‘Vohémar / Madagascar [p] // Holotypus [p, red label] // Holotypus [p] / Rhagovelia / abalienata / sp. n. f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. HOLOTYPE ( apter.): ‘Vohémar / Madagascar [p] // Holotypus [p, red label] // Holotypus [p] / Rhagovelia / abalienata / sp. n. f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. PARATYPE ( apter.): ‘Vohémar / Madagascar [p] // Paratypus [p, red label] // Paratypus [p] / Rhagovelia / abalienata / sp. n. f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. (The specimen is card-mounted; left hind leg, and right middle and hind leg missing. Dissected male genitalia mounted on a microslide on a piece of card attached to the same pin; one middle and one hind leg mounted on two separate slides stored in Slide Box 3). PARATYPE ( apter.): ‘Paratypus [p, red label] // Vohémar / Madagascar [p] // Paratypus [p] / Rhagovelia / abalienata / sp. n. f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. PARATYPE ( apter.): ‘Vohémar / Madagascar [p] // Allotypus [p, red label] // Allotypus [p] / Rhagovelia / abalienata / sp. n. f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. PARATYPES (4 apter.): ‘Vohémar / Madagascar [p] // Paratypus [p, red label] // Paratypus [p] / Rhagovelia / abalienata / sp. n. f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. Current status. Valid species. Rhagovelia abalienata Hoberlandt, 1951 Rhagovelia abalienata Hoberlandt, 1951: 5–7, 10–11, Figs 7–15 (original description, keys to males and fema- les). Rhagovelia abalienata Hoberlandt, 1951: 5–7, 10–11, Figs 7–15 (original description, keys to males and fema- les). Perittopus borneensis Zettel, 2001 Perittopus borneensis Zettel, 2001: 111–116, 118, Figs 1, 12–13, 17, 23, 27 (original description, key). erittopus borneensis Zettel, 2001: 111–116, 118, Figs 1, 12–13, 17, 23, 27 (original description, key). Three paratypes are deposited in NMPC (exchange with Natural History Museum, Vien- na): PARATYPE ( macr.): ‘Malaysia, Sabah, Crocker / Range, Kaningau env., stream near / Liang cave 13.6. 1998 / J. Kodada & F. Čiampor lgt. [p] // [p] // P a r a t y p u s / Perittopus / borneensis sp.n. / des. H. Zettel 2000 [p, red label]’. PARATYPE ( apter.): ‘Malaysia, Sabah, Batu / Punggul Resort env., 24.VI.- / 1.VII.1995, 11b, shaded stream / 1.5- 2.0 m wide, ﬂ owing through / dense primary forest [p] // [p] // P a r a t y p u s / Perittopus / borneensis sp.n. / des. H. Zettel 2000 [p, red label]’. [p ] PARATYPE ( apter.): ‘Malaysia, Sabah, Batu / Punggul Resort env., 24.VI.- / 1.VII.1995, 11b, shaded stream / 1.5- 2.0 m wide, ﬂ owing through / dense primary forest [p] // [p] // P a r a t y p u s / Perittopus / borneensis sp.n. / des. H. Zettel 2000 [p, red label]’. o paratypes are deposited in NMPC (exchange with Natural History Museum, Vienn Two paratypes are deposited in NMPC (exchange with Natural History Museum, Vienna): Two paratypes are deposited in NMPC (exchange with Natural History Museum, Vienna): PARATYPE ( apter.): ‘PHILIPPINEN: Negros / SE Bacolod,Mambucal / Seven Falls, 15.-16.3. / 900 m,lg.Zettel 1994(39a) [p] // [p] // P a r a t y p u s / Rhagovelia / canlaonensis sp. n. / des. H. Zettel 1994 [p, red label]’. PARATYPE ( apter.): ‘PHILIPPINEN: Negros / Umg. Canlaon City / Pula, 9.2.1994 / leg. Seyfert & Graindl [p] // PARATYPE ( apter.): ‘PHILIPPINEN: Negros / SE Bacolod,Mambucal / Seven Falls, 15.-16.3. / 900 m,lg.Zettel 1994(39a) [p] // [p] // P a r a t y p u s / Rhagovelia / canlaonensis sp. n. / des. H. Zettel 1994 [p, red label]’. PARATYPE ( apter.): ‘PHILIPPINEN: Negros / SE Bacolod,Mambucal / Seven Falls, 15.-16.3. / 900 m,lg.Zettel 1994(39a) [p] // [p] // P a r a t y p u s / Rhagovelia / canlaonensis sp. n. / des. H. Zettel 1994 [p, red label]’. PARATYPE ( apter.): ‘PHILIPPINEN: Negros / Umg. Canlaon City / Pula, 9.2.1994 / leg. Seyfert & Graindl [p] // 30.11.1992 [p] // [p] // P a r a t y p u s / Rhagovelia / canlaonensis sp. n. / des. H. Zettel 1994 [p, red label]’. PARATYPE ( apter.): ‘PHILIPPINEN: Negros / Umg. Canlaon City / Pula, 9.2.1994 / leg. Seyfert & Graindl [p] // 30.11.1992 [p] // [p] // P a r a t y p u s / Rhagovelia / canlaonensis sp. n. / des. H. Zettel 1994 [p, red label]’. Current status. Valid species (see CHEN et al. 2005). Rhagovelia atrispina J. T. Polhemus, 1977 Rhagovelia atrispina Polhemus, 1977: 645–646 (original description). Four paratypes are deposited in NMPC: PARATYPES (2 apter.): ‘MEX. ,Mich. / Tacambaro / CL1033; 23-IV-64 / J.T. &M.S. Polhemus [p] // [p] // PARATYPE / Rhagovelia / atrispina / J.T.Polhemus [p, red label]. PARATYPES (2 apter.): ‘MEX. ,Mich. / Tacambaro / CL1033; 23-IV-64 / J.T. &M.S. Polhemus [p] // [p] PARATYPE / Rhagovelia / atrispina / J.T.Polhemus [p, red label]. PARATYPES (2 apter.): ‘MEX. ,Mich. / Tacambaro / CL1033; 23-IV-64 / J.T. &M.S. Polhemus [p] // [p] // PARATYPE / Rhagovelia / atrispina / J.T.Polhemus [p, red label]. Current status. Valid species (see POLHEMUS 1997, PADILLA-GIL & MOREIRA 2013). Current status. Valid species (see POLHEMUS 1997, PADILLA-GIL & MOREIRA 2013). 851 Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 Rhagovelia excellentis Drake & Harris, 1927: 134−135 (original description). Rhagovelia excellentis Drake & Harris, 1927: 134−135 (original description). Rhagovelia excellentis Drake & Harris, 1927: 134−135 (original description). Two paratypes are deposited in NMPC: PARATYPES (2 apter.): ‘Dolores, Colo. / Aug. 15, 1925 / C. J. Drake // [p] // PARATYPE [p] / Rhagovelia / excellentis / Drake + Harris [hw, red label]’. Current status. Junior synonym of Rhagovelia distincta Champion, 1898 (see GOULD 1931, SMITH 1988a, POLHEMUS 1997, PADILLA-GIL & MOREIRA 2013). Current status. Junior synonym of Rhagovelia distincta Champion, 1898 (see GOULD 1931, SMITH 1988a, POLHEMUS 1997, PADILLA-GIL & MOREIRA 2013). Rhagovelia graindli Zettel, 2012 Rhagovelia graindli Zettel, 2012c: 84, 89, 93–97, Figs 9–11, 32–33, 43 (original description). Two paratypes are deposited in NMPC (exchange with Natural History Museum, Vi p yp p ( g y , ) PARATYPE ( apter.): ‘PHILIPPINEN: Mindanao / Lanao del Norte, W. Iligan / Tinago Falls, 13.11.1996 / leg. H. Zettel (95) [p] // [p] // P A R A T Y P U S / Rhagovelia / graindli sp.n. / des. H. Zettel 2012 [p, red label]’. PARATYPE ( apter.): ‘PHILIPPINEN: Mindanao / Lanao del Norte, W. Iligan / Tinago Falls, 13.11.1996 / leg. H. Zettel (95) [p] // [p] // P A R A T Y P U S / Rhagovelia / graindli sp.n. / des. H. Zettel 2012 [p, red label]’. PARATYPE ( apter.): ‘PHILIPPINEN: Mindanao / Lanao del Norte, W. Iligan / Tinago Falls, 13.11.1996 / leg. H. Zettel (95) [p] // [p] // P A R A T Y P U S / Rhagovelia / graindli sp.n. / des. H. Zettel 2012 [p, red label]’. PARATYPE ( apter.): ‘PHILIPPINEN: Mindanao / Lanao del Norte, W. Iligan / Tinago Falls, 13.11.1996 / leg. H. ( p ) , g g , g Zettel (95) [p] // [p] // P A R A T Y P U S / Rhagovelia / graindli sp.n. / des. H. Zettel 2012 [p, red label]’. PARATYPE ( apter.): ‘PHILIPPINEN: Mindanao / Lanao del Norte, W. Iligan / Tinago Falls, 13.11.1996 / leg. H. Zettel (95) [p] // [p] // P A R A T Y P U S / Rhagovelia / graindli sp.n. / des. H. Zettel 2012 [p, red label]’. PARATYPE ( apter.): ‘PHILIPPINEN: Mindanao / Lanao del Norte, W. Iligan / Tinago Falls, 13.11.1996 / leg. H. Zettel (95) [p] // [p] // P A R A T Y P U S / Rhagovelia / graindli sp.n. / des. H. Zettel 2012 [p, red label]’. Current status. Valid species. Rhagovelia canlaonensis Zettel, 1996 Rhagovelia canlaonensis Zettel, 1996: 113, 118–120, 130–134, 136, 139, Figs 6, 11, 18, 32, 37, 56, 63 (original description, key). Rhagovelia heissi Zettel & Bongo, 2006 g g , Rhagovelia heissi Zettel & Bongo, 2006: 737–741, Figs 1–10 (original description). Two paratypes are deposited in NMPC (exchange with Natural History Museum, Vienna): PARATYPES (2 apter.): ‘Philippines: Cebu, Boljoon / (Pobl.), river at Lusapon / Bridge, freshwater, 5. 12. / 2005, leg. H. Zettel (437) [p] // [p] // Paratypus / Rhagovelia / heissi sp.n., des. / Zettel & Bongo 2005 [p, red label]’. (Both paratypes are mounted on triangular cards and attached to the same pin with two locality labels). Current status. Valid species. Rhagovelia hovana Hoberlandt, 1941 Rhagovelia hovana Hoberlandt, 1941a: 72–73, Figs 7–8 (original description). The holotype and 8 paratypes (including the allotype) are deposited in NMPC: Two paratypes are deposited in NMPC (exchange with Natural History Museum, Vienna): p yp p ( g y , ) PARATYPES (2 apter.): ‘Philippines: Cebu, Boljoon / (Pobl.), river at Lusapon / Bridge, freshwater, 5. 12. / 2005, leg. H. Zettel (437) [p] // [p] // Paratypus / Rhagovelia / heissi sp.n., des. / Zettel & Bongo 2005 [p, red label]’. (Both paratypes are mounted on triangular cards and attached to the same pin with two locality labels). Current status. Valid species. Rhagovelia hovana Hoberlandt, 1941 Rhagovelia hovana Hoberlandt, 1941a: 72–73, Figs 7–8 (original description). Th h l d 8 (i l di h ll ) d i d i NMPC Rhagovelia hovana Hoberlandt, 1941 Rhagovelia hovana Hoberlandt, 1941a: 72–73, Figs 7–8 (original description). Rhagovelia impensa Bacon, 1956: 817, 827–829, Pl. 6: Fig. 8 (original description, key). Rhagovelia impensa Bacon, 1956: 817, 827–829, Pl. 6: Fig. 8 (original description, key). Two paratypes are deposited in NMPC: PARATYPE ( apter.): ‘Peru S. A. / Lagunas Villa / Dept. Lima / Je. 8 to Ju. 1-34 [p] // Collected by / F. Woytkowski [p] // P A R A T Y P E / Rhagovelia [p] / impensa [hw] / John A. Bacon [p, blue label] // Ragovelia / impensa / Bacon [hw] / Det H. B. Hungerford [p, white label with black frame submarginally] // [p]’. PARATYPE ( apter.): ‘Peru S. A. / Lagunas Villa / Dept. Lima / Je. 8 to Ju. 1-34 [p] // Collected by / F. Woytkowski [p] // P A R A T Y P E / Rhagovelia [p] / impensa [hw] / John A. Bacon [p, blue label] // Ragovelia / impensa / Bacon [hw] / Det H. B. Hungerford [p, white label with black frame submarginally] // [p]’. PARATYPE ( apter.): ‘Peru S. A. / Lagunas Villa / Dept. Lima / Je. 8 to Ju. 1-34 [p] // Collected by / F. Woytkowski [p] // [p] // P A R A T Y P E / Rhagovelia [p] / impensa [hw] / John A. Bacon [p, blue label]’. Current status. Valid species (see POLHEMUS 1997, HECKMAN 2011, PADILLA-GIL & MOREIRA 2013). Rhagovelia hovana Hoberlandt, 1941 Rhagovelia hovana Hoberlandt, 1941a: 72–73, Figs 7–8 (original description). PARATYPES (3 apter.): ‘Vohémar / Madagascar [p] // Paratypus [p, red label with black margins] // Rhagovelia / hovana n. sp. [hw] / L. Hoberlandt det. [p]’. PARATYPES (3 apter.): ‘Vohémar / Madagascar [p] // Paratypus [p, red label with black margins] // Rhagovelia / hovana n. sp. [hw] / L. Hoberlandt det. [p]’. PARATYPE (): ‘Madagascar / Env. de Rogez [p] // Paratypus [p, red label with black margins] // Rhagovelia / hovana n. sp. [hw] / L. Hoberlandt det. [p]’. PARATYPE (): ‘Madagascar / Env. de Rogez [p] // Paratypus [p, red label with black margins] // Rhagovelia / hovana n. sp. [hw] / L. Hoberlandt det. [p]’. Current status. Valid species (see HOBERLANDT 1951c, POISSON 1963). Rhagovelia hovana Hoberlandt, 1941 Rhagovelia hovana Hoberlandt, 1941a: 72–73, Figs 7–8 (original description). The holotype and 8 paratypes (including the allotype) are deposited in NMPC: The holotype and 8 paratypes (including the allotype) are deposited in NMPC: The holotype and 8 paratypes (including the allotype) are deposited in NMPC: 852 KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. HOLOTYPE ( apter.): ‘Vohémar / Madagascar [p] // Holotypus [p, red label with black margins] // Rhagovelia / hovana n. sp. [hw] / L. Hoberlandt det. [p]’. (The specimen is card-mounted; both antennae, all legs on the right side, and pygophore dissected, mounted on four separate slides stored in Slide Box 3). HOLOTYPE ( apter.): ‘Vohémar / Madagascar [p] // Holotypus [p, red label with black margins] // Rhagovelia / hovana n. sp. [hw] / L. Hoberlandt det. [p]’. (The specimen is card-mounted; both antennae, all legs on the right side, and pygophore dissected, mounted on four separate slides stored in Slide Box 3). pyg p p ) PARATYPES (3 apter.): ‘Vohémar / Madagascar [p] // Paratypus [p, red label with black margins] // Rhagovelia / hovana n. sp. [hw] / L. Hoberlandt det. [p]’. pyg p p ) PARATYPES (3 apter.): ‘Vohémar / Madagascar [p] // Paratypus [p, red label with black margins] // Rhagovelia / hovana n. sp. [hw] / L. Hoberlandt det. [p]’. PARATYPE ( apter.): ‘Vohémar / Madagascar [p] // Allotypus [p, red label with black margins] // Rhagovelia / hovana n. sp. [hw] / L. Hoberlandt det. [p]’. (The specimen is card-mounted; left antenna and all middle and hind legs missing. The antenna and one middle and one hind leg mounted on a separate slide stored in Slide Box 3). PARATYPES (3 apter.): ‘Vohémar / Madagascar [p] // Paratypus [p, red label with black margins] // Rhagovelia / h [h ] / L H b l d d [ ]’ PARATYPE ( apter.): ‘Vohémar / Madagascar [p] // Allotypus [p, red label with black margins] // Rhagovelia / hovana n. sp. [hw] / L. Hoberlandt det. [p]’. (The specimen is card-mounted; left antenna and all middle and hind legs missing. The antenna and one middle and one hind leg mounted on a separate slide stored in Slide Box 3). legs missing. The antenna and one middle and one hind leg mounted on a separate slide stored in Slide Box 3). Rhagovelia impensa Bacon, 1956 g p Rhagovelia impensa Bacon, 1956: 817, 827–829, Pl. 6: Fig. 8 (original description, key). Rhagovelia inexpectata Zettel, 2000 Rhagovelia inexpectata Zettel, 2000b: 176–178, Figs 1–7 (original description). Two paratypes are deposited in NMPC (exchange with Natural History Museum, Vienna): Two paratypes are deposited in NMPC (exchange with Natural History Museum, Vienna): PARATYPE ( macr.): ‘Thailand: Phrae Prov., / 50km NE Phrae, Hua Kaet / Kaet river, 17.-18.11. / 1995,leg.H.Zettel (17b) [p] // [p] // P a r a t y p u s / Rhagovelia / inexpectata sp.n. / des. H. Zettel 1998 [p, red label]’. PARATYPE ( macr.): ‘Thailand: Phetchabun Prov., / Nam Nao NP, Heo Sai / 25.11.1995 / leg. H. Zettel (24) [p] // [p] // P a r a t y p u s / Rhagovelia / inexpectata sp n / des H Zettel 1998 [p red label]’ PARATYPE ( macr.): ‘Thailand: Phrae Prov., / 50km NE Phrae, Hua Kaet / Kaet river, 17.-18.11. / 1995,leg.H.Zettel (17b) [p] // [p] // P a r a t y p u s / Rhagovelia / inexpectata sp.n. / des. H. Zettel 1998 [p, red label]’. PARATYPE ( macr.): ‘Thailand: Phetchabun Prov., / Nam Nao NP, Heo Sai / 25.11.1995 / leg. H. Zettel (24) [p] // [p] // P a r a t y p u s / Rhagovelia / inexpectata sp.n. / des. H. Zettel 1998 [p, red label]’. Current status. Valid species The holotype and 3 paratypes are deposited in NMPC: The holotype and 3 paratypes are deposited in NMPC: HOLOTYPE ( apter.): ‘Vohémar / Madagascar [p] // Holotypus [p, red label with black margins] // Rhagovelia / madari n. sp. [hw] / L. Hoberlandt det. [p]’. (The specimen is card-mounted; left antenna, all legs on left side, and fore and middle tibia and tarsus on right side, and pygophore dissected, mounted on four separate slides deposited in Slide Box 3). PARATYPES (2 apter.): ‘Vohémar / Madagascar [p] // Paratypus [p, red label with black margins] // Rhagovelia / madari n. sp. [hw] / L. Hoberlandt det. [p]’. PARATYPE ( apter.): ‘Madagascar / Env. de Rogez [p] // Paratypus [p, red label with black margins] // Rhagovelia / madari n. sp. [hw] / L. Hoberlandt det. [p]’. Current status. Valid species (see HOBERLANDT 1951c). Rhagovelia madagascariensis Hoberlandt, 1941 Rhagovelia madagascariensis Hoberlandt, 1941a: 71–72, Figs 5–6 (original description). PARATYPES (12 apter.): ‘Vohémar / Madagascar [p] // Paratypus [p, red label with black margins] // Rhagovelia / madagascariensis / n. sp. [hw] / L. Hoberlandt det. [p]’. Note. Collection also includes seven slides containing various body parts originating from paratypes (Slide Box 3). Note. Collection also includes seven slides containing various body parts originating from paratypes (Slide Box 3). Current status. Valid species (see HOBERLANDT 1951c, POISSON 1963). Current status. Valid species (see HOBERLANDT 1951c, POISSON 1963) Rhagovelia mindoroensis Zettel, 1994 Rhagovelia mindoroensis Zettel, 1994: 226–227, 229–230, 232–233, Figs 1–5, 9–10, 13, 15 (original description). o paratypes are deposited in NMPC (exchange with Natural History Museum, Vienn PARATYPE ( apter.): ‘PHILIPPINEN: Mindoro or. / Tamaraw Beach, Talipanan / River, W Puerto Galera / leg. H.ZETTEL (19) [p] // 30.11.1992 [p] // [p] // P A R A T Y P U S / Rhagovelia / mindoroensis sp. n. / des. H.Zettel 1993 [p, red label]’. PARATYPE ( apter.): ‘PHILIPPINEN: Mindoro or. / Tamaraw Beach, Talipanan / River, W Puerto Galera / leg. H.ZETTEL (19) [p] // 30.11.1992 [p] // [p] // P A R A T Y P U S / Rhagovelia / mindoroensis sp. n. / des. H.Zettel 1993 [p, red label]’. Current status. Valid species (see ZETTEL 1996, CHEN et al. 2005). Rhagovelia madari Hoberlandt, 1941 g , Rhagovelia madari Hoberlandt, 1941: 69–70, Figs 3–4, 14 (original description). Rhagovelia madari Hoberlandt, 1941: 69–70, Figs 3–4, 14 (original description). Rhagovelia madagascariensis Hoberlandt, 1941 Rhagovelia madagascariensis Hoberlandt, 1941a: 71–72, Figs 5–6 (original description). The holotype and 18 paratypes (including the allotype) are deposited in NMPC: HOLOTYPE ( apter.): ‘Vohémar / Madagascar [p] // Holotypus [p, red label with black margins] // Rhagovelia / mada- gascariensis / n. sp. [hw] / L. Hoberlandt det. [p]’. (The specimen is card-mounted; antenna and all legs on left side missing. Pygophore dissected, male genitalia mounted on four separate slides deposited in Slide Box 3). HOLOTYPE ( apter.): ‘Vohémar / Madagascar [p] // Holotypus [p, red label with black margins] // Rhagovelia / mada- gascariensis / n. sp. [hw] / L. Hoberlandt det. [p]’. (The specimen is card-mounted; antenna and all legs on left side missing. Pygophore dissected, male genitalia mounted on four separate slides deposited in Slide Box 3). HOLOTYPE ( apter.): ‘Vohémar / Madagascar [p] // Holotypus [p, red label with black margins] // Rhagovelia / mada- gascariensis / n. sp. [hw] / L. Hoberlandt det. [p]’. (The specimen is card-mounted; antenna and all legs on left side missing. Pygophore dissected, male genitalia mounted on four separate slides deposited in Slide Box 3). g yg p , g p p ) PARATYPES (5 apter.): ‘Vohémar / Madagascar [p] // Paratypus [p, red label with black margins] // Rhagovelia / madagascariensis / n. sp. [hw] / L. Hoberlandt det. [p]’. PARATYPES (5 apter.): ‘Vohémar / Madagascar [p] // Paratypus [p, red label with black margins] // Rhagovelia / madagascariensis / n. sp. [hw] / L. Hoberlandt det. [p]’. 853 Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 PARATYPE ( apter.): ‘Vohémar / Madagascar [p] // Allotypus [p, red label with black margins] // Rhagovelia / madagascariensis / n. sp. [hw] / L. Hoberlandt det. [p]’. (The specimen is card-mounted; left antenna and all legs on left side dissected and mounted on a separate slide deposited in Slide Box 3). PARATYPE ( apter.): ‘Vohémar / Madagascar [p] // Allotypus [p, red label with black margins] // Rhagovelia / madagascariensis / n. sp. [hw] / L. Hoberlandt det. [p]’. (The specimen is card-mounted; left antenna and all legs on left side dissected and mounted on a separate slide deposited in Slide Box 3). g p p ) PARATYPES (12 apter.): ‘Vohémar / Madagascar [p] // Paratypus [p, red label with black margins] // Rhagovelia / madagascariensis / n. sp. [hw] / L. Hoberlandt det. [p]’. Four paratypes are deposited in NMPC: Four paratypes are deposited in NMPC: PARATYPE ( apter.): ‘Bolivia S.A. / Miguelito / May, 1938 / A. M. Olalla [p] // P A R A T Y P E / Rhagovelia / ornata / John A. Bacon [p, blue label] // Rhagovelia / ornata / Bacon [hw] / Det. H. B. Hungerford [p, white label with black frame submarginally] // [p]’. PARATYPE ( apter.): ‘Bolivia S.A. / Miguelito / May, 1938 / A. M. Olalla [p] // P A R A T Y P E / Rhagovelia / ornata / John A. Bacon [p, blue label] // Rhagovelia / ornata / Bacon [hw] / Det. H. B. Hungerford [p, white label with black frame submarginally] // [p]’. PARATYPE ( apter.): ‘Bolivia S.A. / Miguelito / May, 1938 / A. M. Olalla [p] // P A R A T Y P E / Rhagovelia / ornata / John A. Bacon [p, blue label] // [p]’. PARATYPES (2 apter.): ‘Bolivia S.A. / Miguelito / May, 1938 / A. M. Olalla [p] // P A R A T Y P E / Rhagovelia / ornata / John A. Bacon [p, blue label] // [p]’. Current status. Valid species (see POLHEMUS 1997, HECKMAN 2011, PADILLA-GIL & MOREIRA 2013). Rhagovelia orba Hoberlandt, 1941 Rhagovelia orba Hoberlandt, 1941a: 71 (original description). The holotype and one paratype are deposited in NMPC: HOLOTYPE ( apter.): ‘Vohémar / Madagascar [p] // Holotypus [p, red label with black margins] // Rhagovelia / orba n. sp. [hw] / L. Hoberlandt det. [p]’. (The specimen is card-mounted; right antenna and all legs on left side dissected, mounted on a separate slide stored in Slide Box 3). PARATYPE ( apter.): ‘Vohémar / Madagascar [p] // Paratypus [p, red label with black margins] // Rhagovelia / orba n. sp. [hw] / L. Hoberlandt det. [p]’. Current status. Junior synonym of Rhagovelia pexa Hoberlandt, 1941 (see HOBERLANDT 1951c, POLHEMUS & ANDERSEN 2010). KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. 854 Rhagovelia ornata Bacon, 1948 Rhagovelia ornata Bacon, 1948: 80–81, Fig. 9 (original description). Rhagovelia ornata Bacon, 1948: 80–81, Fig. 9 (original description). Rhagovelia pexa Hoberlandt, 1941 Rhagovelia pexa Hoberlandt, 1941a: 68–69, Figs 1–2, 13 (original description). Rhagovelia pexa Hoberlandt, 1941a: 68–69, Figs 1–2, 13 (original description). The holotype and six paratypes (including the allotype) are deposited in NMPC: HOLOTYPE ( apter.): ‘Vohémar / Madagascar [p] // Holotypus [p, red label with black margins] // Rhagovelia / pexa n. sp. [hw] / L. Hoberlandt det. [p]’. (The specimen is card-mounted; right antenna, middle and hind leg, and pygophore dissected, mounted on four separate slides deposited in Slide Box 3). PARATYPE ( apter.): ‘Vohémar / Madagascar [p] // Paratypus [p, red label with black margins] // Rhagovelia / pexa n. sp. [hw] / L. Hoberlandt det. [p]’. PARATYPE ( apter.): ‘Ambania / Madagascar / Mus Praha // Paratypus [p, red label with black margins] // Rhagovelia / pexa n. sp. [hw] / L. Hoberlandt det. [p]’. PARATYPE ( apter.): ‘Madagascar / Env. de Rogez // Paratypus [p, red label with black margins] // Rhagovelia / pexa n. sp. [hw] / L. Hoberlandt det. [p]’. PARATYPE ( apter.): ‘Vohémar / Madagascar [p] // Allotypus [p, red label with black margins] // Rhagovelia / pexa n. sp. [hw] / L. Hoberlandt det. [p]’. (The specimen is card-mounted; left metatibia and metatarsus missing. Right antenna and all legs on right side dissected, mounted on a separate microscopic slide deposited in Slide Box 3). PARATYPE ( apter.): ‘Vohémar / Madagascar [p] // Allotypus [p, red label with black margins] // Rhagovelia / pexa n. sp. [hw] / L. Hoberlandt det. [p]’. (The specimen is card-mounted; left metatibia and metatarsus missing. Right antenna and all legs on right side dissected, mounted on a separate microscopic slide deposited in Slide Box 3). PARATYPES (2 apter.): ‘Vohémar / Madagascar [p] // Paratypus [p, red label with black margins] // Rhagovelia / pexa n. sp. [hw] / L. Hoberlandt det. [p]’. PARATYPES (2 apter.): ‘Vohémar / Madagascar [p] // Paratypus [p, red label with black margins] // Rhagovelia / pexa n. sp. [hw] / L. Hoberlandt det. [p]’. Note. Collection also includes ten slides with various body parts from paratypes (Slide Box 3). Note. Collection also includes ten slides with various body parts from paratypes (Slide Box 3). tatus. Valid species (see HOBERLANDT 1951c). Current status. Valid species (see HOBERLANDT 1951c). Current status. Valid species (see POLHEMUS & ANDERSEN 2010). Rhagovelia tesari Hoberlandt, 1941 Rhagovelia tesaři Hoberlandt, 1941: 73–75, Figs 9–12, 15 (original description). The holotype and 21 paratypes (including the allotype) are deposited in NMPC: HOLOTYPE ( apter.): ‘Vohémar / Madagascar [p] // Holotypus [p, red label with black margins] // Rhagovelia / tesaři n. sp. [hw] / L. Hoberlandt det. [p] // \| [p]’. (The specimen is card-mounted; left mesotibia and tar- sus missing. Right antenna and all legs on right side, and pygophore dissected, mounted on four separate slides deposited in Slide Box 3). HOLOTYPE ( apter.): ‘Vohémar / Madagascar [p] // Holotypus [p, red label with black margins] // Rhagovelia / tesaři n. sp. [hw] / L. Hoberlandt det. [p] // \| [p]’. (The specimen is card-mounted; left mesotibia and tar- sus missing. Right antenna and all legs on right side, and pygophore dissected, mounted on four separate slides deposited in Slide Box 3). PARATYPES (13 : 2 apter., 11 macr.): ‘Vohémar / Madagascar [p] // Holotypus [p, red label with black margins] // Rhagovelia / tesaři n. sp. [hw] / L. Hoberlandt det. [p] // \| [p]’. PARATYPES (13 : 2 apter., 11 macr.): ‘Vohémar / Madagascar [p] // Holotypus [p, red label with black margins] // Rhagovelia / tesaři n. sp. [hw] / L. Hoberlandt det. [p] // \| [p]’. PARATYPE ( apter.): ‘Vohémar / Madagascar [p] // Allotypus [p, red label with black margins] // Rhagovelia / tesaři n. sp. [hw] / L. Hoberlandt det. [p] // \|\| [p]’. PARATYPES (7 macr.): ‘Vohémar / Madagascar [p] // Paratypus [p, red label with black margins] // Rhagovelia / tesaři n. sp. [hw] / L. Hoberlandt det. [p]’. PARATYPES (7 macr.): ‘Vohémar / Madagascar [p] // Paratypus [p, red label with black margins] // Rhagovelia / tesaři n. sp. [hw] / L. Hoberlandt det. [p]’. Note. Collection also contains eleven slides with various body parts originating from para- types (Slide Box 3). Note. Collection also contains eleven slides with various body parts originating from para- types (Slide Box 3). Current status. Valid species: Rhagovelia tesari tesari Hoberlandt, 1941 (see HOBERLANDT 1951c, POLHEMUS & ANDERSEN 2010). Current status. Valid species: Rhagovelia tesari tesari Hoberlandt, 1941 (see HOBERLANDT 1951c, POLHEMUS & ANDERSEN 2010). Rhagovelia reuteri Hoberlandt, 1951 hagovelia reuteri Hoberlandt, 1951c: 2–4, 10–11, Figs 1–6 (original description, keys to males and fema The holotype and two paratypes (including the allotype) are deposited in NMPC The holotype and two paratypes (including the allotype) are deposited in NMPC: HOLOTYPE ( apter.): ‘Holotypus [p, red label] // Rogez / Madagascar / Mus. Praha [p] / 800 m [hw] // Holotypus [p] / Rhagovelia / reuteri / sp. n. f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw]’. (The specimen is card-mounted; left fore leg missing: right hind leg and pygophore dissected, mounted on two separate slides deposited in Slide Box 3). HOLOTYPE ( apter.): ‘Holotypus [p, red label] // Rogez / Madagascar / Mus. Praha [p] / 800 m [hw] // Holotypus [p] / Rhagovelia / reuteri / sp. n. f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw]’. (The specimen is card-mounted; left fore leg missing: right hind leg and pygophore dissected, mounted on two separate slides deposited in Slide Box 3). HOLOTYPE ( apter.): ‘Holotypus [p, red label] // Rogez / Madagascar / Mus. Praha [p] / 800 m [hw] // Holotypus [p] / Rhagovelia / reuteri / sp. n. f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw]’. (The specimen is card-mounted; left fore leg missing: right hind leg and pygophore dissected, mounted on two separate slides deposited in Slide Box 3). PARATYPE ( apter.): ‘Rogez / Madagascar / Mus. Praha [p] / 800 m [hw] // Allotypus [p, red label] // Allotypus [p] / Rhagovelia / reuteri / sp. n. f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw]’. g p p p PARATYPE ( apter.): ‘Paratypus [p, red label] // Rogez / Madagascar / Mus. Praha [p] / 800 m [hw] // Paratypus [p] / Rhagovelia / reuteri / sp. n. f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw]’. Current status. Valid species (see POLHEMUS & ANDERSEN 2010). 855 Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 Rhagovelia rigovae Zettel, 2012 g g Rhagovelia rigovae Zettel, 2012c: 84, 89, 93, 97–100, Figs 12–14, 34–36, 44–45 (original description). Rhagovelia rigovae Zettel, 2012c: 84, 89, 93, 97–100, Figs 12–14, 34–36, 44–45 (original description). Two paratypes are deposited in NMPC (exchange with Natural History Museum, Vienna): Current status. Valid subspecies (see POISSON 1963). Two paratypes are deposited in NMPC (exchange with Natural History Museum, Vie Two paratypes are deposited in NMPC (exchange with Natural History Museum, Vienna): PARATYPE ( apter.): ‘Philippines,Luzon:Lagunas, / Mt.Banahaw above Kinabu- / hayan, 800 m, creek in degra / ded rainforest, 25.XI. 1995, / J. Kodada & B. Rigová lgt. [p] // [p] // P A R A T Y P U S / Rhagovelia / rigovae sp.n. / des. H. Zettel 2012 [p, red label]’. PARATYPE ( apter.): ‘Philippines,Luzon:Lagunas, / Mt.Banahaw above Kinabu- / hayan, 800 m, creek in degra / ded rainforest, 25.XI. 1995, / J. Kodada & B. Rigová lgt. [p] // [p] // P A R A T Y P U S / Rhagovelia / rigovae sp.n. / des. H. Zettel 2012 [p, red label]’. PARATYPE ( apter.): ‘Philippines,Luzon:Lagunas, / Mt.Banahaw above Kinabu- / hayan, 800 m, creek in degra / ded rainforest, 25.XI. 1995, / J. Kodada & B. Rigová lgt. [p] // [p] // P A R A T Y P U S / Rhagovelia / rigovae sp.n. / des. H. Zettel 2012 [p, red label]’. PARATYPE ( apter.): ‘Philippines,Luzon:Lagunas, / Mt.Banahaw above Kinabu- / hayan, 800 m, creek in degra / ded rainforest, 25.XI. 1995, / J. Kodada & B. Rigová lgt. [p] // [p] // P A R A T Y P U S / Rhagovelia / rigovae sp.n. / des. H. Zettel 2012 [p, red label]’. Current status. Valid species. Rhagovelia tesari ﬂ avomarginata Hoberlandt, 1951 Rhagovelia tesari ﬂ avomarginata Hoberlandt, 1951c: 7–11, Figs 16–23 (original description, keys to males and females). The holotype and 3 paratypes (including the allotype) are deposited in NMPC: HOLOTYPE ( macr.): ‘Rogez / Madagascar / Mus Praha [p] / 800 m [hw] // Holotypus [p, red label] // Holotypus [p] / Rhagovelia / ﬂ avomarginata / sp. n. f. macr. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // [p]’. (The specimen is card-mounted; right hind leg is missing). PARATYPES (2 macr.): ‘Rogez / Madagascar / Mus Praha [p] / 800 m [hw] // Paratypus [p, red label] // Paratypus [p] / Rhagovelia / ﬂ avomarginata / sp. n. f. macr. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. (Both specimens card-mounted; one of them with left hemelytron, all legs on right side, and pygophore dissected, mounted on two separate slides deposited in Slide Box 3). PARATYPES (2 macr.): ‘Rogez / Madagascar / Mus Praha [p] / 800 m [hw] // Paratypus [p, red label] // Paratypus [p] / Rhagovelia / ﬂ avomarginata / sp. n. f. macr. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. (Both specimens card-mounted; one of them with left hemelytron, all legs on right side, and pygophore dissected, mounted on two separate slides deposited in Slide Box 3). PARATYPES (2 macr.): ‘Rogez / Madagascar / Mus Praha [p] / 800 m [hw] // Paratypus [p, red label] // Paratypus [p] / Rhagovelia / ﬂ avomarginata / sp. n. f. macr. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. (Both specimens card-mounted; one of them with left hemelytron, all legs on right side, and pygophore dissected, mounted on two separate slides deposited in Slide Box 3). PARATYPE ( macr.): ‘Rogez / Madagascar / Mus Praha [p] / 800 m [hw] // Allotypus [p, red label] // Allotypus [p] / Rha- govelia / ﬂ avomarginata / sp. n. f. macr. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // \|\|\|\| [p]’. PARATYPE ( macr.): ‘Rogez / Madagascar / Mus Praha [p] / 800 m [hw] // Allotypus [p, red label] // Allotypus [p] / Rha- govelia / ﬂ avomarginata / sp. n. f. macr. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // \|\|\|\| [p]’. Current status. Valid subspecies (see POISSON 1963). KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. 856 Paravelia bullialata J. T. Polhemus & D. A. Polhemus, 1984 Paravelia bullialata J. T. Polhemus & D. A. Polhemus, 1984 Paravelia bullialata J. T. Polhemus & D. A. Polhemus, 1984 Paravelia bullialata Polhemus & Polhemus, 1984: 342−343, 348−349, Figs 4, 5a (original description) Paravelia bullialata Polhemus & Polhemus, 1984: 342−343, 348−349, Figs 4, 5a (original descript Four paratypes are deposited in NMPC: Four paratypes are deposited in NMPC: PARATYPES (2 macr.): ‘SURINAME / N. Nieser [p] // 1st Trib. Colakreek / 8-IX-´69 SN[p] 118 A [hw] / 55°14′ 5°27′ [p] // PARATYPE / Paravelia / bullialata / J. & D. Polhemus [p, pink label] // [p]’. PARATYPES (2 macr.): ‘SURINAME / N. Nieser [p] // 1st Trib. Colakreek / 8-IX-´69 SN[p] 118 A [hw] / 55°14′ 5°27′ [p] // PARATYPE / Paravelia / bullialata / J. & D. Polhemus [p, pink label] // [p]’. PARATYPES (2 macr.): ‘SURINAME / N. Nieser [p] // 1st Trib. Colakreek / 8-IX-´69 SN[p] 118 A [hw] / 55°14′ PARATYPES (2 macr.): ‘SURINAME / N. Nieser [p] // 1st Trib. Colakreek / 8-IX-´69 SN[p] 118 A [hw] / 55°14′ 5°27′ [p] // PARATYPE / Paravelia / bullialata / J. & D. Polhemus [p, pink label] // [p]’. Current status. Valid species (see HECKMAN 2011, MOREIRA et al. 2011). Rhagovelia trepida Bacon, 1948: 84–85, Fig. 4 (original description). Rhagovelia trepida Bacon, 1948: 84–85, Fig. 4 (original description). Four paratypes are deposited in NMPC: Four paratypes are deposited in NMPC: PARATYPES (3 apter.): ‘Sao Paulo Braz. [p] / XI. II. [hw] 193[p]5 [hw] / Nat. Sci Mus. / Via A. M. Olalla // P A R A T Y P E / Rhagovelia / trepida / John A. Bacon [p, blue label] // [p]’. PARATYPES (3 apter.): ‘Sao Paulo Braz. [p] / XI. II. [hw] 193[p]5 [hw] / Nat. Sci Mus. / Via A. M. Olalla // P A R A T Y P E / Rhagovelia / trepida / John A. Bacon [p, blue label] // [p]’. PARATYPE ( apter.): ‘Sao Paulo Braz. [p] / XI. II. [hw] 193[p]5 [hw] / Nat. Sci Mus. / Via A. M. Olalla // P A R A T Y P E / Rhagovelia / trepida / John A. Bacon [p, blue label] // Rhagovelia / trepida / Bacon [hw] / Det H B Hungerford [p, white label with black frame submarginally] // [p]’. PARATYPE ( apter.): ‘Sao Paulo Braz. [p] / XI. II. [hw] 193[p]5 [hw] / Nat. Sci Mus. / Via A. M. Olalla // P A R A T Y P E / Rhagovelia / trepida / John A. Bacon [p, blue label] // Rhagovelia / trepida / Bacon [hw] / Det H B Hungerford [p, white label with black frame submarginally] // [p]’. PARATYPE ( apter.): ‘Sao Paulo Braz. [p] / XI. II. [hw] 193[p]5 [hw] / Nat. Sci Mus. / Via A. M. Olalla // P A R A T Y P E / Rhagovelia / trepida / John A. Bacon [p, blue label] // Rhagovelia / trepida / Bacon [hw] / Det H B Hungerford [p, white label with black frame submarginally] // [p]’. Current status. Valid species (see POLHEMUS 1997, HECKMAN 2011, MOREIRA et al. 2011, PADILLA-GIL & MOREIRA 2013). Current status. Valid species (see POLHEMUS 1997, HECKMAN 2011, MOREIRA et al. 2011, PADILLA-GIL & MOREIRA 2013). Current status. Valid species (see POLHEMUS 1997, HECKMAN 2011, MOREIRA et al. 2011, PADILLA-GIL & MOREIRA 2013). Angilia (Angilia) aeterna Hoberlandt, 1946 Angilia (Angilia) aeterna Hoberlandt, 1946: 55–58, Figs 1a–b, 2a–e (original description). Angilia (Angilia) aeterna Hoberlandt, 1946: 55–58, Figs 1a–b, 2a–e (original description). Four paratypes are deposited in NMPC: Four paratypes are deposited in NMPC: HOLOTYPE ( macr.): ‘Maradi, Nigérie / Saha[r]a mer. / X 1936, Škulina [p] // Holotypus [p, red label] // Angilia / aeterna n. sp. / [hw] / L. Hoberlandt det. [p]’. (Holotype card-mounted; left antennal segments II–IV, protarsus, middle leg, metatarsus and hemelytron, and right antennal segments III–IV, apical protarsomeres, middle and hind leg, and pygophore dissected, mounted on six separate slides stored in Slide Box 2). Current status. Valid species (see LINNAVUORI 1971). Paravelia bullialata J. T. Polhemus & D. A. Polhemus, 1984 Paravelia bullialata Polhemus & Polhemus, 1984: 342−343, 348−349, Figs 4, 5a (original description Rhagovelia trepida Bacon, 1948 Rhagovelia trepida Bacon, 1948: 84–85, Fig. 4 (original description). Velia ﬁ lippii anatolica Tamanini, 1951 [p] // [p] // PA VELIA / FILIPPII ANATOLICA / Tamanini, 1951 / labelled: P. KMENT 2013 [p, red label]’. PARATYPE ( macr.): ‘Sultan dağ [hw] / Anat. [p] 4.IX.47 2000 m [hw] / Exp. N. Mus. ČSR. [p] // Velia [hw] / 19[p]48 nervosa Horv. [hw] / L. Hoberlandt det. [p] [hw] // PARATYPUS / VELIA / FILIPPII ANATOLICA / Tamanini, 1951 / labelled: P. KMENT 2013 [p, red label]’. PARATYPE ( macr.): ‘Bürücek, Toros / Anat. 29-31 7. 47 / Exp. N. Mus. ČSR. [p] // Allotypus [p] / Velia Filippii / T. / subsp. anatolica / nov. subsp. [hw] / DET- L. TAMANINI 19 [p, red label] // Mus. Nat. Pragae / Inv [p] 1279 [hw, orange label]’. PARATYPE ( macr.): ‘Bürücek, Toros / Anat. 29-31 7. 47 / Exp. N. Mus. ČSR. [p] // PARATYPUS [p] / Velia Filip- pii. T. / sbsp. anatolica / n. sbsp. [hw] / DET- L. TAMANINI 19 [p, red label] // Mus. Nat. Pragae / Inv [p] 1280 [hw, orange label]’. PARATYPES (13 macr.): ‘Bürücek, Toros / Anat. 29-31 7. 47 / Exp. N. Mus. ČSR. [p] // Paratypus [p] [hw, red label] // Mus. Nat. Pragae / Inv [p] 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293 [respectively; hw, orange label] // PARATYPUS / VELIA / FILIPPII ANATOLICA / Tamanini, 1951 / labelled: P. KMENT 2013 [p, red label]’. PARATYPES (2 macr.): ‘Bürücek, Toros / Anat. 29-31 7. 47 / Exp. N. Mus. ČSR. [p] // Velia [hw] / 19[p]48 nervosa Horv. [hw] / L. Hoberlandt det. [p] [hw] // PARATYPUS / VELIA / FILIPPII ANATOLICA / Tamanini, 1951 / labelled: P. KMENT 2013 [p, red label]’. p PARATYPE ( macr.): ‘Sultan dağ [hw] / Anat. [p] 4.IX.47 2000 m [hw] / Exp. N. Mus. ČSR. [p] // Velia [hw] / 19[p]48 nervosa Horv. [hw] / L. Hoberlandt det. [p] [hw] // PARATYPUS / VELIA / FILIPPII ANATOLICA / Tamanini, 1951 / labelled: P. KMENT 2013 [p, red label]’. PARATYPE ( macr.): ‘Sultan dağ [hw] / Anat. [p] 4.IX.47 2000 m [hw] / Exp. N. Mus. ČSR. [p] // [p] // PARATY- PUS / VELIA / FILIPPII ANATOLICA / Tamanini, 1951 / labelled: P. KMENT 2013 [p, red label]’. PARATYPE ( macr.): ‘Sultan dağ, 2000 m [hw] / Anat. [p] 4.IX.47 [hw] / Exp. N. Velia ﬁ lippii anatolica Tamanini, 1951 Mus. ČSR. [p] // [p] // PARA- TYPUS / VELIA / FILIPPII ANATOLICA / Tamanini, 1951 / labelled: P. KMENT 2013 [p, red label]’. Current status. Junior synonym of Velia (Plesiovelia) afﬁ nis afﬁ nis Kolenati, 1857 (see TAMANINI 1952 1953; ANDERSEN 1995a) Current status. Junior synonym of Velia (Plesiovelia) afﬁ nis afﬁ nis Kolenati, 1857 (see TAMANINI 1952, 1953; ANDERSEN 1995a). Velia ﬁ lippii anatolica Tamanini, 1951 Velia ﬁ lippii anatolica Tamanini, 1951a: 1–3, Figs B, D–F, L, N–Q (original description). Velia ﬁ lippii anatolica Tamanini, 1951a: 1–3, Figs B, D–F, L, N–Q (original description). The holotype and 93 paratypes (including the allotype) are deposited in NMPC: HOLOTYPE ( macr.): ‘Bürücek, Toros / Anat. 29-31 7 47 / Exp. N Mus. ČSR. [p] // V. anatolica / holot. - / Toros .29. VII. 47. [hw, card with attached microslides containing dissected male genitalia] // Holotypus [p] / Velia Filippii. T. / subsp. anatolica / n. subsp. [hw] / DET- L. TAMANINI 19 [p, red label] // Mus. Nat. Pragae / Inv [p] 1216 [hw, orange label]’. PARATYPE (): ‘Bürücek, Toros / Anat. 29-31 7. 47 / Exp. N. Mus. ČSR. [p] // PARATYPUS [p] / Velia Filippii. T. / sbsp. / anatolica / n. sbsp. [hw] / DET- L. TAMANINI 19 [p, red label] // Mus. Nat. Pragae / Inv [p] 1220 [hw, orange label]’. 857 Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 PARATYPES (31 macr.): ‘Bürücek, Toros / Anat. 29-31 7. 47 / Exp. N. Mus. ČSR. [p] // Paratypus [p, red label] // Mus. Nat. Pragae / Inv [p] 1217, 1218, 1219, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244, 1245, 1246, 1247, 1248 [respectively; hw, orange label] // [p] // PARATYPUS / VELIA / FILIPPII ANATOLICA / Tamanini, 1951 / labelled: P. KMENT 2013 [p, red label]’. PARATYPES (29 macr.): ‘Bürücek, Toros / Anat. 29-31 7. 47 / Exp. N. Mus. ČSR. [p] // Paratypus [p] [hw, red label] // Mus. Nat. Pragae / Inv [p] 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277 [respectively; hw, orange label] // PARATYPUS / VELIA / FILIPPII ANATOLICA / Tamanini, 1951 / labelled: P. KMENT 2013 [p, red label]’. PARATYPE ( macr.): ‘Bürücek, Toros / Anat. 29-31 7. 47 / Exp. N. Mus. ČSR. [p] // Mus. Nat. Pragae / Inv [p] 1278 [hw, orange label] // [p] // PARATYPUS / VELIA / FILIPPII ANATOLICA / Tamanini, 1951 / labelled: P. KMENT 2013 [p, red label]’. PARATYPES (10 macr.): ‘Bürücek, Toros / Anat. 29-31 7. 47 / Exp. N. Mus. ČSR. Velia afﬁ nis var. obscura Tamanini, 1953 Velia afﬁ nis var. obscura Tamanini, 1953: 140–141 (original description). Three paratypes are deposited in NMPC: Velia gridellii Tamanini, 1947 g , Velia gridellii Tamanini, 1947: 23, 25, 28, 30–31, 35, 41, 52, 54–56, Figs 33–34, 75–78, 100 (original description, key). T d i d i NMPC Velia gridellii Tamanini, 1947: 23, 25, 28, 30–31, 35, 41, 52, 54–56, Figs 33–34, 75–78, 100 (original description, key). Two paratypes are deposited in NMPC: PARATYPE ( apter.): ‘TRENTINO / Val Lagarina [p] / Bordala 8.9.46 [hw] // Velia / Gridelii Tam. [hw] / paratypus [hw, red ink] / Det. Tamanini L. [hw]’. PARATYPE ( apter.): ‘TRENTINO / Val Lagarina [p] / Bordala 8.9.46 [hw] // Velia / Gridelii Tam. [hw] / paratypus [hw, red ink] / Det. Tamanini L. [hw]’. PARATYPE ( apter.): ‘TRENTINO / Val Lagarina [p] / Bordala 8.9.46 [hw] // Velia / Gridelii Tam. [hw] / paratypus [hw, red ink] / Det. Tamanini L. [hw]’. PARATYPE ( apter.): ‘TRENTINO / Val Lagarina [p] / Bordala 8.9.46 [hw] // Velia / Gridelii Tam. [hw] / paratypus [hw, red ink] / Det. Tamanini L. [hw]’. Current status. Valid species: Velia (Plesiovelia) gridellii Tamanini, 1947 (see ANDERSEN 1995a). Current status. Valid species: Velia (Plesiovelia) gridellii Tamanini, 1947 (see ANDERSEN 1995a). Velia hoberlandti Tamanini, 1951 Velia hoberlandti Tamanini, 1951b: 5–10, Figs 2E–I, 3A–C, 4I–N (original description). Velia hoberlandti Tamanini, 1951b: 5–10, Figs 2E–I, 3A–C, 4I–N (original description). The holotype and three paratypes (including the allotype) are deposited in NMPC: HOLOTYPE ( macr.): ‘Is. Baleares [hw] / Palma. [p] // Velia / Hoberlandti / Palma. [hw; card with attached microslides] // Coll. L. Salvator [p] // Holotypus [p] / Velia / Hoberlandti / n. sp. [hw] / DET- L. TAMANINI 19[p]49 [hw, red label]’. (The specimen is card-mounted, right middle leg missing; genitalia dissected, mounted on microslides attached to the same pin). HOLOTYPE ( macr.): ‘Is. Baleares [hw] / Palma. [p] // Velia / Hoberlandti / Palma. [hw; card with attached microslides] // Coll. L. Salvator [p] // Holotypus [p] / Velia / Hoberlandti / n. sp. [hw] / DET- L. TAMANINI 19[p]49 [hw, red label]’. (The specimen is card-mounted, right middle leg missing; genitalia dissected, mounted on microslides attached to the same pin). PARATYPE ( macr.): ‘Is. Baleares [hw] / Alfabia. [p] // Coll. L. Salvator [p] // Allotypus [p] / Velia / Hoberlandti / n. sp. [hw] / DET- L. TAMANINI 19[p]49 [hw, red label]’. PARATYPE ( macr.): ‘Is. Baleares [hw] / Inea. [p] // Coll. L. Salvator [p] // PARATYPUS [p] / Velia / Hoberlandti / n. sp. [hw] / DET- L. TAMANINI [p] 49 [hw, red label]’. PARATYPE ( apter.): ‘Is. Baleares [hw] / Es Prat / de S. Jordi. [p] // Coll. L. Salvator [p] // PARATYPUS [p] / Velia / Hoberlandti / n. sp. [hw] / DET- L. TAMANINI [p] 49 [hw, red label]’. PARATYPE ( apter.): ‘Is. Baleares [hw] / Es Prat / de S. Jordi. [p] // Coll. L. Salvator [p] // PARATYPUS [p] / Velia / Hoberlandti / n. sp. [hw] / DET- L. TAMANINI [p] 49 [hw, red label]’. Current status. Valid species: Velia (Plesiovelia) hoberlandti Tamanini, 1951 (see ANDERSEN 1995a). Current status. Valid species: Velia (Plesiovelia) hoberlandti Tamanini, 1951 (see ANDERSEN 1995a). Three paratypes are deposited in NMPC: PARATYPE ( macr.): ‘Jerusalem, Palestine [hw, pencil, illegible] // [p] // Paratypus [p] / Velia afﬁ nis / Kol. / f. obscura f n. [hw] / DET.. L. TAMANINI 195[p]3 [hw, red label]’. PARATYPE ( apter.): ‘Jerusalem / Palestine [p in blue] 26/II. [hw] / Houška 94[p in blue]6 [hw] // Velia [hw] 19[p]51 ﬁ lippii Tam [hw] / L. Hoberlandt det. [p] [hw] // Paratypus [p] / Velia afﬁ nis / f. obscura f. n. [hw] / DET.. L. 858 KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. TAMANINI 195[p] 3 [hw, red label]’. PARATYPE ( apter.): ‘Jerusalem / Palestine [p in blue] 26/II. [hw] / Houška 94[p in blue]6 [hw] // Paratypus [p] / Velia afﬁ nis Kol. / f. obscura f. n. [hw] / DET.. L. TAMANINI 195[p] 3 [hw, red label]’. Current status. As the name was intentionally proposed merely for the dark coloured variety (TAMANINI 1953) it is an unavailable name according to ICZN (1999) (see ANDERSEN 1995a). Current status. As the name was intentionally proposed merely for the dark coloured variety (TAMANINI 1953) it is an unavailable name according to ICZN (1999) (see ANDERSEN 1995a). One syntype is deposited in NMPC: One syntype is deposited in NMPC: SYNTYPE ( apter.): ‘La Chiffah / Algérie [p, blue label with black frame submarginally] // M. Noualhier. [p] // [p] // Velia / Noualhieri / Put. [hw] // SYNTYPUS / VELIA / NOUALHIERI / Puton, 1889 / labelled: P. KMENT 2013 [p, red label]’. SYNTYPE ( apter.): ‘La Chiffah / Algérie [p, blue label with black frame submarginally] // M. Noualhier. [p] // [p] // Velia / Noualhieri / Put. [hw] // SYNTYPUS / VELIA / NOUALHIERI / Puton, 1889 / labelled: P. KMENT 2013 [p, red label]’. Current status. Valid species: Velia (Plesiovelia) noualhieri noualhieri Puton, 1889 (see TAMANINI 1968, ANDERSEN 1995a). Current status. Valid species: Velia (Plesiovelia) noualhieri noualhieri Puton, 1889 (see TAMANINI 1968, ANDERSEN 1995a). Velia mancinii Tamanini, 1947 Velia mancinii Tamanini, 1947: 25, 27–28, 30–31, 53, 57, 63, 66–69, Figs 47–49, 107–108, 113–114, 133, 136 (original description, key). he holotype and three paratypes (including the allotype) are deposited in NMPC: PARATYPE (): ‘Kresnensko def. / 24. 6. 38 Bulg. Maced. / lgt. L. Hoberlandt. [p] // Collectio / L. Hoberlandt [p] // Velia [hw] / 19[p]41 rivulorum Fab. / apt. [hw] / L. Hoberlandt det. [p] [hw] // Velia / Mancinii [hw] / paratypus. [hw, red ink] Tam. [hw] / DET. TAMANINI L. [p] // Mus. Nat. Pragae / Inv. [p] 1215 [hw, orange label]’. Current status. Valid species: Velia (Plesiovelia) mancinii mancinii Tamanini, 1947 (see TAMANINI 1955, ANDERSEN 1995a, KANYUKOVA 2006). 859 Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 Velia obenbergeri cresia Hoberlandt, 1941 Velia obenbergeri cresia Hoberlandt, 1941b: 163 (original description). Velia obenbergeri cresia Hoberlandt, 1941b: 163 (original description). The holotype is deposited in NMPC: Velia noualhieri Puton, 1889 Velia noualhieri Puton, 1889 Velia noualhieri Puton, 1889: 307−308 (original description). Velia noualhieri Puton, 1889: 307−308 (original description). Velia obenbergeri Hoberlandt, 1941 1938 / Táborský [hw] // Allotypus [p, red label with black margins] // Velia [hw] / 19[p]41 / obenbergeri n sp. [hw] / L. Hoberlandt det. [p] [hw]’. PARATYPES (5 apter.): ‘Insula / Gaudos [p] // 15. V. 1938 / Táborský [hw] // Paratypus [p, red label with black margins] // Velia [hw] / 19[p]41 / obenbergeri n sp. [hw] / L. Hoberlandt det. [p] [hw]’. PARATYPES (5 apter.): ‘Insula / Gaudos [p] // 15. V. 1938 / Táborský [hw] // Paratypus [p, red label with black margins] // Velia [hw] / 19[p]41 / obenbergeri n sp. [hw] / L. Hoberlandt det. [p] [hw]’. Note. Collection also includes seven additional slides containing various body parts origi- nating from paratypes, and one slide containing head with antennae, and one fore, middle and hind leg, indicated as originating from the holotype, evidently by mistake; the slides are deposited in Slide Box 4. Current status. Junior synonym of Velia (Plesiovelia) rhadamantha rhadamantha Hoberlandt, 1941 (see TAMANINI 1947, ANDERSEN 1995a). Velia obenbergeri Hoberlandt, 1941 Velia obenbergeri Hoberlandt, 1941b: 162–163, Figs 6, 19–20 (original description). The holotype and 10 paratypes (including the allotype) are deposited in NMPC: HOLOTYPE ( apter.): ‘Insula / Gaudos [p] // 15. V. 1938 / Táborský [hw] // Holotypus [p, red label with black margins] // Velia [hw] / 19[p]41 / obenbergeri n sp. [hw] / L. Hoberlandt det. [p] [hw]’. (The specimen is card-mounted; right antenna, all legs on right side, and pygophore dissected, mounted on two separate slides in Slide Box 4). ) PARATYPES (3 apter.): ‘Insula / Gaudos [p] // 15. V. 1938 / Táborský [hw] // Paratypus [p, red label with black margins] // Velia [hw] / 19[p]41 / obenbergeri n sp. [hw] / L. Hoberlandt det. [p] [hw]’. ) PARATYPES (3 apter.): ‘Insula / Gaudos [p] // 15. V. 1938 / Táborský [hw] // Paratypus [p, red label with black margins] // Velia [hw] / 19[p]41 / obenbergeri n sp. [hw] / L. Hoberlandt det. [p] [hw]’. PARATYPES (3 apter.): ‘Insula / Gaudos [p] // 15. V. 1938 / Táborský [hw] // Paratypus [p, red label with black margins] // Velia [hw] / 19[p]41 / obenbergeri n sp. [hw] / L. Hoberlandt det. [p] [hw]’. PARATYPE ( apter.): ‘Insula / Gaudos [p] // 15. V. 1938 / Dr. K. Táborský [hw] // Paratypus [p, red label with black margins] // Collectio / L. Hoberlandt [p] // Velia [hw] / 19[p]41 / obenbergeri n sp. [hw] / L. Hoberlandt det. [p]’. PARATYPE ( apter.): ‘Insula / Gaudos [p] // 15. V. 1938 / Dr. K. Táborský [hw] // Paratypus [p, red label with black margins] // Collectio / L. Hoberlandt [p] // Velia [hw] / 19[p]41 / obenbergeri n sp. [hw] / L. Hoberlandt det. [p]’. PARATYPE ( apter.): ‘Insula / Gaudos [p] // 15. V. 1938 / Dr. K. Táborský [hw] // Paratypus [p, red label with black margins] // Collectio / L. Hoberlandt [p] // Velia [hw] / 19[p]41 / obenbergeri n sp. [hw] / L. Hoberlandt det. [p]’. PARATYPE ( apter.): ‘Insula / Gaudos [p] // 15. V. 1938 / Táborský [hw] // Allotypus [p, red label with black margins] // Velia [hw] / 19[p]41 / obenbergeri n sp. [hw] / L. Hoberlandt det. [p] [hw]’. PARATYPE ( apter.): ‘Insula / Gaudos [p] // 15. V. The holotype is deposited in NMPC: HOLOTYPE ( apter.): ‘Knossos, Creta / Mařan et Štěp. / 1934 Coll. Bartoň [p] // Holotypus [p, red label with black margins] // Velia [hw] 19[p]41 obenbergeri / cresia n. [hw] / L. Hoberlandt det. [p] // Velia [hw] [hw, red ink] / rhadamantha [hw] / immatura [hw, i in black ink, rest in red ink] Hoberl. [hw] / DET. TAMANINI L. [p]’. (The specimen is card-mounted, immature; left mesotibia, mesotarsus and hind leg, and apical segments of both antennae missing). Current status. Junior synonym of Velia (Plesiovelia) rhadamantha rhadamantha Hoberlandt, 1941 (see TAMANINI 1947, ANDERSEN 1995a). KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. 860 Velia pueblana Drake, 1951 Velia pueblana Drake, 1951c: 376−378 (original description). Velia pueblana Drake, 1951c: 376−378 (original description). Two paratypes are deposited in NMPC: PARATYPE ( apter.): ‘Tampico, Mexico / July 16, 1950 / Drake & Hottes [p] // PARATYPE / By C. J. Drake [p] / Velia / bueblana [sic!] / Drake [hw, red label] // [p]’. PARATYPE ( apter.): ‘Valles, Mexico / July 17, 1950 / Drake & Hottes [p] // PARATYPE / By C. J. Drake [p] / Velia / pueblana / Drake [hw, red label] // [p] // Velia / pueblana / Drake [hw, white label with black frame submarginally]‘. Current status. Valid species: Stridulivelia (Aenictovelia) pueblana (Drake, 1951) (DRAKE & MENKE 1962; POLHEMUS 1976, 1979). Current status. Valid species: Velia (Plesiovelia) pelagonensis Hoberlandt, 1941 (see ANDERSEN 1995a). ANDERSEN 1995a). Velia pelagonensis Hoberlandt, 1941b: 163–164, Figs 7, 21–23 (original description). Velia pelagonensis Hoberlandt, 1941b: 163–164, Figs 7, 21–23 (original description). Velia rhadamantha Hoberlandt, 1941 Velia rhadamantha Hoberlandt, 1941b: 160–162, Figs 4–5, 16–18 (original description). Velia rhadamantha Hoberlandt, 1941b: 160–162, Figs 4–5, 16–18 (original description). The holotype and 7 paratypes (including the allotype) are deposited in NMPC: holotype and 7 paratypes (including the allotype) are deposited in NMPC: HOLOTYPE ( macr.): ‘Knossos, Creta / Mařan et Štěp. / 1934. Coll Bartoň [p] // Holotypus [p, red label with black margins] // Velia [hw] / 19[p]41 rhadamantha / n. sp. [hw] / L. Hoberlandt det. [p]’ (see Figs 1−2). Š PARATYPE ( macr.): ‘Knossos, Creta / Mařan et Štěp. / 1934. Coll Bartoň [p] // Paratypus [p, red label with black margins] // Velia [hw] / 19[p]41 rhadamantha / n. sp. [hw] / L. Hoberlandt det. [p]’. (Male genitalia dissected, glued on a separate piece of card attached to the same pin). PARATYPE ( macr.): ‘Nida - Ida montes / Creta, V, 1934 / Coll Bartoň [p] // Paratypus [p, red label with upper and bottom margins black] // Velia [hw] / 19[p]41 rhadamantha / n. sp. [hw] / L. Hoberlandt det. [p] [hw]’. (The specimen is card-mounted, left antenna, all legs on left side, and pygophore are dissceted, mounted in three separate slides stored in Slide Box 4). p ) PARATYPE ( macr.): ‘Knossos, Creta / Mařan et Štěp. / 1934 Coll Bartoň [p] // Allotypus [p, red label margins] // Velia [hw] / 19[p]41 rhadamantha / L. Hoberlandt det. [p] / n. sp. [hw]’. Š PARATYPE ( macr.): ‘Knossos, Creta / Mařan et Štěp. / 1934. Coll Bartoň [p] // Paratypus [p, red label with black margins] // Collectio / L. Hoberlandt [p] // Velia [hw] / 19[p]41 rhadamantha / n. sp. [hw] / L. Hoberlandt det. [p] [hw]’. PARATYPE ( macr.): ‘Knossos, Creta / Mařan et Štěp. / 1934. Coll Bartoň [p] // Paratypus [p, red label with black margins] // Collectio / L. Hoberlandt [p] // Velia [hw] / 19[p]41 rhadamantha / n. sp. [hw] / L. Hoberlandt det. [p] [hw]’. [p] [ ] PARATYPES (3 macr.): ‘Knossos, Creta / Mařan et Štěp. / 1934. Coll Bartoň [p] // Paratypus [p, red label with black margins] // Velia [hw] / 19[p]41 rhadamantha / n. sp. [hw] / L. Hoberlandt det. [p] [hw]’. PARATYPES (3 macr.): ‘Knossos, Creta / Mařan et Štěp. / 1934. The holotype and one paratype is deposited in NMPC: The holotype and one paratype is deposited in NMPC: HOLOTYPE ( apter.): ‘Kresnensko def. / 26. 6. 38 Bulg. Maced. / lgt. L. Hoberlandt. [p] // Collectio / L. Hoberlandt [p] // Holotypus [p, red label with black margins] // Velia [hw] / 19[p]41 pelagonensis / n. sp. [hw] / L. Hober- landt det. [p]’. (The specimen is card-mounted; right antenna, all legs on right side, and pygophore dissected, mounted on four separate slides deposited in Slide Box 4). HOLOTYPE ( apter.): ‘Kresnensko def. / 26. 6. 38 Bulg. Maced. / lgt. L. Hoberlandt. [p] // Collectio / L. Hoberlandt [p] // Holotypus [p, red label with black margins] // Velia [hw] / 19[p]41 pelagonensis / n. sp. [hw] / L. Hober- landt det. [p]’. (The specimen is card-mounted; right antenna, all legs on right side, and pygophore dissected, mounted on four separate slides deposited in Slide Box 4). PARATYPE ( apter.): ‘Issari, Pelopon. / Mařan et Štěp. / 1935 Coll. Bartoň [p] // Paratypus [p, red label with black margins] // Velia / pelagonensis n. sp. / [hw] / L. Hoberlandt det. [p]’. PARATYPE ( apter.): ‘Issari, Pelopon. / Mařan et Štěp. / 1935 Coll. Bartoň [p] // Paratypus [p, red label with black margins] // Velia / pelagonensis n. sp. / [hw] / L. Hoberlandt det. [p]’. Current status. Valid species: Velia (Plesiovelia) pelagonensis Hoberlandt, 1941 (see ANDERSEN 1995a). Brachymetra anduze Drake & Harris, 1942 Brachymetra anduze Drake & Harris, 1942: 95−96 (original description); RUHOFF (1968): 30 (list of taxa described by Drake); ARISTIZÁBAL (2002): 127, 136 (diagnosis). Brachymetra anduzeei (incorrect subsequent spelling): HARRIS & DRAKE (1945): 212 (list); DRAKE & ROZE (1954): 227 (distribution); DRAKE (1957): 128 (differential diagnosis). Brachymetra anduzei (incorrect subsequent spelling): MATSUDA (1960): 236–237, 240, 524–525, Fig. 538 (morpho- logy, variability); HECKMAN (2011): 132 (key). Brachymetra anduze Drake & Harris, 1942: 95−96 (original description); RUHOFF (1968): 30 (list of taxa described by Drake); ARISTIZÁBAL (2002): 127, 136 (diagnosis). Brachymetra anduzeei (incorrect subsequent spelling): HARRIS & DRAKE (1945): 212 (list); DRAKE & ROZE (1954): 227 (distribution); DRAKE (1957): 128 (differential diagnosis). Brachymetra anduzei (incorrect subsequent spelling): MATSUDA (1960): 236–237, 240, 524–525, Fig. 538 (morpho- logy, variability); HECKMAN (2011): 132 (key). by Drake); ARISTIZÁBAL (2002): 127, 136 (diagnosis). Brachymetra anduzeei (incorrect subsequent spelling): HARRIS & DRAKE (1945): 212 (list); DRAKE & ROZE (1954): 227 (distribution); DRAKE (1957): 128 (differential diagnosis). Brachymetra anduzei (incorrect subsequent spelling): MATSUDA (1960): 236–237, 240, 524–525, Fig. 538 (morpho- logy, variability); HECKMAN (2011): 132 (key). 227 (distribution); DRAKE (1957): 128 (differential diagnosis). Brachymetra anduzei (incorrect subsequent spelling): MATSUDA (1960): 236–237, 240, 524–525, Fig. 538 (morpho- logy, variability); HECKMAN (2011): 132 (key). Velia tersa Drake & Harris, 1941: 338−339 (original description). PARATYPE ( apter.): ‘Trinidad. B. W. I. / Oct. 27-29, 1938 / Carl J. Drake [p] // [p] // PARATYPE [p] / Velia / tersa / D & H [hw, red label]’. PARATYPES (2 apter.): ‘Trinidad. B. W. I. / Oct. 27-29, 1938 / Carl J. Drake [p] // [p] // PARATYPE [p] / Velia / tersa / D & H [hw, red label]’. Current status. Valid species: Stridulivelia (Stridulivelia) tersa (Drake & Harris, 1941) (see DRAKE & MENKE 1962; POLHEMUS 1976, 1979; POLHEMUS & SPANGLER 1995; HECKMAN 2011; MOREIRA et al. 2011). Family GERRIDAE Leach, 1815 Subfamily Charmatometrinae Matsuda, 1960 Family GERRIDAE Leach, 1815 Subfamily Charmatometrinae Matsuda, 1960 Family GERRIDAE Leach, 1815 Subfamily Charmatometrinae Matsuda, 1960 Velia saulii var. serbica Tamanini, 1951 Velia saulii Tam. var. serbica Tamanini, 1951b: 2–3, Figs 1A–B (original description). Velia tersa Drake & Harris, 1941 Velia tersa Drake & Harris, 1941: 338−339 (original description). The holotype is deposited in NMPC: The holotype is deposited in NMPC: HOLOTYPE ( apter.): ‘Peristeri / Serbia mer. / Dr. Purkyně [p] // Holotypus [p] / Velia / Saulii Tam. / v. serbica / nov. var. [hw] / DET- L. TAMANINI 19[p]49 [hw, red label]. Current status. Valid species: Velia (Plesiovelia) serbica Tamanini, 1951; upgraded to subspecies rank by TAMANINI (1955, 1959) and to species rank by TAMANINI (1970) (see also ANDERSEN 1995a). Velia rhadamantha Hoberlandt, 1941 Coll Bartoň [p] // Paratypus [p, red label with black margins] // Velia [hw] / 19[p]41 rhadamantha / n. sp. [hw] / L. Hoberlandt det. [p] [hw]’. Note. Collection also includes three separate slides containing head with antennae, all legs and male genitalia, originally indicated as originating from the holotype, clearly in mistake (Slide Box 4). Note. Collection also includes three separate slides containing head with antennae, all legs and male genitalia, originally indicated as originating from the holotype, clearly in mistake (Slide Box 4). Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 861 Current status. Valid species: Velia (Plesiovelia) rhadamantha rhadamantha Hoberlandt, 1941 (see TAMANINI 1947, 1965; ANDERSEN 1995a). Current status. Valid species: Velia (Plesiovelia) rhadamantha rhadamantha Hoberlandt, 1941 (see TAMANINI 1947, 1965; ANDERSEN 1995a). Remark. The status of the single male paratype from Nida-Ida Mts. is controversial. HOBER- LANDT (1941b) did not explicitely mention the specimen as paratype in the original description, as he did in case of paratypes from Knossos. However, the original labels of the specimen and the slides of its dissected body parts clearly state Hoberlandt’s intention to include it as paratype, and absence of the statement is merely an omission in the original text. Velia saulii var. serbica Tamanini, 1951 Velia saulii Tam. var. serbica Tamanini, 1951b: 2–3, Figs 1A–B (original description). One paratype is deposited in NMPC: PARATYPE ( apter.): ‘Los Lechozos [hw] D. F. [p] / Venezuela [p] 3-13-38 [hw] / G. Vivas Berthier [p] // [p] // Brachymetra / anduzeii [hw] / PARATYPE [p, red label]’. (Male genitalia dissected, glued on a separate triangular piece of card attached to the same pin). PARATYPE ( apter.): ‘Los Lechozos [hw] D. F. [p] / Venezuela [p] 3-13-38 [hw] / G. Vivas Berthier [p] // [p] // Brachymetra / anduzeii [hw] / PARATYPE [p, red label]’. (Male genitalia dissected, glued on a separate triangular piece of card attached to the same pin). 862 KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. Cu e s us. V d spec es (see 00 , 0 ). Remark. DRAKE & HARRIS (1942) dedicated the newly described species to Dr. Pablo J. Anduze, mentioning the name three times as Brachymetra anduze, which is therefore the only original spelling. The species epithet, anduze, thus represents a noun in apposition, which contradicts the Recommendation 31A of the ICZN (1999) which however does not make the name unavailable. Subsequently, only RUHOFF (1968) and ARISTIZÁBAL (2002) listed the name as B. anduze. HARRIS & DRAKE (1945), DRAKE & ROZE (1954), and DRAKE (1957) listed the name as B. anduzeei, while MATSUDA (1960) and HECKMAN (2011) used the name as B. anduzei, but neither of them commented on the change of the spelling or mentioned the original spelling at all so their actions cannot be regarded as emendations. As an incorrect transliteration or latinization of the name are not considered inadvertent errors by ICZN (1999: 32.5.1), Brachymetra anduze must be accepted as the correct original spelling, B. anduzeei and B. anduzei being incorrect subsequent spellings. Subfamily Eotrechinae Matsuda, 1960 Amemboa (Amemboa) aquafrigida Zettel & Chen, 1997 Amemboa (Amemboa) aquafrigida Zettel & Chen, 1997 Amemboa (Amemboa) aquafrigida Zettel & Chen, 1997: 94–97, 100, Figs 2, 5–6, 9 (original description). Amemboa (Amemboa) aquafrigida Zettel & Chen, 1997: 94–97, 100, Figs 2, 5–6, 9 (original descripti our paratypes are deposited in NMPC (exchange with Natural History Museum, Vie Four paratypes are deposited in NMPC (exchange with Natural History Museum, Vienna): PARATYPE ( apter.): ‘Thailand: Petchabun / Nam Nao NP, Prom Laeng / 22.III.1994 / leg. W.D.Shepard (1040) [p] // [p] // P a r a t y p u s / Amemboa (s.str.) / aquafrigida sp.n. / des. Zettel & Chen 1996 [p, red label]’. PARATYPE ( apter.): ‘Thailand:Phetchabun Prov. / Nam Nao NP, Huai Phrom / Laeng. 24.11.1995 / leg. H. Zettel (22) [p] // [p] // P a r a t y p u s / Amemboa (s.str.) / aquafrigida sp.n. / des. Zettel & Chen 1996 [p, red label]’. PARATYPE ( apter.): ‘Thailand:Phetchabun Prov. / Nam Nao NP, Huai Phrom / Laeng. 24.11.1995 / leg. H. Zettel (22) [p] // [p] // P a r a t y p u s / Amemboa (s.str.) / aquafrigida sp.n. / des. Zettel & Chen 1996 [p, red label]’. PARATYPE ( apter.): ‘Thailand: Petchabun / Nam Nao NP, Huai Ya / Krue, 14.III.1994 / leg. W.D.Shepard (1039) [p] // [p] // P a r a t y p u s / Amemboa (s.str.) / aquafrigida sp.n. / des. Zettel & Chen 1996 [p, red label]’. ARATYPE ( apter.): ‘Thailand: Petchabun / Nam Nao NP, Prom Laeng / 22.III.1994 / leg. W.D.Shepard (1 // [p] // P a r a t y p u s / Amemboa (s.str.) / aquafrigida sp.n. / des. Zettel & Chen 1996 [p, red label] Current status. Valid species. Gerris (Gerris) caucasicus Kanyukova, 1982 s (Gerris) caucasicus Kanyukova, 1982: 66, 72, 74, 88–90, 92, Figs 29, 82–85 (original description, key PARATYPE ( macr.): ‘Кумбаши, мοрце / Ленкоран. у. [p] 23.IV.[hw] / Кириченко 909 [p] [= Kumbashi, mortse, Lenkoran. u., Kiritshenko, 23.iv.1909] // Paratypus [p, red label] // Gerris / caucasicus Kan. / Kanyukova det. [hw] // [p] // PARATYPUS / GERRIS / CAUCASICUS / Kanyukova, 1982, labelled: P. KMENT 2013 [p, red label]’. PARATYPE ( macr.): ‘Кумбаши, мοрце / Ленкоран. у. [p] 23.IV.[hw] / Кириченко 909 [p] [= Kumbashi, mortse, Lenkoran. u., Kiritshenko, 23.iv.1909] // Paratypus [p, red label] // Gerris / caucasicus Kan. / Kanyukova det. [hw] // [p] // PARATYPUS / GERRIS / CAUCASICUS / Kanyukova, 1982, labelled: P. KMENT 2013 [p, red label]’. PARATYPE ( macr.): ‘Кумбаши, мοрце / Ленкоран. у. [p] 23.IV.[hw] / Кириченко 909 [p] [= Kumbashi, mortse, Lenkoran. u., Kiritshenko, 23.iv.1909] // Paratypus [p, red label] // Gerris / caucasicus Kan. / Kanyukova det. [hw] // [p] // PARATYPUS / GERRIS / CAUCASICUS / Kanyukova, 1982, labelled: P. KMENT 2013 [p, red label]’. PARATYPE ( macr.): ‘Кумбаши, мοрце / Ленкоран. у. / Кириченко [p] 23.IV.[hw] 909 [p] [= Kumbashi, mortse, Lenkoran. u., Kiritshenko, 23.iv.1909] // Paratypus [p, red label] [p] // PARATYPUS / GERRIS / CAUCASICUS / Kanyukova, 1982, labelled: P. KMENT 2013 [p, red label]’. PARATYPE ( macr.): ‘Кумбаши, мοрце / Ленкоран. у. / Кириченко [p] 23.IV.[hw] 909 [p] [= Kumbashi, mortse, Lenkoran. u., Kiritshenko, 23.iv.1909] // Paratypus [p, red label] [p] // PARATYPUS / GERRIS / CAUCASICUS / Kanyukova, 1982, labelled: P. KMENT 2013 [p, red label]’. Current status. Valid species (see ANDERSEN 1994, 1995a,b; KANYUKOVA 2006; FENT et al. 2011; AUKEMA et al. 2013; DAMGAARD et al. in press). 863 Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 Gerris (Gerris) comatus Drake & Hottes, 1925: 48–49 (original description). Gerris (Gerris) comatus Drake & Hottes, 1925: 48–49 (original description). One paratype is deposited in NMPC: PARATYPE ( macr.): ‘Ames. Iowa [p] / X-18 [hw] 192[p]4 [hw] / C. [p] J. [hw] Drake [p] // H G Barber / Colln 1950 [p] // PARATYPE [p] / Gerris / comatus / D + H [hw, red label] // Gerris / comatus / D. & H. [hw, white label with black frame submarginally] // [p]’. One paratype is deposited in NMPC: One paratype is deposited in NMPC: PARATYPE ( macr.): ‘Ames. Iowa [p] / X-18 [hw] 192[p]4 [hw] / C. [p] J. [hw] Drake [p] // H G Barber / Colln 1950 [p] // PARATYPE [p] / Gerris / comatus / D + H [hw, red label] // Gerris / comatus / D. & H. [hw, white label with black frame submarginally] // [p]’. PARATYPE ( macr.): ‘Ames. Iowa [p] / X-18 [hw] 192[p]4 [hw] / C. [p] J. [hw] Drake [p] // H G Barber / Colln 1950 [p] // PARATYPE [p] / Gerris / comatus / D + H [hw, red label] // Gerris / comatus / D. & H. [hw, white label with black frame submarginally] // [p]’. Current status. Valid species: Gerris (Gerris) comatus Drake & Hottes, 1925 (see SMITH 1988b; ANDERSEN 1994, 1995b; DAMGAARD et al. in press). Current status. Valid species: Gerris (Gerris) comatus Drake & Hottes, 1925 (see SMITH 1988b; ANDERSEN 1994, 1995b; DAMGAARD et al. in press). Two paratypes are deposited in NMPC: PARATYPE ( apter.): ‘Алексеевка Лен- / кор. р-н, Талыш [p] / 13 [hw] / Вельтищев [p] IX [hw] 938 [p] [= Alek- seevka Lenkor. r-n, Talysh, 13.xi.1938, Vel’tishchev lgt.] // Г. Hacияку. [hw] [= G. Nasiyaku.] // [p] // Paratypus [p] Gerris (G.) / kiritshenkoi / Kanyukova E. [hw, red label]’. PARATYPE ( apter.): ‘Алексеевка Лен- / кор. р-н, Талыш [p] / 13 [hw] / Вельтищев [p] IX [hw] 938 [p] [= Alek- seevka Lenkor. r-n, Talysh, 13.xi.1938, Vel’tishchev lgt.] // Г. Hacияку. [hw] [= G. Nasiyaku.] // [p] // Paratypus [p] Gerris (G.) / kiritshenkoi / Kanyukova E. [hw, red label]’. Current status. Valid species: Gerris (Gerriselloides) kiritshenkoi Kanyukova, 1979 (see ANDERSEN 1994, 1995a,b; KANYUKOVA 2006; DAMGAARD et al. in press). Gerris dissortis Drake & Harris, 1930: 145−146 (original description). Gerris dissortis Drake & Harris, 1930: 145−146 (original description). Gerris dissortis Drake & Harris, 1930: 145−146 (original description). One paratype is deposited in NMPC: PARATYPE ( macr.): ‘Jack River [hw] Ont. [p] / 21. v. [hw] 1927 / G. S. Walley [p] // PARATYPE [p] / Gerris / dissortis / D. & H. [hw, red label] // [p]’. Current status. Valid species: Limnoporus dissortis (Drake & Harris, 1930) (see SMITH 1988b, ANDERSEN & SPENCE 1992, ANDERSEN 1995b, DAMGAARD et al. in press). Gerris kiritshenkoi Kanyukova, 1979 Gerris kiritshenkoi Kanyukova, 1979: 51–53, Figs 1–9 (original description). Two paratypes are deposited in NMPC: Gerris paludum palmonii Wagner, 1954 ris paludum palmonii Wagner, 1954: 205–207, Figs 1–3 (upper row), 4, 6, 8, 10, 13 (original description) Gerris paludum palmonii Wagner, 1954: 205–207, Figs 1–3 (upper row), 4, 6, 8, 10, 13 (original description). Two paratypes are deposited in NMPC: Limnogonus (Limnogonus) hungerfordi Andersen, 1975 Limnogonus (Limnogonus) hungerfordi Andersen, 1975: 28–30, 44–47, Figs 115, 120–122 (original description, key). One paratype is deposited in NMPC: PARATYPE ( macr.): ‘E.DUTCH NEW GUINEA: / Jutefa Bay.Pim. / Sealevel-100 ft. ii.1936. / L.E.Cheesman. / B.M.1936-271. [p] // PARATYPE / Limnogonus / hungerfordi Andersen [p] / [hw] 19[p]70[hw] / Det. N. Møller Andersen [p]’. Current status. Valid species (see ANDERSEN 1995b, ANDERSEN & WEIR 2004, CHEN et al. 2005, DAMGAARD et al. in press). Two paratypes are deposited in NMPC: PARATYPE ( brach.): ‘Tabgha 24.8.42 / (Genezareth Sea) [hw] / Palestina / J. Houška lg. [p] // Gerris (Aquarius) / [hw] 19[p]51 paludum Fab. [hw] / L. Hoberlandt det. [p] [hw] // PARATYPOID [p] / G. paludum / palmonii n. sbsp. [hw] / F. Wagner det. [p, pink label]’. PARATYPE ( brach.): ‘Tabgha 24.8.42 / (Genezareth sea) [hw] / Palestina / J. Houška lg. [p] // Gerris (Aquarius) / [hw] 19[p]51 paludum Fab. [hw] / L. Hoberlandt det. [p] [hw] // PARATYPOID [p] / G. paludum / palmonii PARATYPE ( brach.): ‘Tabgha 24.8.42 / (Genezareth Sea) [hw] / Palestina / J. Houška lg. [p] // Gerris (Aquarius) / [hw] 19[p]51 paludum Fab. [hw] / L. Hoberlandt det. [p] [hw] // PARATYPOID [p] / G. paludum / palmonii n. sbsp. [hw] / F. Wagner det. [p, pink label]’. PARATYPE ( brach.): ‘Tabgha 24.8.42 / (Genezareth sea) [hw] / Palestina / J. Houška lg. [p] // Gerris (Aquarius) / [hw] 19[p]51 paludum Fab. [hw] / L. Hoberlandt det. [p] [hw] // PARATYPOID [p] / G. paludum / palmonii n. sbsp. [hw] / F. Wagner det. [p, pink label]’. Current status. Junior synonym of Aquarius paludum paludum (Fabricius, 1794) (see POISSON 1965b; KANYUKOVA 1982; ANDERSEN 1990, 1995a,b; DAMGAARD et al. in press). Current status. Junior synonym of Aquarius paludum paludum (Fabricius, 1794) (see POISSON 1965b; KANYUKOVA 1982; ANDERSEN 1990, 1995a,b; DAMGAARD et al. in press). KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. 864 One paratype is deposited in NMPC: One paratype is deposited in NMPC: PARATYPE ( apter.): ‘Vitilevu / Fiji [p] // Vatuthere, Nr. / Nandarivatu [p] / IX.8-38 2200′ [hw] // Stream [p] // ECZimmerman / Collector [p] // [hw, yellow label] // PARATYPE / Limnogonus / buxtoni Esaki ssp. / ﬁ jiensis Andersen [p] / [hw] 19[p]70[hw] / Det. N. Møller Andersen [p]’. Current status. Valid subspecies (see ANDERSEN 1995b, DAMGAARD et al. in press). Limnogonus (Limnogonus) buxtoni ﬁ jiensis Andersen, 1975 Limnogonus (Limnogonus) buxtoni ﬁ jiensis Andersen, 1975: 29–30, 38, 49, 56–59, Figs 128–129, 164–165, 167, 178–179 (original description, key). Tenagogonus madagascariensis Hoberlandt, 1947 [p] / 46 [hw] // Mus. Nat. Pragae / Inv. [p] 1358, 1359, 1362, 1364, 1365, 1366, 1367, 1369, 1372, 1375, 1399 [respectively; hw, orange label]’. [ p y g ] PARATYPES (3 apter.): ‘Madagascar / Inv. č. [p] // Paratypus [p, red label with black upper and bottom margin] // Tenagogonus / madagasca- [hw] / 19[p]46 [hw] / riensis n. sp. [hw] / L. Hoberlandt det. [p] // Mus. Nat. Pragae / Inv. [p] 1381, 1385, 1386 [respectively; hw, orange label]’. PARATYPE ( apter.): ‘Madagascar / Muš [sic!] Pragense [p] // Paratypus [p, red label with black upper and bottom margin] // Tenagogonus / madagascariensis / n. sp. [hw] / L. Hoberlandt det. [p] / 46 [hw] // Mus. Nat. Pragae / Inv. [p] 1363 [hw, orange label]’. PARATYPES (9 apter.): ’Vohémar / Madagascar [p] // Paratypus [p, red label with black upper and bottom margin] // Tenagogonus / madagascariensis / n. sp. [hw] / L. Hoberlandt det. [p] / 46 [hw] // Mus. Nat. Pragae / Inv. [p] 1368, 1370, 1371, 1374, 1376, 1377, 1378, 1379, 1383 [respectively; hw, orange label]’. PARATYPES (9 apter.): ‘Vohémar / Madagascar [p] // Paratypus [p, red label with black upper and bottom margin] // Tenagogonus / madagasca- [hw] / 19[p]46 [hw] / riensis n. sp. [hw] / L. Hoberlandt det. [p] // Mus. Nat. Pragae / Inv. [p] 1380, 1382, 1387, 1388, 1389, 1390, 1391, 1392, 1393 [respectively; hw, orange label]’. PARATYPE ( apter.): ‘Vohémar / Madagascar [p] // Paratypus [p, red label with black upper and bottom margin] // Tenagogonus / madagascarien [hw] / 19[p]46 / sis n. sp. [hw] / L. Hoberlandt det. [p] // Mus. Nat. Pragae / Inv. [p] 1384 [hw, orange label]’. PARATYPE ( apter.): ‘Vohémar / Madagascar [p] // Paratypus [p, red label with black upper and bottom margin] // Tenagogonus / madagascariensis / n. sp. [hw] / L. Hoberlandt det. [p] 1946 [hw] // Mus. Nat. Pragae / Inv. [p] 1395 [hw, orange label]’. PARATYPE ( apter.): ‘Vohémar / Madagascar [p] // Paratypus [p, red label with black upper and bottom margin] // Tenagogonus / madagascariensis / n. sp. [hw] / L. Hoberlandt det. [p] [hw] // Mus. Nat. Pragae / Inv. [p] 1398 [hw, orange label]’. Tenagogonus madagascariensis Hoberlandt, 1947 Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 865 PARATYPES (4 apter.): ‘Vohémar / Madagascar [p] // Paratypus [p, red label with black upper and bottom margin] // Tenagogonus / madagascariensis / n. sp. [hw] / L. Hoberlandt det. [p] /1946 [hw] // Mus. Nat. Pragae / Inv. [p] 1330, 1333, 1336, 1337 [respectively; hw, orange label]’. PARATYPES (4 apter.): ‘Vohémar / Madagascar [p] // Paratypus [p, red label with black upper and bottom margin] // Tenagogonus / madagascariensis / n. sp. [hw] / L. Hoberlandt det. [p] /1946 [hw] // Mus. Nat. Pragae / Inv. [p] 1330, 1333, 1336, 1337 [respectively; hw, orange label]’. PARATYPES (6 apter.): ‘Ambanja / Madagascar [p] // Paratypus [p, red label with black upper and bottom margin] // Tenagogonus / madagascariensis / n. sp. [hw] / L. Hoberlandt det. [p] 1946 [hw] // Mus. Nat. Pragae / Inv. [p] 1348, 1349, 1350, 1351, 1353, 1354 [respectively; hw, orange label]’. PARATYPES (6 apter.): ‘Ambanja / Madagascar [p] // Paratypus [p, red label with black upper and bottom margin] // Tenagogonus / madagascariensis / n. sp. [hw] / L. Hoberlandt det. [p] 1946 [hw] // Mus. Nat. Pragae / Inv. [p] 1348, 1349, 1350, 1351, 1353, 1354 [respectively; hw, orange label]’. PARATYPE ( apter.): ‘Madagascar [p] // Paratypus [p, red label with black upper and bottom margin] // Tenagogonus / madagascariensis / n. sp. [hw] / L. Hoberlandt det. [p] 1946 [hw] // Mus. Nat. Pragae / Inv. [p] 1342 [hw, orange label]’. (Male genitalia dissected, mounted on two pointed pieces of card attached to the same pin). PARATYPE ( apter.): ‘Madagascar / Inv, č, [p] // Allotypus [p, red label with black upper and bottom margin] // Tenagogonus / madagascariensis / n. sp. [hw] / L. Hoberlandt det. [p] [hw] // Mus. Nat. Pragae / Inv. [p] 1356 [hw, orange label]’. PARATYPES (4 apter.): ‘Madagascar / Inv, č, [p] // Paratypus [p, red label with black upper and bottom margin] // Tenagogonus / madagascariensis / n. sp. [hw] / L. Hoberlandt det. [p] 1946 [hw] // Mus. Nat. Pragae / Inv. [p] 1357, 1360, 1361, 1396 [respectively; hw, orange label]’. PARATYPES (11 apter.): ‘Madagascar / Inv, č, [p] // Paratypus [p, red label with black upper and bottom margin] // Tenagogonus / madagascariensis / n. sp. [hw] / L. Hoberlandt det. Tenagogonus madagascariensis Hoberlandt, 1947 Tenagogonus madagascariensis Hoberlandt, 1947: 105–112, 3 unpaginated plates with 14 ﬁ gures without consecutive numbering (original description). The holotype and 73 paratypes (including the allotype) are deposited in NMPC: HOLOTYPE ( apter.): ‘Madagascar / Inv, č, [p] // Holotypus [p, red label with black upper and bottom margin] // Tenagogonus / madagascariensis n. sp. [hw, blue ink] / L. Hoberlandt det. [p] [hw, blue ink] // Mus. Nat. Pragae / Inv. [p] 1322 [hw, orange label]’. PARATYPES (2 apter.): ‘Madagascar / Inv, č, [p] // Paratypus [p, red label with black upper and bottom margin] // Tenagogonus / madagascariensis / n. sp. [hw, blue ink] / L. Hoberlandt det. [p] // Mus. Nat. Pragae / Inv. [p] 1324, 1347 [respectively; hw, orange label]’. PARATYPES (6 apter.): ‘Madagascar / Inv, č, [p] // Paratypus [p, red label with black upper and bottom margin] // Tenagogonus / madagascariensis / n. sp. [hw] / L. Hoberlandt det. [p] / 1946 [hw] // Mus. Nat. Pragae / Inv. [p] 1327, 1328, 1331, 1334, 1338, 1339 [respectively; hw, orange label]’. PARATYPE ( apter.): ‘Madagascar [p] // Paratypus [p, red label with black upper and bottom margin] // Tenagogonus / madagascariensis / n. sp. [hw] / L. Hoberlandt det. [p] /1946 [hw] // Mus. Nat. Pragae / Inv. [p] 1332 [hw, orange label]’. PARATYPE ( apter.): ‘Madagascar / Inv, č, [p] Vohémar [hw] // Paratypus [p, red label with black upper and bottom margin] // Tenagogonus / madagascariensis / n. sp. [hw] / L. Hoberlandt det. [p] // Mus. Nat. Pragae / Inv. [p] 1346 [hw, orange label]’. PARATYPES (4 apter.): ‘Vohémar / Madagascar [p] // Paratypus [p, red label with black upper and bottom margin] // Tenagogonus / madagascariensis / n. sp. [hw] / L. Hoberlandt det. [p] // Mus. Nat. Pragae / Inv. [p] 1325, 1343, 1344, 1345 [respectively; hw, orange label]’. PARATYPES (4 apter.): ‘Vohémar / Madagascar [p] // Paratypus [p, red label with black upper and bottom margin] // Tenagogonus / madagascariensis / n. sp. [hw] / L. Hoberlandt det. [p] 1946 [hw] // Mus. Nat. Pragae / Inv. [p] 1323, 1326, 1335, 1340, [respectively; hw, orange label]’. Tenagogonus madagascariensis Hoberlandt, 1947 PARATYPE ( apter.): ‘Ambanja / Madagascar [p] // Paratypus [p, red label with black upper and bottom margin] // Tenagogonus / madagascariensis / n. sp. [hw] / L. Hoberlandt det. [p] / 46 [hw] // Mus. Nat. Pragae / Inv. [p] 1401 [hw, orange label]’. PARATYPE ( apter.): ‘Ambanja / Madagascar [p] // Paratypus [p, red label with black upper and bottom margin] // Tenagogonus / madagascariensis / n. sp. [hw] / L. Hoberlandt det. [p] [hw] // Mus. Nat. Pragae / Inv. [p] 1402 [hw, orange label]’. g PARATYPE ( apter.): ‘Madagascar / Mus. Pragense [p] // Paratypus [p, red label with black upper and bottom margin] // Tenagogonus / 19[p]46 madagasca- / riensis n. sp. [hw] / L. Hoberlandt det. [p] // Mus. Nat. Pragae / Inv. [p] 1400 [hw, orange label]’. Current status. Valid species (see POISSON 1965b, ANDERSEN 1995b, DAMGAARD et al. in press). KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. 866 Eurymetra angolensis Hoberlandt, 1951a: 10–14, Figs 6–12 (original description). Seven paratypes are deposited in NMPC: PARATYPE ( apter.): ‘16.I.48 Kamassangu [hw] / – Dundo, Angola / A. B. Machado lgt [p] // Angola [p] No. 333.4 [hw] / A. B. Machado lgt. [p] // Paratypus [p, red label] // Paratypus [p] / Eurymetra / angolensis / sp. n. f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 13 331 [hw, orange label]’. PARATYPE ( apter.): ‘16.I.48 Kamassangu [hw] / – Dundo, Angola / A. B. Machado lgt [p] // Angola [p] No. 333.4 [hw] / A. B. Machado lgt. [p] // Paratypus [p, red label] // Paratypus [p] / Eurymetra / angolensis / sp. n. f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 13 331 [hw, orange label]’. PARATYPE ( apter.): ‘16.I.48 Kamassangu [hw] / – Dundo, Angola / A. B. Machado lgt [p] // Angola [p] No. 333.4 [hw] / A. B. Machado lgt. [p] // Paratypus [p, red label] // Paratypus [p] / Eurymetra / angolensis / sp. n. f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 13 331 [hw, orange label]’. PARATYPE ( apter.): ‘16.I.48 Kamassangu [hw] / – Dundo, Angola / A. B. Machado lgt [p] // Ang. [p] No. 333.4 [hw] / coll.A.B.Machado [p] // Paratypus [p, red label] // Paratypus [p] / Eurymetra / angolensis / sp. n. f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 13 330 [hw, orange label]’. PARATYPE ( apter.): ‘16.I.48 Kamassangu [hw] / – Dundo, Angola / A. B. Machado lgt [p] // Ang. [p] No. 333.4 [hw] / coll.A.B.Machado [p] // Paratypus [p, red label] // Paratypus [p] / Eurymetra / angolensis / sp. n. f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 13 330 [hw, orange label]’. p g p g PARATYPE ( macr.): ‘2.II.48 Tshikapa [hw] / Dundo, Angola / A. B. Machado lg. [p] // Ang. [p] No. 354.8 [hw] / coll.A.B.Machado [p] // Paratypus [p, red label] // Paratypus [p] / Eurymetra / angolensis / sp. n. f. macr. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // Mus. Nat. Pragae / Inv. Seventeen syntypes are deposited in NMPC: Seventeen syntypes are deposited in NMPC: SYNTYPE ( apter.): ‘Ambanja / Madagascar [p] // TYPUS [p, red label with black frame submarginally] // Tenag. mad. [hw] / 19[p]46[hw] / f. fusca n. [hw] / L. Hoberlandt det. [p] // Mus. Nat. Pragae / Inv. [p] 1414 [hw, orange label]’. SYNTYPE ( apter.): ‘Ambanja / Madagascar [p] // TYPUS [p, red label with black frame submarginally] // Tenag. mad. [hw] / 19[p]46[hw] / f. fusca n. [hw] / L. Hoberlandt det. [p] // Mus. Nat. Pragae / Inv. [p] 1414 [hw, orange label]’. SYNTYPE ( apter.): ‘Ambanja / Madagascar [p] // TYPUS [p, red label with black frame submarginally] // Tenag. mad. [hw] / 19[p]46[hw] / f. fusca n. [hw] / L. Hoberlandt det. [p] // Mus. Nat. Pragae / Inv. [p] 1414 [hw, orange label]’. g SYNTYPES (8 apter.): ‘Madagascar / Inv. č. [p] // TYPUS [p, red label with black frame submarginally] // Tenag. mad. [hw] / 19[p]46[hw] / f. fusca n. [hw] / L. Hoberlandt det. [p] // Mus. Nat. Pragae / Inv. [p] 1405, 1406, 1407, 1409, 1411, 1413, 1416, 1418 [respectively; hw, orange label]’. SYNTYPES (8 apter.): ‘Madagascar / Inv. č. [p] // TYPUS [p, red label with black frame submarginally] // Tenag. mad. [hw] / 19[p]46[hw] / f. fusca n. [hw] / L. Hoberlandt det. [p] // Mus. Nat. Pragae / Inv. [p] 1405, 1406, 1407, 1409, 1411, 1413, 1416, 1418 [respectively; hw, orange label]’. SYNTYPES (8 apter.): ‘Madagascar / Inv. č. [p] // TYPUS [p, red label with black frame submarginally] // Tenag. mad. [hw] / 19[p]46[hw] / f. fusca n. [hw] / L. Hoberlandt det. [p] // Mus. Nat. Pragae / Inv. [p] 1405, 1406, 1407, 1409, 1411, 1413, 1416, 1418 [respectively; hw, orange label]’. p y g SYNTYPES (8 apter.): ‘Vohémar / Madagascar [p] // TYPUS [p, red label with black frame submarginally] // Tenag. mad. [hw] / 19[p]46[hw] / f. fusca n. [hw] / L. Hoberlandt det. [p] // Mus. Nat. Pragae / Inv. [p] 1408, 1410, 1412, 1415, 1417, 1419, 1420, 1421 [respectively; hw, orange label]’. SYNTYPES (8 apter.): ‘Vohémar / Madagascar [p] // TYPUS [p, red label with black frame submarginally] // Tenag. mad. [hw] / 19[p]46[hw] / f. fusca n. [hw] / L. Hoberlandt det. [p] // Mus. Nat. Pragae / Inv. Gerris kahli Drake & Harris, 1934 Gerris kahli Drake & Harris, 1934: 185, 199–200 (original description, key). One paratype is deposited in NMPC: One paratype is deposited in NMPC: PARATYPE ( apter.): ‘Elvecia near / Mt. Turumquire, / Sucre, Venez. / G. Netting. [p] // Jan. [p] 22 [hw] / 19[p]30[hw] // Carn. Mus. / Acc. [p] 8696 [hw] // [p] // PARATYPE [p] / Gerris / kahli / D & H [hw, red label]’. One paratype is deposited in NMPC: One paratype is deposited in NMPC: PARATYPE ( apter.): ‘Elvecia near / Mt. Turumquire, / Sucre, Venez. / G. Netting. [p] // Jan. [p] 22 [hw] / 19[p]30[hw] // Carn. Mus. / Acc. [p] 8696 [hw] // [p] // PARATYPE [p] / Gerris / kahli / D & H [hw, red label]’. p yp p PARATYPE ( apter.): ‘Elvecia near / Mt. Turumquire, / Sucre, Venez. / G. Netting. [p] // Jan. [p] 22 [hw] / 19[p]30[hw] // Carn. Mus. / Acc. [p] 8696 [hw] // [p] // PARATYPE [p] / Gerris / kahli / D & H [hw, red label]’. Current status. Junior synonym of Eurygerris fuscinervis Berg, 1898 (see MORALES-CASTAÑO & MOLANO-RENDÓN 2010, DAMGAARD et al. in press). Current status. Junior synonym of Eurygerris fuscinervis Berg, 1898 (see MORALES-CASTAÑO & MOLANO-RENDÓN 2010, DAMGAARD et al. in press). Seventeen syntypes are deposited in NMPC: [p] 1408, 1410, 1412, 1415, 1417, 1419, 1420, 1421 [respectively; hw, orange label]’. Current status. Unavailable name. It is clear from the original description that Hoberlandt (1947) proposed this name merely for the colour variant occurring sympatrically with the typical form and therefore there is no doubt he meant an infraspeciﬁ c entity within Tenago- gonus madagascariensis Hoberlandt, 1947. Tenagogonus madagascariensis f. fusca Hoberlandt, 1947 Tenagogonus madagascariensis forma fusca Hoberlandt, 1947: 108 (original description). Seventeen syntypes are deposited in NMPC: Eurymetra angolensis Hoberlandt, 1951 Eurymetra angolensis Hoberlandt, 1951a: 10–14, Figs 6–12 (original description). Eurymetra angolensis Hoberlandt, 1951a: 10–14, Figs 6–12 (original description). [p] 13 336 [hw, orange label]’. PARATYPE ( macr.): ‘2.II.48 Tshikapa [hw] / Dundo, Angola / A. B. Machado lg. [p] // Ang. [p] No. 354.8 [hw] / coll.A.B.Machado [p] // Paratypus [p, red label] // Paratypus [p] / Eurymetra / angolensis / sp. n. f. macr. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 13 336 [hw, orange label]’. g g PARATYPES (2 apter.): ‘16.I.48 Kamassangu [hw] / – Dundo, Angola / A. B. Machado lgt. // Angola [p] No. 333.4 [hw] / A B Machado lgt // Paratypus [p, red label] // Paratypus [p] / Eurymetra / angolensis / sp. n. f. apt. PARATYPES (2 apter.): ‘16.I.48 Kamassangu [hw] / – Dundo, Angola / A. B. Machado lgt. // Angola [p] No. 333.4 [hw] / A B Machado lgt // Paratypus [p, red label] // Paratypus [p] / Eurymetra / angolensis / sp. n. f. apt. Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 867 [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 13 333, 13 335 [respectively; hw, orange label]’. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 13 333, 13 335 [respectively; hw, orange label]’. PARATYPES (2 apter.): ‘16.I.48 Kamassangu [hw] / – Dundo, Angola / A. B. Machado lgt // Ang. [p] No. 333.4 [hw] / coll.A.B.Machado [p] // Paratypus [p, red label] // Paratypus [p] / Eurymetra / angolensis / sp. n. f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 13 332, 13 334 [respectively; hw, orange label]’. PARATYPES (2 apter.): ‘16.I.48 Kamassangu [hw] / – Dundo, Angola / A. B. Machado lgt // Ang. [p] No. 333.4 [hw] / coll.A.B.Machado [p] // Paratypus [p, red label] // Paratypus [p] / Eurymetra / angolensis / sp. n. f. apt. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 13 332, 13 334 [respectively; hw, orange label]’. Note. Collection also includes three separate slides containing dissected legs and male geni- talia originating from an additional paratype (Slide Box 4). Note. Collection also includes three separate slides containing dissected legs and male geni- talia originating from an additional paratype (Slide Box 4). Current status. Metrocoris nieseri Chen & Zettel, 1999 Metrocoris nieseri Chen & Zettel, 1999: 14–19, 29–31, Figs 1–3, 5–9, 12, 14, 30–31, 35 (original descrip One paratype is deposited in NMPC (exchange with Natural History Museum, Vienn PARATYPE ( apter.): ‘Thailand: Chiang Mai / Doi Sutep NP, Wang / Bua Boon, 24.III.1994 / leg. W.D.Shepard(1042) [p] // paratype [hw, red label] // [p] // Metrocoris nieseri / Chen & Zettel [p]’. Current status. Valid species. Eurymetra angolensis Hoberlandt, 1951a: 10–14, Figs 6–12 (original description). Valid subspecies: Eurymetra natalensis angolensis (see POISSON 1965b; LINNAVUORI 1971, 1981). Eurymetra manengolensis Hoberlandt, 1952 Eurymetra manengolensis Hoberlandt, 1952 Eurymetra manengolensis Hoberlandt, 1952b: 1–3, Figs 1–5 (original description). Eurymetra manengolensis Hoberlandt, 1952b: 1–3, Figs 1–5 (original description). The holotype is deposited in NMPC: HOLOTYPE ( apter.): ‘Manengole / 800 m, IV. 49 / Cameroun / lgt. Tesárek [hw] // Holotypus [p, red label] // Holo- typus [p] / Eurymetra / manengolensis n. sp. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. Current status. Valid species: Eurymetropsiella manengolensis (Hoberlandt, 1952) (see POISSON 1965b, LINNAVUORI 1981). Potamometra macrokosos Drake & Hoberlandt, 1965 Potamometra macrokosos Drake & Hoberlandt, 1965: 304, 306, 308–309, Figs 2b, 3e–f (original description, key). One paratype is deposited in NMPC: PARATYPE ( apter.): ‘Suisape, 1000 M. / Lichuan Distr. / W. Hupeh, China / VIII-[p] 6 [hw] -48 [p] // Gressitt & / Djou Collrs. // [p] // Paratype [p] / Potamometra / macrocola [sic!] / Drake [hw, red label]’. Current status. Valid species (see CHEN & ANDERSEN 1993, ANDERSEN 1995). Rheumatogonus intermedius Hungerford, 1933 Rheumatogonus intermedius Hungerford, 1933: 3−4 (original description). Subfamily Trepobatinae Matsuda, 1960 Tribe Metrobatini J. T. Polhemus & D. A. Polhemus, 1993 Tribe Metrobatini J. T. Polhemus & D. A. Polhemus, 1993 Tribe Metrobatini J. T. Polhemus & D. A. Polhemus, 1993 Rheumatobates clanis Drake & Harris, 1932 Rheumatobates clanis Drake & Harris, 1932: 157−158 (original description). Rheumatobates clanis Drake & Harris, 1932: 157−158 (original description). Five paratypes are deposited in NMPC: Rheumatogonus intermedius Hungerford, 1933 PARATYPE ( apter.): ‘Baboera River / E Coast Sumatra / 9. 24. 31 / v d. Meer Mohr [p] // P A R A T Y P E / Rheumatogonus / intermedius / H. B. Hungerford [p, blue label] // Purch. from / Meer Mohr / See Pub. 1933 PARATYPE ( apter.): ‘Baboera River / E Coast Sumatra / 9. 24. 31 / v d. Meer Mohr [p] // P A R A T Y P E / Rheumatogonus / intermedius / H. B. Hungerford [p, blue label] // Purch. from / Meer Mohr / See Pub. 1933 KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. 868 [p] // [p]’. PARATYPE ( apter.): ‘Baboera River / E Coast Sumatra / 9. 24. 31 / v d. Meer Mohr [p] // P A R A T Y P E / Rheumatogonus / intermedius / H. B. Hungerford [p, blue label] // Purch. from / Meer Mohr / See Pub. 1933 [p] // [p]’. [p] // [p] . PARATYPE ( apter.): ‘Baboera River / E Coast Sumatra / 9. 24. 31 / v d. Meer Mohr [p] // P A R A T Y P E / Rheumatogonus / intermedius / H. B. Hungerford [p, blue label] // Purch. from / Meer Mohr / See Pub. 1933 [p] // [p]’. Current status. Valid species (see CHEN & NIESER 2002, CHEN et al. 2005). Subfamily Rhagadotarsinae Lundblad, 1933 Tribe Naboandelini J. T. Polhemus & D. A. Polhemus, 1993 Tribe Naboandelini J. T. Polhemus & D. A. Polhemus, 1993 Tribe Naboandelini J. T. Polhemus & D. A. Polhemus, 1993 Naboandelus africanus Hoberlandt, 1951 Naboandelus africanus Hoberlandt, 1951a: 12, 14–16, Figs 13–16 (original description). Rheumatometra philarete Kirkaldy, 1902 Rheumatometra philarete Kirkaldy, 1902 Rheumatometra philarete Kirkaldy, 1902: 281 (original description). Rheumatometra philarete: ANDERSEN & WEIR (1998): 517 (lectotype designation). Two paralectotypes are deposited in NMPC: PARALECTOTYPES (+ apter.): ‘VICTORIA / Alexandra / F. L. Billinghurst. [p] // [p] // [p] // R [hw] Paratype [p] / philarete / Kirk [Drake’s hw, red label] // PARALECTOTYPI / RHEUMATOMETRA / PHILARETE / Kirkaldy, 1902 / labelled: P. KMENT 2013 [p, red label]’. (Both specimens mounted on a single piece of card). Current status. Valid species (see POLHEMUS & POLHEMUS 1993; ANDERSEN & WEIR 1998, 2004). Tribe Naboandelini J. T. Polhemus & D. A. Polhemus, 1993 Naboandelus africanus Hoberlandt, 1951 Naboandelus africanus Hoberlandt, 1951a: 12, 14–16, Figs 13–16 (original description). The holotype and one paratype (allotype) are deposited in NMPC: HOLOTYPE ( apter.): ‘Dundo, Lunda / Angola [p] II- 48 [hw] / A B Machado lg . [p] // Ang. [p] No 398.4 [hw] / coll. A.B.Machado [p] // Holotypus [p, red label] // Holotypus [p] / Naboandelus / africanus / n. sp. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. Rheumatometra philarete Kirkaldy, 1902: 281 (original description). Rheumatometra philarete Kirkaldy, 1902: 281 (original description). p y ( g p ) Rheumatometra philarete: ANDERSEN & WEIR (1998): 517 (lectotype designation). Two paralectotypes are deposited in NMPC: PARALECTOTYPES (+ apter.): ‘VICTORIA / Alexandra / F. L. Billinghurst. [p] // [p] // [p] // R [hw] Paratype [p] / philarete / Kirk [Drake’s hw, red label] // PARALECTOTYPI / RHEUMATOMETRA / PHILARETE / Kirkaldy, 1902 / labelled: P. KMENT 2013 [p, red label]’. (Both specimens mounted on a single piece of card). Current status. Valid species (see POLHEMUS & POLHEMUS 1993; ANDERSEN & WEIR 1998, 2004). Five paratypes are deposited in NMPC: PARATYPES (2 apter.): ‘Rio Grande / British Honduras / Nov. 1931 [p] // PARATYPE [p] / Rheumatobates / clanis / D+H [hw, red label] // [p] // PARATYPUS / RHEUMATOBATES / CLANIS / Drake & Harris, 1932 / labelled: P. KMENT 2013 [p, red label]’. PARATYPES (2 apter.): ‘Rio Grande / British Honduras / Nov. 1931 [p] // PARATYPE [p] / Rheumatobates / clanis / D+H [hw, red label] // [p] // PARATYPUS / RHEUMATOBATES / CLANIS / Drake & Harris, 1932 / labelled: P. KMENT 2013 [p, red label]’. [p ] PARATYPE ( apter.): ‘Rio Grande / British Honduras / Nov. 1931 [p] // PARATYPE [p] / Rheumatobates / clanis / D+H [hw, red label] // [p]’. PARATYPE ( apter.): ‘Rio Grande / British Honduras / Nov. 1931 [p] // PARATYPE [p] / Rheumatobates / clanis / D+H [hw, red label] // [p]’. PARATYPES (2 apter.): ‘Rio Grande / British Honduras / Nov. 1931 [p] // PARATYPE [p] / Rheumatobates / clanis / D+H [hw, red label] // [p] // PARATYPUS / RHEUMATOBATES / CLANIS / Drake & Harris, 1932 / labelled: P. KMENT 2013 [p, red label]’. Current status. Valid species (see SMITH 1988b, CASTRO-VARGAS & MORALES-CASTAÑO 2011). Nine paratypes are deposited in NMPC: PARATYPE ( apter.): ‘Sudan. Kordofan / Lake Keilak / 8–11. II. 63 / Linnavuori [p] // [p] // PARATYPUS / NABO- ANDELUS / BERGEVINI PYGMAEA / Linnavuori, 1971 / labelled: P. KMENT 2013 [p, red label]’. PARATYPE ( apter.): ‘Sudan. Kordofan / Lake Keilak / 8–11. II. 63 / Linnavuori [p] // [p] // PARATYPUS / NABO- ANDELUS / BERGEVINI PYGMAEA / Linnavuori, 1971 / labelled: P. KMENT 2013 [p, red label]’. PARATYPES (2 apter.): ‘Sudan. Kordofan / Lake Keilak / 8–11. 2. 63 / Linnavuori [p] // [p] // PARATYPUS / NABOANDELUS / BERGEVINI PYGMAEA / Linnavuori, 1971 / labelled: P. KMENT 2013 [p, red label]’. PARATYPES (2 apter.): ‘Sudan. Kordofan / Lake Keilak / 8–11. 2. 63 / Linnavuori [p] // [p] // PARATYPUS / NABOANDELUS / BERGEVINI PYGMAEA / Linnavuori, 1971 / labelled: P. KMENT 2013 [p, red label]’. PARATYPE ( apter.): ‘Sudan. Kordofan / Lake Keilak / 8–11. II. 63 / Linnavuori [p] // Naboandelus / bergevini ssp. / pygmaeus Lv. / paratypes [hw] // [p] // PARATYPUS / NABOANDELUS / BERGEVINI PYGMAEA / Linnavuori, 1971 / labelled: P. KMENT 2013 [p, red label]’. PARATYPE ( apter.): ‘Sudan. Kordofan / Lake Keilak / 8–11. II. 63 / Linnavuori [p] // Naboandelus / bergevini ssp. / pygmaeus Lv. / paratypes [hw] // [p] // PARATYPUS / NABOANDELUS / BERGEVINI PYGMAEA / Linnavuori, 1971 / labelled: P. KMENT 2013 [p, red label]’. [p ] PARATYPES (3 apter.): ‘Sudan. Kordofan / Lake Keilak / 8–11. II. 63 / Linnavuori [p] // [p] // PARATYPUS / NABOANDELUS / BERGEVINI PYGMAEA / Linnavuori, 1971 / labelled: P. KMENT 2013 [p, red label]’. [p ] PARATYPES (3 apter.): ‘Sudan. Kordofan / Lake Keilak / 8–11. II. 63 / Linnavuori [p] // [p] // PARATYPUS / NABOANDELUS / BERGEVINI PYGMAEA / Linnavuori, 1971 / labelled: P. KMENT 2013 [p, red label]’. PARATYPE ( apter.): ‘Sudan. Kordofan / Lake Keilak / 8–11. 2. 63 / Linnavuori [p] // [p] // PARATYPUS / NABO- ANDELUS / BERGEVINI PYGMAEA / Linnavuori, 1971 / labelled: P. KMENT 2013 [p, red label]’. PARATYPE ( apter.): ‘Sudan. Kordofan / Lake Keilak / 8–11. 2. 63 / Linnavuori [p] // [p] // PARATYPUS / NABO- ANDELUS / BERGEVINI PYGMAEA / Linnavuori, 1971 / labelled: P. KMENT 2013 [p, red label]’. PARATYPE ( apter.): ‘Sudan. Naboandelus africanus Hoberlandt, 1951 Naboandelus africanus Hoberlandt, 1951a: 12, 14–16, Figs 13–16 (original description). HOLOTYPE ( apter.): ‘Dundo, Lunda / Angola [p] II- 48 [hw] / A B Machado lg . [p] // Ang. [p] No 398.4 [hw] / coll. A.B.Machado [p] // Holotypus [p, red label] // Holotypus [p] / Naboandelus / africanus / n. sp. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. 869 Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 PARATYPE ( apter.): ‘Dundo, Lunda / Angola [p] II- 48 [hw] / A. B. Machado lgt. [p] // Ang. [p] No 398.4 [hw] / coll.A.B.Machado [p] // Allotypus [p, red label] // Allotypus [p] / Naboandelus / africanus / n. sp. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. PARATYPE ( apter.): ‘Dundo, Lunda / Angola [p] II- 48 [hw] / A. B. Machado lgt. [p] // Ang. [p] No 398.4 [hw] / coll.A.B.Machado [p] // Allotypus [p, red label] // Allotypus [p] / Naboandelus / africanus / n. sp. [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. Current status. Valid species (see POISSON 1965b, LINNAVUORI 1981, POLHEMUS & POLHEMUS 2002). Current status. Valid species (see POISSON 1965b, LINNAVUORI 1981, POLHEMUS & POLHEMUS 2002). Nine paratypes are deposited in NMPC: Kordofan / Lake Keilak / 8–11. 2. 63 / Linnavuori [p] // [p] // Naboandelus / ber- gevini / pygmaeus / Lv. [hw] // PARATYPUS / NABOANDELUS / BERGEVINI PYGMAEA / Linnavuori, 1971 / labelled: P. KMENT 2013 [p, red label]’. Current status. Valid subspecies: Naboandelus bergevini pygmaeus Linnavuori, 1971 (see LINNAVUORI 1975, 1981; ANDERSEN 1995; POLHEMUS & POLHEMUS 2002). Current status. Valid subspecies: Naboandelus bergevini pygmaeus Linnavuori, 1971 (see LINNAVUORI 1975, 1981; ANDERSEN 1995; POLHEMUS & POLHEMUS 2002). ) Remark. LINNAVUORI (1971) cited the subspecies epithet as pygmaea; pygmaeus (-a, -um) is a Latin adjective meaning ‘of or pertaining to a pygmy or dwarf, especially to the mythical Pygmaei of Africa; short; of reduced statue’. This spelling was accepted in all the subsequent papers (LINNAVUORI 1975 1981; ANDERSEN 1995; POLHEMUS & POLHEMUS 2002). However, as the genus name Naboandelus Distant, 1910 (type species N. signatus Distant, 1910 – see DISTANT 1910) is undoubtedly a masculine, the correct spelling of the subspecies must be Naboandelus bergevini pygmaeus (see ICZN 1999: 31.2, 34.2). Naboandelus bergevini pygmaeus Linnavuori, 1971 g g p pyg , ( g p ) Naboandelus bergevini pygmaea: LINNAVUORI (1975): 45–46 (diagnosis), LINNAVUORI (1981): 17 (list), ANDERSEN (1995): 97 (catalogue), POLHEMUS & POLHEMUS (2002): 293 (catalogue). Nine paratypes are deposited in NMPC: Nine paratypes are deposited in NMPC: Naboandelus patrizii migrans Linnavuori, 1971 Naboandelus patrizii Mc. ssp. migrans Linnavuori, 1971: 361 (original description). One paratype is deposited in NMPC: PARATYPE ( apter.): ‘Sudan, Khartoum / 30. VI. - 3. VII. 1961 / R. Linnavuori leg [p, blue label] // [p] // Nabo- andelus / patrizii / migrans / Lv. [hw] // PARATYPUS / NABOANDELUS / PATRIZII MIGRANS / Linnavuori, 1971 / labelled: P. KMENT 2013 [p, red label]’. Current status. Valid subspecies (see LINNAVUORI 1981, POLHEMUS & POLHEMUS 2002). KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. 870 Telmatometra indentata Kenaga, 1941 Telmatometra indentata Kenaga, 1941: 173, 179−180, 183, Figs 2a−c (original description, key). Telmatometra indentata Kenaga, 1941: 173, 179−180, 183, Figs 2a−c (original description, key). Three paratypes deposited in NMPC: PARATYPES (2 apter.): ‘Bolivia S. A. / R. Beni Cachuela / Esperanza 9-37 / A. M. Olalla [p] // [p, yellow label] // P A R A T Y P E / Telmatometra / indentata / Eugena Kenaga [p, blue label]’. PARATYPES (2 apter.): ‘Bolivia S. A. / R. Beni Cachuela / Esperanza 9-37 / A. M. Olalla [p] // [p, yellow label] // P A R A T Y P E / Telmatometra / indentata / Eugena Kenaga [p, blue label]’. PARATYPE ( apter.): ‘Bolivia S. A. / R. Beni Cachuela / Esperanza 9-37 / A. M. Olalla [p] // [p] // P A R A T Y P E / Telmatometra / indentata / Eugena Kenaga [p, blue label]’. label] // P A R A T Y P E / Telmatometra / indentata / Eugena Kenaga [p, blue label]’. PARATYPE ( apter.): ‘Bolivia S. A. / R. Beni Cachuela / Esperanza 9-37 / A. M. Olalla [p] // [p] // P A R A T Y P E / Telmatometra / indentata / Eugena Kenaga [p, blue label]’. Current status. Valid species (see MATSUDA 1960; POLHEMUS & POLHEMUS 1995b, 2002; HECKMAN 2011). Trepobates citatus Drake & Chapman, 1953 Trepobates citatus Drake & Chapman, 1953: 111–112 (original description). Four paratypes are deposited in NMPC: PARATYPE ( apter.): ‘Apopka, Fla. [p] / XII - 17 - [hw] 195[p]2 [hw] / H. C. Chapman [p] // [p] // Paratype [p] / Trepobates / citatus / D. & C. [hw, red label]’. PARATYPE ( apter): ‘Apopka, Fla. [p] / XI - 10 [hw] 195[p]2 [hw] / H. C. Chapman [p] // [p] // Paratype [p] / Trepobates / citatus / D. & C. [hw, red label]’. PARATYPE ( apter.): ‘Apopka, Fla. [p] / XI - 11 - [hw] 195[p]2 [hw] / H. C. Chapman [p] // [p] // Paratype [p] / Trepobates / citatus / D. & C. [hw, red label]’. p PARATYPE ( apter): ‘Apopka, Fla. [p] / XI - 10 [hw] 195[p]2 [hw] / H. C. Chapman [p] // [p] // Paratype [p] / Trepobates / citatus / D. & C. [hw, red label]’. Current status. Junior synonym of Trepobates subnitidus Esaki, 1926 (see KITTLE 1991; POLHEMUS & POLHEMUS 1995b, 2002). Trepobates knighti Drake & Harris, 1928 Trepobates knighti Drake & Harris, 1928 Trepobates knighti Drake & Harris, 1928: 28−29 (original description). Trepobates knighti Drake & Harris, 1928: 28−29 (original description). Trepobates knighti Drake & Harris, 1928: 28−29 (original description). Trepobates knighti Drake & Harris, 1928: 28−29 (original description). One paratype is deposited in NMPC: One paratype is deposited in NMPC: PARATYPE ( macr.): ‘Ames. Iowa [p] / 7-29- [hw] 192[p]4 [hw] / C. J. Drake [p] // [p] // PARATYPE [p] / Trepo- bates / knighti n. sp. / Drake + Harris [hw, red label]’. Current status. Valid species (see SMITH 1988b; POLHEMUS & POLHEMUS 1995b, 2002). Trepobates panamensis Drake & Hottes, 1952 Trepobates panamensis Drake & Hottes, 1952 Trepobates panamensis Drake & Hottes, 1952 Trepobates panamensis Drake & Hottes, 1952b: 35–37 (original description). Two paratypes are deposited in NMPC: Two paratypes are deposited in NMPC: PARATYPE ( apter.): ‘Panama, Pan. / Aug. 13. 1934 / L. J. Rozeboom [p] // [p] // Paratype [p] / Trepobates / panamensis / D. + H. [hw, red label]’. PARATYPE ( macr.): ‘Canal Zone / Pan. 2 – 10. 39 / C. J. Drake // [p] // Paratype [p] / Trepobates / panamensis / D. + H. [hw, red label]’. Current status. Valid species (see FROESCHNER 1999; POLHEMUS & POLHEMUS 1995b, 2002; HECKMAN 2011). 871 Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 Subfamily Saldinae Amyot & Serville, 1843 Tribe Saldoidini Reuter, 1912 Subfamily Saldinae Amyot & Serville, 1843 Tribe Saldoidini Reuter, 1912 Family SALDIDAE Amyot & Serville, 1843 Chiloxanthus pilosus var. zaitzevi Kiritshenko, 1916 Two paralectotypes are deposited in NMPC: PARALECTOTYPE (): ‘Карская тундра / с. Тобольс. губ. / ФЗайцев [p] 27 [hw] VII 09 [p] [= Karskaya tundra / s. Tobol’s. gub., F. Zaytsev, 27.vii.09] // COTYPUS / Acanthia (Chiloxanthus) / pilosus Fall. var. / zaitzevi Kir. 1916 [hw, violet label] // PARALECTOTYPUS / CHILOXANTHUS / PILOSUS var. ZAITZEVI / Kiritshenko, 1916 / labelled: P. KMENT 2013 [p, red label]’. PARALECTOTYPE (): ‘Карская тундра / с. Тобольс. губ. / ФЗайцев [p] 27 [hw] VII 09 [p] [= Karskaya tundra / s. Tobol’s. gub., F. Zaytsev, 27.vii.09] // COTYPUS / Acanthia (Chiloxanthus) / pilosus Fall. var. / zaitzevi Kir. 1916 [hw, violet label] // PARALECTOTYPUS / CHILOXANTHUS / PILOSUS var. ZAITZEVI / Kiritshenko, 1916 / labelled: P. KMENT 2013 [p, red label]’. Current status. Junior synonym of Chiloxanthus arcticus (J. Sahlberg, 1878) (see BIANCHI & KIRITSHENKO 1923, SCHUH et al. 1987, LINDSKOG 1995, VINOKUROV 2005, AUKEMA et al. 2013). Chiloxanthus pilosus var. zaitzevi Kiritshenko, 1916 Chiloxanthus pilosus var. zaitzevi Kiritshenko, 1916: 2 (original description). Chiloxanthus pilosus var. zaitzevi Kiritshenko, 1916: 2 (original description). Chiloxanthus pilosus var. zaitzevi: VINOKUROV (2006): 704 (lectotype designation). Chiloxanthus pilosus var. zaitzevi: VINOKUROV (2006): 704 (lectotype designation Two paralectotypes are deposited in NMPC: Two paralectotypes are deposited in NMPC: Trepobates vazquezae Drake & Hottes, 1951 Trepobates vazquezae Drake & Hottes, 1951a: 141–143 (original description). Nine paratypes are deposited in NMPC: Nine paratypes are deposited in NMPC: PARATYPES (4 macr.): ‘Acapulco, Mex. / Aug. 3, 1951 / Drake & Hottes [p] // [p] // Paratype [p] / Trepobates / vazquezae / D + H [hw, red label]’. PARATYPES (4 macr.): ‘Acapulco, Mex. / Aug. 3, 1951 / Drake & Hottes [p] // [p] // Paratype [p] / Trepobates / vazquezae / D + H [hw, red label]’. PARATYPE ( apter.): ‘Acapulco, Mex. / Aug. 3, 1951 / Drake & Hottes [p] // [p] // Paratype [p] / Trepobates / vazquezae / D + H [hw, red label] // Trepobates / vazquezae / D. & H. [hw, white label with black frame sub- marginally]’. PARATYPE ( apter.): ‘Acapulco, Mex. / Aug. 3, 1951 / Drake & Hottes [p] // [p] // Paratype [p] / Trepobates / vazquezae / D + H [hw, red label] // Trepobates / vazquezae / D. & H. [hw, white label with black frame sub- marginally]’. PARATYPES (4 : 2 apter., 2 macr.): ‘Acapulco, Mex. / Aug. 3, 1951 / Drake & Hottes [p] // [p] // Paratype [p] / Trepobates / vazquezae / D + H [hw, red label]’. PARATYPES (4 : 2 apter., 2 macr.): ‘Acapulco, Mex. / Aug. 3, 1951 / Drake & Hottes [p] // [p] // Paratype [p] / Trepobates / vazquezae / D + H [hw, red label]’. Current status. Valid species (see MATSUDA 1960; POLHEMUS & POLHEMUS 1995b, 2002). Current status. Valid species (see MATSUDA 1960; POLHEMUS & POLHEMUS 1995b, 2002). Acanthia (Acanthia) angulosa Kiritshenko, 1912 ( ) g Acanthia (s. str.) angulosa Kiritshenko, 1912: 548−549 (original description). Acanthia angulosa: VINOKUROV (2006): 703 (lectotype designation). Acanthia (s. str.) angulosa Kiritshenko, 1912: 548−549 (original description). Acanthia angulosa: VINOKUROV (2006): 703 (lectotype designation). Acanthia (s. str.) angulosa Kiritshenko, 1912: 548−549 (original description). Acanthia angulosa: VINOKUROV (2006): 703 (lectotype designation). One paralectotype is deposited in NMPC: One paralectotype is deposited in NMPC: PARALECTOTYPE (): ‘ВЕРХ. Р. ХИ – ЧЮ, БАС / ΓОЛУБОЙ 13-14000´ / КОЗЛОВЪ. cep.VII 00 [p] [= verkh. r. Khi-Chyu, bas Goluboy, 13-14,000 ft, Kozlov’.ser.vii 00] // Type [p in red, white label with black frame submarginally] // COTYPUS / Acanthia (s. str.) / angulosa Kir. 1911 [hw, violet label] // PARALECTOTYPUS / ACANTHIA / ANGULOSA / Kiritshenko, 1912 / labelled: P. KMENT 2013 [p, red label]’. PARALECTOTYPE (): ‘ВЕРХ. Р. ХИ – ЧЮ, БАС / ΓОЛУБОЙ 13-14000´ / КОЗЛОВЪ. cep.VII 00 [p] [= verkh. r. Khi-Chyu, bas Goluboy, 13-14,000 ft, Kozlov’.ser.vii 00] // Type [p in red, white label with black frame submarginally] // COTYPUS / Acanthia (s. str.) / angulosa Kir. 1911 [hw, violet label] // PARALECTOTYPUS / ACANTHIA / ANGULOSA / Kiritshenko, 1912 / labelled: P. KMENT 2013 [p, red label]’. Current status. Valid species: Calacanthia angulosa (Kiritshenko, 1912) (LINDSKOG 1975, 1995; COBBEN 1985; SCHUH et al. 1987; AUKEMA et al. 2013). Current status. Valid species: Calacanthia angulosa (Kiritshenko, 1912) (LINDSKOG 1975, 1995; COBBEN 1985; SCHUH et al. 1987; AUKEMA et al. 2013). 872 KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. Two paratypes are deposited in NMPC: PARATYPE (): ‘ [hw] // leg. R.H. Cobben [p] / St. Cruz - Krip / 3 - 2 [hw] 195[p]7 [hw] / Curaçao [hw, white label with black frame submarginally] // PARATYPE [p] / Micracanthia / drakei Cobben. [hw, red label]’. PARATYPE (): ‘ [hw] // leg. R.H. Cobben [p] / St. Cruz - Krip / 3 - 2 [hw] 195[p]7 [hw] / Curaçao [hw, white label with black frame submarginally] // PARATYPE [p] / Micracanthia / drakei Cobben. [hw, red label]’. PARATYPE (): ‘ [hw] // leg. R.H. Cobben [p] / Santa Cruz / brak. / 20 - 4 - [hw] 195[p]7 [hw] / Aruba [hw, white label with black frame submarginally] // PARATYPE [p] / Micracanthia / drakei Cobben. [hw, red label]’. PARATYPE (): ‘ [hw] // leg. R.H. Cobben [p] / Santa Cruz / brak. / 20 - 4 - [hw] 195[p]7 [hw] / Aruba [hw, white label with black frame submarginally] // PARATYPE [p] / Micracanthia / drakei Cobben. [hw, red label]’. Current status. Valid species (see POLHEMUS 1985a, SCHUH et al. 1987). Macrosaldula indica Vinokurov, 2013 Macrosaldula indica: VINOKUROV (2012a): 303–305, Figs 22–30 (original description) (unavailable name). M ld l i di V (2012b) 854 857 Fi 22 30 (E li h t l ti f i i l d i ti ) ( Macrosaldula indica: VINOKUROV (2012a): 303–305, Figs 22–30 (original description) (unavailable name). Macrosaldula indica: VINOKUROV (2012b): 854–857, Figs 22–30 (English translation of original description) (una- Macrosaldula indica: VINOKUROV (2012a): 303–305, Figs 22–30 (original description) (unavailable name). Macrosaldula indica: VINOKUROV (2012b): 854–857, Figs 22–30 (English translation of original description) (una- vailable name). Macrosaldula indica: VINOKUROV (2012b): 854–857, Figs 22–30 (English translation of original description) (una- vailable name). ) Macrosaldula indica Vinokurov, 2013: 58 (diagnosis, valid holotype designation). Micracanthia drakei Cobben, 1960 Micracanthia drakei Cobben, 1960: 56–59, Figs 45, 51, 53 (original description). Two paratypes are deposited in NMPC: Two paratypes are deposited in NMPC: Micracanthia husseyi Drake & Chapman, 1952 Micracanthia husseyi Drake & Chapman, 1952: 148–150 (original description). Micracanthia husseyi Drake & Chapman, 1952: 148–150 (original description Two paratypes are deposited in NMPC: PARATYPE (): ‘Cocoa Fla / 20.XII-1952 / H.C.Chapman [hw] // Paratype [p] / Micracanthia / saltaica / D & C [hw, red label] // [p] // Micracanthia / husseyi / D & C [hw, white label with black frame submarginally]’. PARATYPE (): ‘Cocoa Fla / 20.XII-1952 / H.C.Chapman [hw] // Paratype [p] / Micracanthia / saltaica / D & C [hw, red label] // [p]’. red label] // [p] // Micracanthia / husseyi / D & C [hw, white label with black frame submarginally]’. PARATYPE (): ‘Cocoa Fla / 20.XII-1952 / H.C.Chapman [hw] // Paratype [p] / Micracanthia / saltaica / D & C [hw, red label] // [p]’. Current status. Valid species (see POLHEMUS 1985a, 1988; SCHUH et al. 1987). Three paratypes are deposited in NMPC: Three paratypes are deposited in NMPC: PARATYPE (): ‘INDIA [IN-11] Himachal Pradesh, / Kothi Vill. and environs, 32°18′N / 77°11′E, 2300-2600m, 29.05- / 8.06. 1999 Yu.M.Marusik [p] // [p] // Paratypus / Macrosaldula indica / Vinokur, 2011 [p, red label]’. 8.06. 1999 Yu.M.Marusik [p] // [p] // Paratypus / Macrosaldula indica / Vinokur, 2011 [p, red label] . PARATYPE (): ‘India, Kashmir [p] / Srinagar 5.000 ft [hw] / 22. V. – 9. VI. 52 / F A. Kincl leg [p] // COLLECTIO / NATIONAL MUSEUM / Praha, Czech Republic [p] // [p] // Paratypus / Macrosaldula indica / Vinokurov, 2011 [p, red label]’. (Male genitalia dissected, glued on the same card as the specimen). PARATYPE (): ‘India, Kashmir [p] / Srinagar 5.000 ft [hw] / 22. V. – 9. VI. 52 / F A. Kincl leg [p] // COLLECTIO / NATIONAL MUSEUM / Praha, Czech Republic [p] // [p] // Paratypus / Macrosaldula indica / Vinokurov, 2011 [p, red label]’. (Male genitalia dissected, glued on the same card as the specimen). PARATYPE (): ‘India, Kashmir [p] / Srinagar 5.000 ft [hw] / 22. V. – 9. VI. 52 / F A. Kincl leg [p] // COLLECTIO / NATIONAL MUSEUM / Praha, Czech Republic [p] // [p] // Paratypus / Macrosaldula indica / Vinokurov, 2011 [p, red label]’. (Male genitalia dissected, glued on the same card as the specimen). PARATYPE (): ‘India, Kashmir [p] / Srinagar 5.000 ft [hw] / 22. V. – 9. VI. 52 / F A. Kincl leg [p] // COLLECTIO / NATIONAL MUSEUM / Praha, Czech Republic [p] // [p] // Paratypus / Macrosaldula indica / Vinokurov, 2011 [p, red label]’. (Female genitalia dissected, glued on the same card as the specimen). Current status. Valid species. VINOKUROV (2012a,b) provided detailed description of the taxon but failed to indicate unequivocally the depository of the holotype, which made the species name unavailable according to the provisions of ICZN (1999). VINOKUROV (2013) provided a diagnose and valid holotype designation, including its depository, making the name available from that publication. Three paratypes are deposited in NMPC: Three paratypes are deposited in NMPC: PARATYPE (): ‘Aztec, N. M. / Aug. 26, 1937 / Drake & Andre [p] // Paratype [p] / Saldula / andrei / D. & C. [p, red label] // Saldula / andrei / C.J.D. Drake [hw, white label with black frame submarginally] // Mus. Nat. Pragae / Inv. [p] 1051 [hw, orange label] // [p]’. PARATYPE (): ‘San Carlos, Ariz. / August 17, 1934 / Carl J. Drake [p] // Paratype [p] / Saldula / andrei / D. & C. [hw, red label] // Mus. Nat. Pragae / Inv. [p] 1052 [hw, orange label] // [p]’. PARATYPE (): ‘Eagle Nest Lake / New Mex IX, 1934 / Carl J. Drake [p] // Paratype [p] / Saldula / andrei / D& C. [hw, red label] // Saldula / andrei / C.J.D. Drake [hw, white label with black frame submarginally] // Mus. Nat. Pragae / Inv. [p] 1053 [hw, orange label] // [p]’. Current status. Valid species: Saldula andrei andrei Drake, 1949 (see POLHEMUS 1985a, 1988; SCHUH et al. 1987). Salda alpicola J. Sahlberg, 1880 Salda alpicola J. Sahlberg, 1880: 8−11 (original description). Two syntypes are deposited in NMPC: SYNTYPE (): ‘Salten / Baadfj. [hw] // J. Sahlb. [p] // Spec. typ. [p] // TYPUS [p, red label with black frame submar- ginally] // [p] // Salda / alpicola / J. Sahlb. / Det. J. Sahlberg [hw]’. 873 Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 SYNTYPE (): ‘Salten / Baadfj. [hw] // J. Sahlb. [hw] // TYPUS [p, red label with black frame submarginally] // [p] // Salda / alpicola J. Sahlb. / Det. J. Sahlberg [hw]’. SYNTYPE (): ‘Salten / Baadfj. [hw] // J. Sahlb. [hw] // TYPUS [p, red label with black frame submarginally] // [p] // Salda / alpicola J. Sahlb. / Det. J. Sahlberg [hw]’. Current status. Valid species: Calacanthia aplicola (J. Sahlberg, 1880) (COBBEN 1959, SCHUH et al. 1987, LINDSKOG 1995). Current status. Valid species: Calacanthia aplicola (J. Sahlberg, 1880) (COBBEN 1959, SCHUH et al. 1987, LINDSKOG 1995). Salda reuteri Jakovlev, 1889 Salda reuteri Jakovlev, 1889: 66−68 (original description). Salda reuteri: VINOKUROV (2006): 704 (lectotype designation). Two paralectotypes are deposited in NMPC: PARALECTOTYPE (): ‘Иркутскъ. / В. Яковлевъ [p] [= Irkutsk’, V. Jakovlev’] // COTYPUS / Salda / reuteri Jak. 1889 [hw, violet label] // Mus. Nat. Pragae / Inv. [p] 11.128 [hw, orange label] // PARALECTOTYPUS / SALDA / REUTERI / Jakovlev, 1889 / labelled: P. KMENT 2013 [p, red label]’. PARALECTOTYPE (): ‘Иркутскъ. / В. Яковлевъ [p] [= Irkutsk’, V. Jakovlev’] // COTYPUS / Salda / reuteri Jak. 1889 [hw, violet label] // Mus. Nat. Pragae / Inv. [p] 11.129 [hw, orange label] // PARALECTOTYPUS / SALDA / REUTERI / Jakovlev, 1889 / labelled: P. KMENT 2013 [p, red label]’. Current status. Junior synonym of Saldula nobilis Horváth, 1884 (see REUTER 1895, SCHUH et al. 1987, LINDSKOG 1995). Saldula azteca Drake & Hottes, 1949 Saldula azteca Drake & Hottes, 1949: 177−179, Pl. X: Fig. III (original description) Saldula andrei Drake, 1949 Saldula andrei Drake, 1949a: 3−4 (original description). Saldula gidshaensis Cobben, 1987 Saldula gidshaensis Cobben, 1987a: 401–404, Figs 1a–c, Table 1 (original description). Saldula gidshaensis Cobben, 1987a: 401–404, Figs 1a–c, Table 1 (original description). One paratype is deposited in NMPC: PARATYPE (): ‘ETHIOPIE / Gidsha vallei [sic!] / Bronbeek 2400M. / 23-11′69 / Leg: R.H. Cobben [p] // [p] // Paratype / Saldula / gidshaensis / MS / R. H. Cobben 1980 [hw, green label]. Current status. Valid species (see also COBBEN 1987b). Saldula (Macrosaldula) kaszabi Hoberlandt, 1971 Saldula (Macrosaldula) kaszabi Hoberlandt, 1971: 145–149, Figs 1–2, 5, 8–11 (original description). Two paratypes are deposited in NMPC: Two paratypes are deposited in NMPC: PARATYPE (): ‘MONGOLIA, Uburchangaj / aimak, zw, Baga Bogd ul / und Arc Bogd ul, 1600m / 60 km WSW v. som. Chovd / Exp. Dr. Z. KASZAB, 1964 [p] // Nr. 172 / 22. VI. 1964 [p] // Paratypus [p] / Saldula / kaszabi, n. sp. [hw] / Det. L. Hoberlandt, 19[p]69 [hw, red label]. (Male genitalia dissected, remnants of pygophore glued on a pointed card, internal genitalia mounted on two microslides attached to separate pieces of card, all the cards pinned on the same pin). PARATYPE (): ‘MONGOLIA, Südgobi aimak / somon Bulgan, 1350 m / Exp. Dr. Z. KASZAB, 1964 [p] // Nr. 159 / 20. VI. 1964 [p] // Paratypus [p] / Saldula / kaszabi, n. sp. [hw] / Det. L. Hoberlandt, 19[p]69 [hw, red label]. Current status. Valid species: Macrosaldula kaszabi (Hoberlandt, 1971) (see COBBEN 1985, SCHUH et al. 1987, LINDSKOG 1995). Saldula bassingeri Drake, 1949b: 190–191 (original description). Saldula bassingeri Drake, 1949b: 190–191 (original description). Two paratypes are deposited in NMPC: Two paratypes are deposited in NMPC: PARATYPE (): ‘Dolores, Colo. / Aug. 15. 1925 / C. J. Drake [p] // Paratype [p] / Saldula / bassingeri / D. & C [hw, red label] // Saldula / bassingeri / Drake [hw, white label with black frame submarginally] // Nat. Mus. Pragae / Inv. [p] 1058 [hw, orange label] // [p]’. PARATYPE (): ‘Dolores, Colo. / Aug. 15. 1925 / C. J. Drake [p] // Paratype [p] / Saldula / bassingeri / D. & C [hw, red label] // Saldula / bassingeri / Drake [hw, white label with black frame submarginally] // Nat. Mus. Pragae / Inv. [p] 1058 [hw, orange label] // [p]’. PARATYPE (): ‘Riverside, Cal. [p] / Aug.16, 1937 / Drake & Andre [p] // Paratype [p] / Saldula / bassingeri / D & C. [hw, red label] // Saldula / bassingeri / C.J.D. Drake [hw, white label with black frame submarginally] // Nat. Mus. Pragae / Inv. [p] 1057 [hw, orange label] // [p]’. Current status. Valid species (see SCHUH et al. 1987, POLHEMUS 1988). Two paratypes are deposited in NMPC: Two paratypes are deposited in NMPC: PARATYPE (): Palisades, Colo. / Sept. Oct. 1949 / F.C.Hottes [p] // Paratype [p] / Saldula / azteca / D& H. [hw, red label] // [p] // Saldula / azteca / C.J.D. Drake [hw, white label with black frame submarginally] // Mus. Nat. Pragae / Inv. [p] 1054 [hw, orange label]’. PARATYPE (): ‘Gateway, Colo. / Sept. 8, 1949 / Drake & Hottes [p] // Paratype [p] / Saldula / azteca / D& H. [hw, red label] // [p] // Mus. Nat. Pragae / Inv. [p] 1055 [hw, orange label]. Current status. Valid subspecies: Saldula andrei azteca Drake & Hottes, 1949 (see POLHEMUS 1985a, 1988; SCHUH et al. 1987). KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. 874 Saldula madonica Seidenstücker, 1961: 41–46, Figs 5 11–12, 14, 16 (original description). Saldula madonica Seidenstücker, 1961: 41–46, Figs 5 11–12, 14, 16 (original description). Two paratypes are deposited in NMPC: Two paratypes are deposited in NMPC: PARATYPE (): ‘ [p] // SICILIA, Cefalu, Castelbuo- / no Statione - Ponte Fiumara / 4. - 12.5.57, Seidenstücker [p] // PARATYPOID [p] / Saldula / madonica [hw] / det. Seidenstücker [p] n. sp. [hw, pink label with black frame submarginally]’. g y] PARATYPE (): ‘ [p] // SICILIA, Cefalu, Castelbuo- / no Statione - Ponte Fiumara / 4. - 12.5.57, Seidenstücker [p] // PARATYPOID [p] / Saldula / madonica [hw] / det. Seidenstücker [p] n. sp. [hw, pink label with black frame submarginally]’. Current status. Valid species: Macrosaldula madonica (Seidenstücker, 1961) (see COBBEN 1985, SCHUH et al. 1987, LINDSKOG 1995). 875 Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 Saldula ourayi Drake & Hottes, 1949 Saldula ourayi Drake & Hottes, 1949: 179−180, Pl. X: Fig. IV (original description). One paratype is deposited in NMPC: PARATYPE (): ‘Soap Lake, Wash. / Aug. 30. 1949 / Drake & Hottes [p] // Paratype [p] / Saldula / ourayi / D&H [hw, red label] // [p] // Saldula / ourayi / C.J.D. D & H. [hw, white label with black frame submarginally] // Mus. Nat. Pragae / Inv. [p] 1056 [hw]’. Current status. Junior synonym of Saldula dispersa Uhler, 1893 (see POLHEMUS 1985b, 1988; SCHUH et al. 1987). Saldula penningtoni Drake & Carvalho, 1948: 479 (original description). One paratype is deposited in NMPC: PARATYPE (): ‘Buenos Aires / Arg. 11-23-1938 / Carl J. Drake [p] // Paratype [p] / Saldula / penningtoni / D & C. [hw, red label] // Saldula / penningtoni / Drake / C.J.D. [hw, white label with black frame submarginally] // Nat. Mus. Pragae / Inv. [p] 1062 [hw, orange label] // [p]’. Current status. Valid species (see SCHUH et al. 1987). One paratype is deposited in NMPC: One paratype is deposited in NMPC: p yp p PARATYPE (): ‘Salt Lake, Utah / Aug. 23, 1949 / Drake & Hottes [p] // Paratype [p] / Saldula / opiparia / D & H. [hw, red label] // [p] // Saldula / opiparia / D. & H. [hw, white label with black frame submarginally] // Nat. Mus. Pragae / Inv. [p] 1059 [hw, orange label]’. PARATYPE (): ‘Salt Lake, Utah / Aug. 23, 1949 / Drake & Hottes [p] // Paratype [p] / Saldula / opiparia / D & H. [hw, red label] // [p] // Saldula / opiparia / D. & H. [hw, white label with black frame submarginally] // Nat. Mus. Pragae / Inv. [p] 1059 [hw, orange label]’. Current status. Valid species (see POLHEMUS 1985a, 1988; SCHUH et al. 1987). Saldula opiparia Drake & Hottes, 1955 Saldula opiparia Drake & Hottes, 1955: 63−66, Fig. 6 (original description). Saldula penningtoni Drake & Carvalho, 1948 Saldula penningtoni Drake & Carvalho, 1948: 479 (original description). Saldula pericarti Vinokurov, 2012 Saldula pericarti Vinokurov, 2012a: 305–308, Figs 43–55 (original description). Saldula pericarti Vinokurov, 2012a: 305–308, Figs 43–55 (original description). The holotype and 50 paratypes are deposited in NMPC: HOLOTYPE (): ‘India, Kashmir [p] / Srinagar 5.000 ft [hw] / 22. V. - 9. VI. 52 / F. A. Kincl leg. [p] // COLLECTIO / NATIONAL MUSEUM / Praha, Czech Republic [p] // [p] // Holotypus / Saldula pericarti / Vinokurov, 2011 [p, red label]’. PARATYPE (): ‘India Kasmir / Srinagar 5.000 ft / 22.V.– 9.VI.52 / F.A.Kincl leg. [hw] // COLLECTIO / NATIONAL MUSEUM / Praha, Czech Republic [p] // [p] // Paratype / Saldula pericarti / Vinokurov sp.n. [p, red label]’. PARATYPES (4 ): ‘India, Kashmir [p] / Srinagar 5.000 ft [hw] / 22. V. – 9. VI. 52 / F. A. Kincl leg [p] // COLLEC- TIO / NATIONAL MUSEUM / Praha, Czech Republic [p] // [p] // Paratype / Saldula pericarti / Vinokurov sp.n. [p, red label]’. p p PARATYPES (3 ): ‘India, Kashmir [p] / Srinagar 5.000 ft [hw] / 22. V. – 9. VI. 52 / F. A. Kincl leg [p] // COLLEC- TIO / NATIONAL MUSEUM / Praha, Czech Republic [p] // [p] // Paratypus / Saldula pericarti / Vinokurov 2011 [p, red label]’. PARATYPES (4 spec.): ‘India, Kashmir [p] / Srinagar 5.000 ft [hw] / 22. V. – 9. VI. 52 / F. A. Kincl leg [p] // COLLEC- TIO / NATIONAL MUSEUM / Praha, Czech Republic [p] // Paratype / Saldula pericarti / Vinokurov sp.n. [p, red label]’. (Sex unidentiﬁ ed, specimens lacking apex of abdomen). KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. 876 PARATYPE (1 spec.): ‘India Kasmir / Srinagar 5.000 ft / 22.V.– 9.VI.52 / F.A.Kincl leg. [hw] // COLLECTIO / NATIO- NAL MUSEUM / Praha, Czech Republic [p] // Paratype / Saldula pericarti / Vinokurov sp.n. [p, red label]’. (Sex unidentiﬁ ed, specimen lacking apex of abdomen). PARATYPES (2 ): ‘India Kasmir / Srinagar 5.000 ft / 22.V. – 9.VI.52 / F.A.Kincl leg. [hw] // COLLECTIO / NATIO NAL MUSEUM / Praha, Czech Republic [p] // [p] // Paratypus / Saldula pericarti / Vinokurov, 2011 [p, red label]’. PARATYPES (2 ): ‘India, Kashmir [p] / Srinagar 5.000 ft [hw] / 22. V. – 9. VI. 52 / F. A. Saldula varionis Drake & Hottes, 1950 Saldula varionis Drake & Hottes, 1950: 57, 59 (original description). One paratype is deposited in NMPC: PARATYPE (): ‘Gateway, Colo. / Sept. Oct. 1949 F. C. Hottes [p] // Paratype [p] / Saldula / varionis / D & H. [hw, red label] // Saldula / varionis / D. & H. [hw, white label with black frame submarginally] // Nat. Mus. Pragae / Inv. [p] 1061 [hw, orange label] // [p]’. Current status. Junior synonym of Saldula balli Drake, 1950 (see POLHEMUS 1985a, 1988; SCHUH et al. 1987). Saldula pericarti Vinokurov, 2012 Kincl leg [p] // COLLEC- TIO / NATIONAL MUSEUM / Praha, Czech Republic [p] // [p] // Paratype / Saldula pericarti / Vinokurov sp.n. [p, red label]’. PARATYPES (34 ): ‘India, Kashmir [p] / Srinagar 5.000 ft [hw] / 22. V. – 9. VI. 52 / F. A. Kincl leg [p] // COLLEC- TIO / NATIONAL MUSEUM / Praha, Czech Republic [p] // [p] // Paratypus / Saldula pericarti / Vinokurov 2011 [p, red label]’. Current status. Valid species (see also VINOKUROV 2012b). Saldula verdica Drake & Hottes, 1951 Saldula verdica Drake & Hottes, 1951b: 380−382, Fig. 1 (original description). Saldula trivialis Cobben, 1961 Saldula trivialis Cobben, 1961: 102–105, Figs 26a–f, 28–29, 31, 32a–b, 35a–b (original description). aldula trivialis Cobben, 1961: 102–105, Figs 26a–f, 28–29, 31, 32a–b, 35a–b (original description). One paratype is deposited in NMPC: One paratype is deposited in NMPC: PARATYPE (): ‘S. Karori / 13 - I - 24 / H. Hamilton [p] // [p] // Paratypus / Saldula / trivialis / R. H. Cobben . 1961 [hw, pink label]’. Current status. Valid species (see SCHUH et al. 1987). Current status. Valid species (see SCHUH et al. 1987). One paratype is deposited in NMPC: One paratype is deposited in NMPC: PARATYPE (): ‘Nova Teutonia / St. Catarina / Bras. [p] 2.18 [hw] 1950 [p] / Fritz Plaumann [p] // Paratype [p] / Saldula / verdica / D. & H. [hw, red label] // [p] // Saldula / penningtoni / D & H [hw, white label with black frame submarginally] // Nat. Mus. Pragae / Inv. [p] 1060 [hw, orange label]’. Current status. Valid species: Rupisalda verdica (Drake & Hottes, 1951) (see POLHEMUS 1985a, SCHUH et al. 1987). 877 Acta Entomologica Musei Nationalis Pragae, 53( 1), 2013 The holotype is deposited in NMPC: The holotype is deposited in NMPC: HOLOTYPE (): ‘Dundo, Lunda / Angola [p] IV. 48 [hw] / A. B. Machado lgt. [p] // Ang. [p] No. 488. 2 [hw] / coll A.B.Machado [p] // Holotypus [p, red label] // Holotypus [p] / Pseudopatapius / angolensis gen. et sp. n. / C.J.Drake et [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 1064 [hw, orange label]’. Current status. Valid species (see POLHEMUS 1981, SCHUH et al. 1987). Patapius (Pseudopatapius) africanus Drake & Hoberlandt, 1951: 10–13, Figs 1, 3–5 (original description) The holotype and two paratypes are deposited in NMPC: HOLOTYPE (): ‘Dundo, Lunda / Angola [p] XI. 48 [hw] / A B.Machado lgt. [p] // Ang. [p] 1197.7 [hw] coll.A.B Machado [p] // Holotypus [p, red label] // Holotypus [p] / Pseudopatapius / africanus gen. et sp. n. / C.J.Drake et [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. HOLOTYPE (): ‘Dundo, Lunda / Angola [p] XI. 48 [hw] / A B.Machado lgt. [p] // Ang. [p] 1197.7 [hw] coll.A.B Machado [p] // Holotypus [p, red label] // Holotypus [p] / Pseudopatapius / africanus gen. et sp. n. / C.J.Drake et [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. PARATYPE (): ‘Dundo, Lunda / Angola [p] XI. 48 [hw] / A B Machado lgt [p] // Ang. [p] 1197. 7 [hw] / coll.A. B.Machado [p] // Paratypus [p, red label] // Paratypus [p] / Pseudopatapius / africanus gen. et sp. n. / C.J.Drake et [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. (Left antenna, right hemelytron, and apex of abdomen missing; male genitalia dissected, mounted on two separate slides deposited in Slide Box 5). PARATYPE (): ‘Dundo, Lunda / Angola [p] XI. 48 [hw] / A B Machado lgt [p] // Ang. [p] 1197. 7 [hw] / coll.A. B.Machado [p] // Paratypus [p, red label] // Paratypus [p] / Pseudopatapius / africanus gen. et sp. n. / C.J.Drake et [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label]’. (Left antenna, right hemelytron, and apex of abdomen missing; male genitalia dissected, mounted on two separate slides deposited in Slide Box 5). PARATYPE (): ‘Dundo, Lunda / Angola [p] VI. 48 [hw] / A. B. Machado lgt [p] // Luachimo [hw] / Ang. [p] 829. 16 [hw] / coll.A.B.Machado [p] // Paratypus [p, red label] // Paratypus [p] / Pseudopatapius / africanus gen. et sp. n. / C.J.Drake et [hw] / Det. L. Hoberlandt, 19[p]50 [hw, red label] // Mus. Nat. Pragae / Inv. [p] 1063 [hw, orange label]’. Current status. Valid species (see POLHEMUS 1981, SCHUH et al. 1987). Patapius (Pseudopatapius) angolensis Drake & Hoberlandt, 1951 Patapius (Pseudopatapius) africanus Drake & Hoberlandt, 1951 Patapius (Pseudopatapius) africanus Drake & Hoberlandt, 1951: 10–13, Figs 1, 3–5 (original desc PARATYPES (5 brach., 2 macr., 9 brach., 1 macr., 5 larvae (1 instar 4, 4 instar 5): ‘E Afghanistan, Nuristan, Bashgultal, 1300 m, 24.v.1953, leg. J. Klapperich’. Missing types Family HELOTREPHIDAE Esaki & China, 1927 Subfamily Helotrephinae Esaki & China, 1927 Tribe Limnotrephini J. T. Polhemus, 1990 Mixotrephes hoberlandti Papáček, Štys & Tonner, 1989 Mixotrephes hoberlandti Papáček, Štys & Tonner, 1989 Patapius (Pseudopatapius) angolensis Drake & Hoberlandt, 1951: 14–16, Fig. 6 (original description Patapius (Pseudopatapius) angolensis Drake & Hoberlandt, 1951: 14–16, Fig. 6 (original description) HOLOTYPE ( brach.): ‘E Afghanistan, Nuristan, Bashgultal, 1300 m, 24.v.1953, leg. J. Klapperich’. (Dry mounted, fully disseceted). Acknowledgements We are very grateful to Ping-Ping Chen and Nico Nieser (Naturalis Biodiversity Centre, Leiden, the Netherlands), Felipe F. F. Moreira (Instituto Oswaldo Cruz, Rio de Janeiro, Brazil), Miroslav Papáček (University of South Bohemia, České Budějovice, Czech Republic), Dan A. Polhemus (Bishop Museum, Honolulu, Hawaii, USA), Pavel Štys (Charles University, Praha, Czech Republic), Nikolay N. Vinokurov (Institute of Biological Problems of the Cryolite Zone, Russian Academy of Sciences, Yakutsk, Russia), and Herbert Zettel (Natural History Museum, Vienna, Austria) for their help and valuable comments on the manuscript. Felipe Moreira, Dan A. Polhemus, and Pavel Štys supported us with numerous hardly obtainable papers, and Jitka Aldhoun (the Natural History Museum, London, U.K.) kindly corrected our English. The present study was supported by the Ministry of Culture of the Czech Republic (grant DF12P01OVV021). Mixotrephes hoberlandti Papáček, Štys & Tonner, 1989 Mixotrephes hoberlandti Papáček, Štys & Tonner, 1989 Mixotrephes hoberlandti Papáček, Štys & Tonner, 1989 HOLOTYPE ( brach.): ‘E Afghanistan, Nuristan, Bashgultal, 1300 m, 24.v.1953, leg. J. Klapperich’. (Dry mounted, fully disseceted). PARATYPES (5 brach., 2 macr., 9 brach., 1 macr., 5 larvae (1 instar 4, 4 instar 5): ‘E Afghanistan, Nuristan, Bashgultal, 1300 m, 24.v.1953, leg. J. Klapperich’. KMENT & KOLÍNOVÁ: Catalogue of types in NMPC, part VIII: Heteroptera I. 878 PARATYPE ( brach.): ‘S. Iran: Makran, 2 km SE Minab, 21.v.1973, leg. Expedition of National Museum (Nat. Hist.) in Prague (locality no. 204)’. (Dry mounted, fully disseceted). PARATYPE ( brach.): ‘S. Iran: Makran, 2 km SE Minab, 21.v.1973, leg. Expedition of National Museum (Nat. Hist.) in Prague (locality no. 204)’. (Dry mounted, fully disseceted). Current status. Valid species: Mixotrephes (Mixotrephes) hoberlandti Papáček, Štys & Tonner, 1989 (see POLHEMUS 1995c, PAPÁČEK & ZETTEL 2004). Current status. Valid species: Mixotrephes (Mixotrephes) hoberlandti Papáček, Štys & Tonner, 1989 (see POLHEMUS 1995c, PAPÁČEK & ZETTEL 2004). Remarks. According to the original description, holotype and larger portion of the paratypes are deposited in NMPC, with multiplicate specimens deposited in collections of P. Štys (Department of Zoology, Charles University, Praha), M. Papáček (Department of Biology, Pedagogical Faculty, České Budějovice), and J. T. Polhemus (University of Colorado Museum, Engelwood, USA) (PAPÁČEK et al. 1989). According to M. Papáček and P. Štys (pers. comm., 2013) the holotype and paratypes belonging to NMPC were returned after the description. Most probably the specimens were borrowed by L. Hoberlandt and taken home (even the collection box is missing) during his unﬁ nished work on Iranian water bugs shortly before his death. So far we have failed to reclaim the material back from the care of his wife. References ANDERSEN N. M. 1975: The Limnogonus and Neogerris of the Old World with character analysis and reclassiﬁ - cation of the Gerrinae (Hemiptera: Gerridae). Entomologica Scandinavica, Supplementum 7: 1–96. ANDERSEN N. M. 1975: The Limnogonus and Neogerris of the Old World with character analysis and reclassiﬁ - cation of the Gerrinae (Hemiptera: Gerridae). Entomologica Scandinavica, Supplementum 7: 1–96. ANDERSEN N. 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ZETTEL H. 2000a: The Helotrephidae (Heteroptera) of Borneo. Entomological Problems 31: 1–22. ZETTEL H. 2000b: Rhagovelia inexpectata sp.nov., a sibling species of R. sumatrensis from Southeast Asia (Hete- roptera: Veliidae). Entomological Problems 31: 175–178. ZETTEL H. 2001: Five new species of Perittopus Fieber, 1861 (Hemiptera: Veliidae) from Southeast Asia. Rafﬂ es Bulletin of Zoology 49: 109–119. ZETTEL H. 2003: The Helotrephidae (Insecta: Heteroptera) of the Philippine Islands. Annalen des Naturhistorisches Museum in Wien, B 104: 45–97. ZETTEL H. 2009: Aphelocheirus (s.str.) gusenleitneri nov.sp. – Erstnachweis der Aphelocheiridae (Heteroptera) aus Myanmar. Linzer Biologische Beiträge 41: 1071–1077. ZETTEL H. 2012a: Aphelocheirus (s. str.) goellnerae sp. nov. from Madagascar (Heteroptera: Aphelocheiridae). Entomologische Zeitschrift (Stuttgart) 122: 111–113. ZETTEL H. 2012b: A new Enithares (Insecta: Heteroptera: Notonectidae) from India. Annalen des Naturhistorischen Museums in Wien, B 113: 23–26. liid ( i ) d hili i h f d b h f ZETTEL H. 2012c: Neue Veliidae (Hemiptera: Heteroptera) von den Philippinen. Zeitschrift der Arbeitsg Österreichischer Entomologen 64: 79–118. ZETTEL H. & BONGO J. P. 2006: Rhagovelia heissi nov.sp., a remarkable new species of the R. orientalis species group (Heteroptera, Veliidae) from Cebu Island, Philippines. Pp. 737–742. In: RABITSCH W. (ed.): Hug the bug – For love of true bugs. Festschrift zum 70. Geburtstag von Ernst Heiss. Denisia 19: 1–1184. ZETTEL H. & CHEN P.-P. 1997: Three new taxa of Amemboa Esaki, 1925, from Thailand and Viet Nam (Hetero- ptera: Gerridae). Annales Historico-Naturales Musei Nationalis Hungarici 89: 93–101. 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https://openalex.org/W3108688960	https://www.frontiersin.org/articles/10.3389/fphar.2020.504624/pdf	English	null	Antioxidant and Anti-Inflammatory Effects of Genus Gynura: A Systematic Review	Frontiers in pharmacology	2,020	cc-by	21,312	Abbreviations: ADI, acceptable daily intake; AOM, azoxymethane; BW, body weight; CAT, catalase; CCl4, carbon tetra- chloride; COX-2, cyclooxygenase-2; CRP, c-reactive protein; DCFH-DA, 2′,7′-dichloroﬂuorescein diacetate; DMSO, dimethyl sulfoxide; EAE, enzyme-assisted extraction; EIA, enzyme immunoassay; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift assay; ESI-MS, electrospray ionization-mass spectrometry; ESI-TOF-MS, electrospray ionization time-of-ﬂight mass spectrometry; EThOS, e-theses online service; FRSA, free radical scavenging activity; GC-MS, gas chromatography-mass spectrometer; GPT, glutame pyruvate transaminase; GR, glutathione reductase; GSH, glutathione; GSH- Px, glutathione peroxidase; GSK3β, glycogen synthase kinase three beta; GSSG, glutathione disulﬁde; GST, glutathione-S- transferase; H, human; H2O2, hydrogen peroxide; HDFs, human dermal ﬁbroblasts; HPLC, high-performance liquid chro- matography; HP-TLC, high-performance thin-layer chromatography; HSOS, sinusoidal obstruction syndrome; huvec, human umbilical vein endothelial cell; HVOD, hepatic veno-occlusive disease; i. p., intraperitoneal; IC50, inhibitory concentration 50%; IFN-γ, interferon-gamma; IL, interleukin; iNOS, inducible nitric oxide synthase; LC-MS/MS, liquid chromatography with tandem mass spectrometry; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; MDA, malondialdehyde; MeOH, methanol; MMP-1, matrix metalloproteinase-1; mRNA, messenger ribonucleic acid; NF-κB, nuclear factor kappa B; NGF, nerve growth factor; NMR, nuclear magnetic resonance; NO, nitric oxide; Nrf2, nuclear factor erythroid 2-related factor 2; OHAT, ofﬁce health assessment and translation for conducting a systematic review and evidence integration; PBMCs, peripheral blood mononuclear cells; PGE2, prostaglandin E2; p-IκBα, phospho-IκB alpha; PMA, phorbol 12-myristate 13-acetate; PMNs, polymorphonuclear cells; PPARγ, peroxisome proliferator-activated receptor gamma; PRISMA, preferred reporting items for systematic reviews and meta-analyses; ROS, reactive oxygen species; RT-PCR, reverse transcription polymerase chain reaction; SIRT, sirtuin; SOD, superoxide dismutase; STZ, streptozotocin; TAS, total antioxidant status; TBARS, thiobarbituric acid reactive substances; TFC, total ﬂavonoid content; TNF-α, tumor necrosis factor alpha; TPC, total phenolic content; UEAE, ultrasonic-enzyme-assisted extraction; UHPLC-Q-TOF-MS/MS, ultra-high-performance liquid chromatography-quadrupole time-of-ﬂight mass spectrometry; UV, ultraviolet; v/v, volume/volume; v/w, volume/weight; Δψm, mitochondrial membrane potential. Antioxidant and Anti-Inﬂammatory Effects of Genus Gynura: A Systematic Review 1Centre for Drug and Herbal Development, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia, 2Centre for Quality Management of Medicines, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia, 3Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia, 4Institute of Systems Biology, Universiti Kebangsaan Malaysia, Bangi, Malaysia Background: Gynura species have been used traditionally to treat various ailments, such as fever, pain, and to control blood glucose level. This systematic review critically discusses studies regarding Gynura species that exhibited antioxidant and anti-inﬂammatory effects, thus providing perspectives and instructions for future research of the plants as a potential source of new dietary supplements or medicinal agents. Methods: A literature search from internet databases of PubMed, Scopus, Science Direct, e-theses Online Service, and ProQuest was carried out using a combination of keywords such as “Gynura,” “antioxidant,” “anti-inﬂammatory,” or other related words. Research articles were included in this study if they were experimental (in vitro and in vivo) or clinical studies on the antioxidant or anti-inﬂammatory effects of Gynura species and if they were articles published in English. Reviewed by: Reviewed by: Sayeed Ahmad, Jamia Hamdard University, India Md. Areeful Haque, International Islamic University Chittagong, Bangladesh *Correspondence: Norsyahida Mohd Fauzi drnorsyahida@ukm.edu.my Specialty section: This article was submitted to Ethnopharmacology, a section of the journal Frontiers in Pharmacology Received: 14 October 2019 Accepted: 13 October 2020 Published: 27 November 2020 SYSTEMATIC REVIEW published: 27 November 2020 doi: 10.3389/fphar.2020.504624 SYSTEMATIC REVIEW Edited by: Marco Leonti, University of Cagliari, Italy INTRODUCTION medicine, G. segetum (Lour.) Merr. is consumed as a decoction or is soaked in wine and orally taken to promote microcirculation or relieve pain (Chen et al., 2011). In Nepal, the juice of G. nepalensis DC. is applied on cuts and wounds as healing treatment (Manandhar, 2002). Incorporation of a plant-based natural antioxidant in daily diet can prevent several human illnesses (Knekt et al., 1996) as the prevalence of many illness is inversely linked to the dietary consumption of antioxidant-rich foods (Sies, 1993). For any herb to be developed into a dietary supplement, it is essential that the evidence for the claimed effects of the herb is scientiﬁcally demonstrated. Ideally, the mechanisms of action should also be understood. The potential use of Gynura as a phytomedicine with antioxidant and anti-inﬂammatory properties has been well documented, but we were unable to identify speciﬁc review articles that focused on the antioxidant and anti- inﬂammatory effects of the Gynura species. This review was undertaken to assess published experimental data that investigated the antioxidant and anti-inﬂammatory activities of the Gynura species to support the design of future studies. It also provided an evaluation of the quality of available information, summarized mechanisms of action data from animal and cell studies following the administration of the Gynura species, and identiﬁed research gaps in the literature. Inﬂammation refers to a complex array of defensive immune responses (Arulselvan et al., 2016), and tissue damage is one of the consequences of an exaggerated or uncontrolled prolonged inﬂammatory process (Biswas, 2016). Inﬂammatory cells, including neutrophils and macrophages, generate free radicals at the inﬂammation site, where reactive oxygen species (ROS) (e.g., hydroxyl radicals, superoxide anion radicals, and hydrogen peroxide) act as both signaling molecules and inﬂammation mediators. Enhancement of pro-inﬂammatory gene expression can be achieved via the initiation of the intracellular signaling cascade by reactive species. The exaggerated generation of reactive species in pathological inﬂammatory conditions may induce localized oxidative stress and tissue injury, thus promoting progression of many inﬂammatory diseases (Mittal et al., 2014). Hence, inﬂammation and oxidative stress are highly interdependent pathophysiological events in various types of chronic diseases (Vaziri and Rodríguez-Iturbe, 2006; Biswas, 2016). A total of 46 species are identiﬁed in the genus Gynura (The Plant List, 2020). They are distributed from tropical Africa to South and East Asia as well as Australia where the highest speciﬁc diversity is found in Southeast Asia (Vanijajiva and Kadereit, 2011). Citation: Tan JN, Mohd Safﬁan S, Buang F, Jubri Z, Jantan I, Husain K and Mohd Fauzi N (2020) Antioxidant and Anti-Inﬂammatory Effects of Genus Gynura: A Systematic Review. Front. Pharmacol. 11:504624. doi: 10.3389/fphar.2020.504624 November 2020 \| Volume 11 \| Article 504624 Frontiers in Pharmacology \| www.frontiersin.org Tan et al. Gynura Antioxidant and Anti-inﬂammatory Effects Results: Altogether, 27 studies on antioxidant and anti-inﬂammatory effects of Gynura species were selected. The antioxidant effects of Gynura species were manifested by inhibition of reactive oxygen species production and lipid peroxidation, modulation of glutathione-related parameters, and enzymatic antioxidant production or activities. The anti-inﬂammatory effects of Gynura species were through the modulation of inﬂammatory cytokine production, inhibition of prostaglandin E2 and nitric oxide production, cellular inﬂammatory-related parameters, and inﬂammation in animal models. The potential anti- inﬂammatory signaling pathways modulated by Gynura species are glycogen synthase kinase-3, nuclear factor erythroid 2-related factor 2, PPARγ, MAPK, NF-κB, and PI3K/Akt. However, most reports on antioxidant and anti-inﬂammatory effects of the plants were on crude extracts, and the chemical constituents contributing to bioactivities were not clearly understood. There is a variation in quality of studies in terms of design, conduct, and interpretation, and in-depth studies on the underlying mechanisms involved in antioxidant and anti-inﬂammatory effects of the plants are in demand. Moreover, there is limited clinical study on antioxidant and anti-inﬂammatory effects of Gynura species. Conclusion: This review highlighted antioxidant and anti-inﬂammatory effects of genus Gynura and supported their traditional uses to treat oxidative stress and inﬂammatory- related diseases. This review is expected to catalyze further studies on genus Gynura. However, extensive preclinical data need to be generated from toxicity and pharmacokinetic studies before clinical studies can be pursued for their development into clinical medicines to treat oxidative stress and inﬂammatory conditions. Keywords: Gynura, medicinal plant, reactive oxygen species, antioxidant, anti-inﬂammatory Keywords: Gynura, medicinal plant, reactive oxygen species, antioxidant, anti-inﬂammatory INTRODUCTION The fresh leaves of G. procumbens (Lour.) Merr. are traditionally consumed to control blood glucose level by Orang Asli in Kampung Bawong, Perak, West Malaysia (Samuel et al., 2010). G. procumbens is also traditionally used to treat kidney discomfort, inﬂammation, rheumatic fever, and viral ailments (Wiart, 2006). G. pseudochina (L.) DC. is traditionally used to treat fever and sore eye (Davies, 1980). In Chinese folk Frontiers in Pharmacology \| www.frontiersin.org Search Strategy gy The information was obtained through a comprehensive literature search using the electronic databases of PubMed, Scopus, and Science Direct from 2000 to March 2020 for journal articles and the databases of e-theses Online Service and ProQuest Dissertations and Theses Global for theses. The combination of keywords used in PubMed was as follows: [Gynura (Title/Abstract)] AND [antioxidant (Title/Abstract)] OR [oxidant (Title/Abstract)] OR [oxidative (Title/Abstract)] OR [oxidation (Title/Abstract)] OR [antiinﬂammatory (Title/Abstract)] OR [antiinﬂammation (Title/ Abstract)] OR [inﬂammation (Title/Abstract)] OR [inﬂammatory (Title/Abstract)] OR [inﬂame (Title/Abstract)]. The reference lists of all included papers were checked for other potentially relevant citations. Studies selection was restricted to articles in English because of language barrier, time efﬁciency, and high cost for translation. However, only four studies were excluded on the basis of not being published in English, which is unlikely to impact our ﬁndings. In order to achieve a comprehensive search of relevant studies, university dissertations, and theses were accessed in the selection process. However, the conﬁdentiality of these tools had possibly veiled some important information and results, for example, negative ﬁndings. Data Extraction and Handling g The following details were extracted from each selected study: 1) the species and part of the Gynura used; 2) methods used, including animal species or cell lines, study design, and treatment details; 3) outcome measures; and 4) ﬁndings on the antioxidant and anti-inﬂammatory effects of Gynura. METHODS This systematic review was carried out according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline (Moher et al., 2010). The quality of November 2020 \| Volume 11 \| Article 504624 Frontiers in Pharmacology \| www.frontiersin.org 2 Gynura Antioxidant and Anti-inﬂammatory Effects Tan et al. references was determined by referring to the Ofﬁce Health Assessment and Translation for Conducting a Systematic Review and Evidence Integration (OHAT) guideline (Rooney et al., 2014). any potential bias such as selection bias, performance bias, attrition or exclusion bias, detection bias, selection reporting bias, and other sources of bias. However, some of the questions in the bias assessment were excluded as they were not applicable for use in the assessment of in vitro, in vivo, and human randomized control study designs. Study Selection S udy Se ec o A total of 183 studies were found from the database search, and 11 additional articles were identiﬁed from other sources. After removing the duplicates, 125 articles were shortlisted, and after title–abstract screening, 57 articles were excluded because of the following reasons: The articles were published in languages other than English (n 4), the articles are non-experimental journal articles (n 10), and the articles reported on the effects of Gynura other than antioxidant and anti-inﬂammatory (n 43). By full-text screening of the remaining 68 articles, a total of 41 articles were eliminated based on the following exclusion criteria: study models used are not accepted as evidence for pharmacological effects (n 25), study intervention does not focus on Gynura (n 1), and a combination of both exclusion criteria (n 15). Thus, 27 articles from year 2002 to 2020 were selected in the ﬁnal qualitative analysis of this systematic review. Three potential dissertations and theses were accessed through the two databases. Only one of the theses was selected, but the result presented in the theses has been published in one of the article selected in this review (Siriwatanametanon et al., 2010). Other two theses were excluded because they did not fulﬁll the inclusion criteria. A ﬂowchart depicting the search process and study selection is presented in Figure 1. Quality Assessment Quality Assessment All selected articles were assessed for their quality and risk of bias. Table 1 shows the quality assessment of studies on the antioxidant and anti-inﬂammatory effects of Gynura species. The reporting completeness of the material in all selected studies was assessed using information of the Gynura species material, voucher specimens, quality control, and chemical analysis. Naming inconsistency between studies was detected as full botanical taxonomic names were not stated in 13 selected studies (Iskander et al., 2002; Siriwatanametanon and Heinrich, 2011; Akowuah et al., 2012; Seow et al., 2014; Chao et al., 2015; Wong et al., 2015; Xu et al., 2015; Rerknimitr et al., 2016; Yin et al., 2017; Rahman et al., 2018; Pai et al., 2019; Yang et al., 2019; Chandradevan et al., 2020). All plant species need to be validated taxonomically, and the full names have to be clearly stated as the incomplete name of plant species can lead to confusion of readers and any recorded uses or properties attributed may, in fact, correlate to different species. Such erroneous publications on the use of plant names are a permanent source of confusion for future research, search engines, and databases (Rivera et al., 2014). An incomplete plant name in some contexts may be ambiguous or may even mislead readers. This error is possible to occur within genus Gynura. Gynura divaricata is no guarantee to solely indicate Gynura divaricata (L.) DC. as Gynura divaricata subsp. barbareifolia (Gagnep.) F.G.Davies Inclusion and Exclusion Criteria Studies were included in this review if they were experimental studies (in vitro and in vivo) or clinical studies on the antioxidant or anti-inﬂammatory effects of Gynura and if they were articles published in English. The Gynura plant can be from any parts of the plant and in any form such as extracts, essential oils, fractions, or isolated compounds. Studies were excluded if they met at least one of the following criteria: 1) study models were not accepted as evidence for pharmacological effects (i.e., antioxidant experiments of FRAP, ABTS, DPPH, and Trolox equivalent antioxidant capacity assays), and 2) intervention was not focused on Gynura, although the antioxidant or anti-inﬂammatory effects were measured. Review articles and book chapters were excluded from this study, but the references were mined to search for further relevant studies. Quality Assessment y The reporting completeness of the material used by each selected study was assessed using the information of the Gynura species material, voucher specimens, report on quality control of extract and chemical analysis. The risk of bias in the included studies was assessed by two investigators based on the OHAT guideline for November 2020 \| Volume 11 \| Article 504624 Frontiers in Pharmacology \| www.frontiersin.org 3 Tan et al. Gynura Antioxidant and Anti-inﬂammatory Effects FIGURE 1 \| The ﬂowchart shows the selection process of (A) the journal articles, (B) university dissertations, and theses in this systematic review bas Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline. FIGURE 1 \| The ﬂowchart shows the selection process of (A) the journal articles, (B) university dissertations, and theses in this systematic review based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline. FIGURE 1 \| The ﬂowchart shows the selection process of (A) the journal articles, (B) university dissertations, and theses in this sy Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline. Frontiers in Pharmacology \| www.frontiersin.org November 2020 \| Volume 11 \| Article 504624 anti-inﬂammatory effects of Gynura species. Plant source Authenticated species Quality control reported? Chemical analysis reported? Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand − No No Penang Island, Malaysia + No No Merr Specialty Natural Products Co. Ltd., Thailand + No Yes—HPLC, electrospray ionization time-of-ﬂight mass spectrometry Merr Ethno Resources Sdn Bhd, Malaysia + No No Green House Facility, Faculty of Science and Technology, Universiti Kebangsaan Malaysia (UKM) + No No Merr Hainan Province, South China + No Yes—GC-MS Merr Jing’an, Jiangxi Province, China − No Yes—ultra-high-performance liquid chromatograph quadrupole time-of-ﬂight mass spectrometry Merr Semenyih, Selangor, Malaysia + No Yes—LC-MS/MS Merr Brightmark Sdn. Bhd, Semenyih, Malaysia + No No Merr Shanxi Jintai Biol, China − No Yes—LC-MS/MS Net house located in the Malaysia Agricultural, Research and development institute (MARDI) + No Yes—1H NMR spectroscopy lld.) DC. Yuanshan Village, Ilan, Taiwan + No Yes—HPLC Farms in Puli Town, Nanton county, Taiwan + No No Farms in Puli Town, Nanton county, Taiwan + No No Farms, unknown location − No No Farms, unknown location − No No var. Quality Assessment Farmland in north-eastern part of Thailand, mainly in Buriram province + No No hispida Not stated − No Yes—electrospray ionization-mass spectrometry, NMR, 13C NMR, 2D-NMR hispida Not stated − No No Koeng Sub-district, Mueang District, Maha Sarakham province, Thailand + No Yes—HP-TLC, HPLC, LC-MS/MS Jabatan Pertanian Relau, Penang, Malaysia + No No Yogyakarta, West Java, Indonesia + No Yes—HPLC Suburb of Shanghai, China + No Yes—HPLC Kendua under Netrokona district of Bangladesh + No Yes—method not stated Silk Biotechnology Laboratory, Soochow University, Suzhou, China − No No Silk biotechnology Lab, Soochow University, Suzhou, China − No Yes—HPLC Plant greenhouse of Longyan University, Longyan, China − No No r specimen; −, a voucher specimen is missing. Gynura Antioxidant and Anti-inﬂammatory Effects Tan et al. is a synonym of Gynura barbareifolia Gagnep and Gynura divaricata subsp. formosana (Kitam.) FG Davies is a synonym of Gynura formosana Kitam (The Plant List, 2020). is a synonym of Gynura barbareifolia Gagnep and Gynura divaricata subsp. formosana (Kitam.) FG Davies is a synonym of Gynura formosana Kitam (The Plant List, 2020). the most commonly used chemical techniques in the identiﬁcation and quality assessment of medicinal plant ingredients, have provided characteristic qualitative and quantitative patterns of the constituents. Spectroscopic techniques, including UV, IR, and nuclear magnetic resonance (NMR), allow the quantitation of single or multiple compounds that share similarities in their UV absorbance, thus providing a more holistic view of herbal medicines in contrast to the quantitation of a single compound (Upton et al., 2019). Chemical analysis of Gynura species were reported in studies conducted by Kim et al. (2011), Siriwatanametanon and Heinrich (2011), Wu et al. (2013), Yu et al. (2016), Yuandani et al. (2017), Rahman et al. (2018), Sukadeetad et al. (2018), Dong et al. (2019), Huang et al. (2019), Liu M. et al. (2019), Liu Y. et al. (2019), Nazri et al. (2019), and Chandradevan et al. (2020) using methods such as HP-TLC, HPLC, gas chromatography-mass spectrometer, electrospray ionization-mass spectrometry, electrospray ionization time-of-ﬂight mass spectrometry, liquid chromatography with tandem mass spectrometry, ultra-high- performance liquid chromatography-quadrupole time-of-ﬂight mass spectrometry, 1H N13C NMR, and 2D-NMR. Quality Assessment y f A total of 17 selected studies (Siriwatanametanon et al., 2010; Kim et al., 2011; Akowuah et al., 2012; Wu et al., 2013; Seow et al., 2014; Shwter et al., 2014; Chao et al., 2015; Wong et al., 2015; Yu et al., 2016; Yin et al., 2017; Yuandani et al., 2017; Rahman et al., 2018; Sukadeetad et al., 2018; Huang et al., 2019; Nazri et al., 2019; Ning et al., 2019; Chandradevan et al., 2020) provided the full information about the botanical material and authenticated the Gynura species by depositing voucher specimens. Six selected studies (Iskander et al., 2002; Xu et al., 2015; Ma et al., 2017; Dong et al., 2019; Liu M. et al., 2019; Liu Y. et al., 2019) reported the information of the Gynura species source but did not provide voucher specimens. Four selected studies (Siriwatanametanon and Heinrich, 2011; Rerknimitr et al., 2016; Pai et al., 2019; Yang et al., 2019) were inadequate on material information as the Gynura species source was not reported and voucher specimens were missing. The absence of voucher specimens seriously makes the reliability of the article suspicious. Sufﬁcient description of the experimental methods used and citation of voucher specimens as evidence of the plants used are critical factors of the repeatability of the ethnopharmacological or any botanical study. Otherwise, the scientiﬁc impact of a study will be drastically diminished. Erroneous identiﬁcation is a serious problem that may jeopardize any recorded uses or properties that may, in fact, relate to different species (Rivera et al., 2014). p y On the basis of the risk of bias score in Table 2, under the domain “other sources of bias,” ﬁve studies (Siriwatanametanon et al., 2010; Kim et al., 2011; Siriwatanametanon and Heinrich, 2011; Chao et al., 2015; Rahman et al., 2018) were rated as “deﬁnitely high” risk of bias because of the unclear or absence of statistical analysis. This ﬁnding raised the validity of the interpretations of these studies in question because the claimed signiﬁcant results are possibly misinterpreted under errors of statistical analysis. Hence, the erroneous statistical methods may contribute to false positive ﬁndings and may successively become misleading literature. Under the domain “Detection bias,” Iskander et al. (2002) and Rahman et al. (2018) were rated as deﬁnitely high risk of bias because the outcome assessment methods used were insensitive to indicate antioxidant or anti-inﬂammatory effects. Quality Assessment All selected studies showed no data on quality control as none of the Gynura preparation was mentioned to follow the monograph of pharmacopeia. The safety, efﬁcacy, and quality control of medicinal plants are getting more attention from both health authorities and the public. Herbal medicines are in widespread use, and the public believes that natural products are safe and devoid of adverse effects. However, medicinal plants are often used in combination, may be contaminated and adulterated, and may contain toxic compounds. The common misconception among the public often leads to inappropriate use and uncontrolled consumption, where poisoning and acute health problems are possible consequences. Hence, quality control of herbal medicines has a direct impact on their safety and efﬁcacy (Wachtel-Galor and Benzie, 2011). According to good manufacturing practice, the crucial requirements for quality control of starting materials include correct identiﬁcation of medicinal plant species, special storage, and special sanitation and cleaning methods for various materials (Ekor, 2014). Frontiers in Pharmacology \| www.frontiersin.org Study Characteristics Species of Gynura p y Of the 27 selected studies, there are seven species of genus Gynura, which are G. procumbens, G. bicolor, G segetum, G. divaricata, G. formosana, G. nepalensis, and G. pseudochina. Among these Gynura species, 11 out of 27 selected studies focused on G. procumbens (Iskander et al., 2002; Kim et al., 2011; Akowuah et al., 2012; Shwter et al., 2014; Wong et al., 2015; Huang et al., 2019; Liu M. et al., 2019; Liu Y. et al., 2019; Nazri et al., 2019; Ning et al., 2019; Chandradevan et al., 2020), ﬁve studies on G. bicolor (Wu et al., 2013; Chao et al., 2015; Yin et al., 2017; Pai et al., 2019; Yang et al., 2019), four studies on G. pseudochina (Siriwatanametanon et al., 2010; Siriwatanametanon and Heinrich, 2011; Rerknimitr et al., 2016; Sukadeetad et al., 2018), two studies on G. segetum (Seow et al., 2014; Yuandani et al., 2017), two studies on G. nepalensis (Yu et al., 2016; Rahman et al., 2018), two studies on G. divaricata (Xu et al., 2015; Dong et al., 2019), and one study on G. formosana (Ma et al., 2017). Identical plant species might exhibit phytochemical or From harvesting to manufacturing, the quality of herbal medicines can be affected by vast factors. Quality evaluation of medicinal plants is possible by detecting the presence of chemical markers within a sample (Li et al., 2008). Qualitative chemical evaluation covers identiﬁcation and characterization of phytochemical constituents in the medicinal plants through different analytical techniques. Phytochemical screening techniques involve botanical identiﬁcation, extraction with suitable solvents, puriﬁcation, and characterization of the active constituents of pharmaceutical importance (Folashade et al., 2012). Chromatographic methods such as thin-layer chromatography/high-performance TLC (TLC/HP-TLC) and high-performance liquid chromatography (HPLC), which are Identical plant species might exhibit phytochemical or bioactivity variations that can possibly be attributed to intrinsic factors (age of the plant and part of the plant used) and extrinsic Identical plant species might exhibit phytochemical or bioactivity variations that can possibly be attributed to intrinsic factors (age of the plant and part of the plant used) and extrinsic November 2020 \| Volume 11 \| Article 504624 Frontiers in Pharmacology \| www.frontiersin.org 6 Xu et al. 2015) 2 3 2 3 3 3 2 2 1 ing to OHAT guideline. andradevan et al. (2020) Wu et al. (2013) Chao et al. (2015) Yin et al. (2017) Pai et al. (2019) Yang et al. Study Characteristics Species of Gynura (2019) Siriwatanametanon et al. (2010) Siriwatanametanon and Heinrich (2011) Rerknimitr et al. (2016) Sukadeetad et al. (2018) Seow et al. (2014) Yuandani et al. (2017) Yu et al. (2016) Rahman et al. (2018) Xu et al. (2015) 2 2 2 2 2 2 2 2 2 2 3 2 2 3 2 3 3 3 3 3 3 3 3 1 3 3 3 3 3 3 2 2 3 1 2 1 2 3 NR 1 1 3 2 2 2 3 3 3 3 3 3 3 3 1 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 4 1 1 1 4 4 2 1 1 2 1 4 1 Gynura Antioxidant and Anti-inﬂammatory Effects Tan et al. as well as the aerial part of G. bicolor and G. divaricata (Chen et al., 2017). Hepatic injury caused by genus Gynura has been reported in few other studies. By daily administration of the root of G. segetum at 1.0 g extract/kg for 40 successive days, hepatic veno-occlusive disease, also called hepatic sinusoidal obstruction syndrome (HSOS), was induced in the mouse model where liver ﬁbrosis- related factors and pro-inﬂammatory cytokines were upregulated (Zhang et al., 2019). The mouse model in which a 30 g/kg decoction of G. segetum dried rhizome was administered by gavage in the morning for 30 days showed features of HSOS, including increased weight ratio of the liver and body, serum transaminase, bilirubin, decreased albumin (Chen et al., 2011), bulging abdomen, a large number of clear ascites, ﬂoating bowels, jelly omentum in the abdominal cavity, and congested and swollen or contracted and hard liver with a grainy surface and blunt edges (Zhu et al., 2011). The potential markers of hepatotoxicity induced by the decoction of G. segetum dried rhizome are veriﬁed in the animal model as differential metabolites of arginine, creatine, valine, glutamine, and citric acid, which are involved in the regulation of multiple metabolic pathways, primarily amino acid metabolism and energy metabolism (Qiu S. et al., 2018). G. Part of Plant Used in Studies Twenty selected studies used the leaves of Gynura to investigate their pharmacological effects. Nazri et al. (2019) and Ning et al. (2019) used the whole plant of G. procumbens, Xu et al. (2015) and Huang et al. (2019) used the leaf and stem parts of Gynura, Iskander et al. (2002) and Dong et al. (2019) used the aerial part of Gynura, and Liu M. et al. (2019) did not mention the part of Gynura used in their study. Hence, the leaf is the dominant part of the plant used in all selected research studies. Notably, the part of the plant that is usually consumed is the leaf. However, compared to the other parts of the plant, the root extract of both G. procumbens and G. bicolor exhibited the highest phenolic content, ﬂavonoid content, ascorbic acid content, and antioxidant capacities (Krishnan et al., 2015). Mice with intraperitoneal injection of G. procumbens leaf extract at 25, 50, 100, and 250 mg/kg/day for four days survived until day 30 of post-injection with no signs of toxicity such as diarrhea, excess urination, and lethargy (Vejanan et al., 2012). On the basis of the study by Zahra et al. (2011) and Shwter et al. (2014), no mortality, or organ toxicity was detected in the animals that were orally administered G. procumbens leaf extract at doses of 2 and 5 g/kg for 14 days. The study carried out by Algariri et al. (2014) demonstrated that the leaf extract of G. procumbens was safe, with no observed acute toxicity effects as there was no treatment-related mortality at 2 g/kg throughout the 14 days observation period. Hence, the oral lethal dose (LD50) of G. procumbens extract for rats was determined to be greater than 2,000 mg/kg, and the acceptable daily intake was 700 mg/kg/day. The same study also showed that consumption of 250, 500, and 1,000 mg/kg of G. procumbens extract for 28 days did not lead to sub-chronic toxicity as no abnormal behavior, disease, or death was observed in the animal model that received the plant extract. Administration of G. procumbens leaf extract at 1,000–5,000 mg/kg did not cause mortality or signiﬁcant changes in the general behavior, bodyweight, or organ gross appearance of rats (Rosidah et al., 2009). At the same time, G. procumbens and G. Study Characteristics Species of Gynura segetum dried rhizome also caused liver injury by dysregulating mitochondrial ROS generation through the SIRT3-SOD2 pathway (Li et al., 2019). The roots and aerial parts of G. segetum were proven to contain large amounts of pyrrolizidine alkaloids, including senecionine, which were responsible for the impaired bile acid homeostasis in the animal model orally administered 1.0 g/kg BW G. segetum extract for 48 h (Xiong et al., 2018). Hence, any contraindications caused by long- term consumption of genus Gynura must be determined prior to the commercialization of genus Gynura products. Appropriate clinical studies are needed to determine the optimal efﬁcacy and minimum toxicity of genus Gynura. The application of genus Gynura must be closely monitored in term of doses and qualities, especially the species with reported cases of hepatotoxicity. factors (geographical climate, nature of soil, season, and processing methods). The study conducted by Muraina et al. (2008) proved that the same plant extract from two different geographical locations varied in their phytochemical contents, cytotoxicity, antimicrobial and antioxidant activities. G. bicolor and G. divaricata extracts derived from plants originating from Nanjing were shown to possess higher free-radical-scavenging activities as compared to other same species extracts from different China origins (Chen et al., 2015). Hence, standardization and quality control of genus Gynura are crucial to gain regulatory approval of genus Gynura preparations as nutraceuticals or therapeutic drugs. Standardization of a drug can be used as a conﬁrmation of its identity, quality, and purity throughout all phases of its cycle (Kumari and Kotecha, 2016). Stability testing and toxicity proﬁles of herbal products are important parameters in improving herbal product safety. Frontiers in Pharmacology \| www.frontiersin.org Part of Plant Used in Studies bicolor leaf extracts both showed a negligible level of toxicity when administered orally at 300, 2,000, and 5,000 mg/kg for 14 days (Teoh et al., 2013, 2016). Sham grou Postmeno subjected cholestero heated pa Positive co Solvents Used for Extraction GP maintained SOD activity from month 3 to month 6 Oral route Daily for 24 weeks In vivo study using postmenopausal rats fed with cholesterol diet enriched with repeatedly heated palm oil Superoxide dismutase (SOD) activity Glutathione peroxidase (GSH-Px) activity Control groups Sham group (rats received basal diet) Postmenopausal (PM) group (rats subjected to ovariectomy, received 2% cholesterol diet fortiﬁed with ﬁve-time heated palm oil (5HPO) catalase (CAT) activity Positive control (10 mg/kg atorvastatin) (Continued on following page) ell line/Animal study model Concentration/dose, control groups Parameter measured and technique used Findings Referenc aT keratinocytes 1, 10, 50 μg/ml Intracellular ROS production level by dichloroﬂuorescein (DCF) content GP extract treatment inhibited UV- induced ROS generation levels about 36% at 50 μg/ml Kim et al. (2011) Control groups Normal control Model control (UV 40 mJ/cm2) Reference group (50, 100, 200 μg/ml vitamin C) dy stimulated by UV liver cell line NCTC-1469 80 and 160 μg/ml (24 h) Intracellular ROS production level ↓ROS level by 80 and 160 μg/ml GP extract Liu Y. et al. (2019) Control groups Normal control (culture medium) Model control (pre-treated with 0.25 mM palmitic acid (PA) + 0.5 mM oleic acid for 24 h) dy stimulated by palmitic leic acid dawley rats (6 rats/group) 1.0 g/kg body weight Plasma lipid peroxidation levels using thiobarbituric acid reactive substances (TBARS) assay ↓plasma TBARS level Akowuah et (2012) Oral route dy using carbon de (CCl4)-induced tress rats Daily single dose for 14 days TAS values of rats fed with the extract (1 g/kg) for 14 days followed by CCl4 administration were comparable to values of control group Control groups Plasma total antioxidant status (TAS) Normal control Model control (single dose of CCl4) e sprague-dawley rats (6 250 and 500 mg/kg body weight Lipid peroxidation levels using TBARS assay ↑GST and SOD activities in treated rats (250 and 500 mg/kg) Shwter et a (2014) Oral route Daily for 10 weeks Glutathione-S-transferase (GST) activity Superoxide dismutase (SOD) activity Control groups Normal control (normal saline subcutaneous injections, 10% tween 20 oral administration) dy using carcinogen- olon cancer rats ↓MDA level in treatment groups 250 and 500 mg/kg GP. e sprague-dawley rats (6 rats/ study using postmenopausal d with cholesterol diet carbon -induced s e-dawley rats (6 carcinogen- cer rats Solvents Used for Extraction Solvents Used for Extraction Five studies used the methanol extracts of G. procumbens, G. pseudochina and G. segetum to investigate their antioxidant or anti-inﬂammatory activities in HeLa cells (Siriwatanametanon et al., 2010), human leukocytes (Siriwatanametanon et al., 2010; Yuandani et al., 2017), murine macrophages (Yuandani et al., 2017), oxidative stress model rats (Akowuah et al., 2012), and granuloma model rats (Seow et al., 2014). As a continuation study of Siriwatanametanon et al. (2010), Siriwatanametanon and Heinrich (2011) as well as Yuandani et al. (2017) isolated compounds from the methanol extract of Gynura and determined their antioxidant or anti- inﬂammatory effects in HeLa cells or macrophages. The extraction of G. procumbens with 80% methanol at a temperature below 60°C would give greater retention of the total phenolic content (TPC) and greater expression of free radical scavenging activity (FRSA) (Akowuah et al., 2009). Methanol was also identiﬁed as a more effective extraction solvent of G. procumbens compared to 95% ethanol and water extracts based on its higher TPC and FRSA (Akowuah et al., 2012). Sukadeetad et al. (2018) demonstrated a higher chlorogenic acid content in G. pseudochina leaf extract by microwave drying as well as good efﬁciency for recovering phenolic compounds by extraction with 50% (v/v) methanol. However, However, the use of genus Gynura requires extra caution on toxic risk because of the presence of hepatotoxic pyrrolizidine alkaloids which was determined in the leaves of G. pseudochina (Siriwatanametanon and Heinrich, 2011; Sukadeetad et al., 2018) November 2020 \| Volume 11 \| Article 504624 8 nce al. et al. t al j , 20 oral administration) g g Carcinogen group (azoxymethane (AOM) subcutaneous injection +10% tween 20 oral administration) Reference group (AOM, ﬂuorouracil intraperitoneal injection) 48 female sprague-dawley rats (6 rats/ group) 250 and 500 mg/kg body weight Plasma malondialdehyde (MDA) level using high-performance liquid chromatography (HPLC) ↓MDA level in treatment groups 250 and 500 mg/kg GP compared to postmenopausal control group at month 3 and month 6 Nazri et al. (2019) ↑SOD, GSH-Px and CAT enzyme activities in treated rats (250 and 500 mg/kg) compared to postmenopausal control group. (2019) (2015) (2019) (2015) ge) ge) Solvents Used for Extraction (2015) 12 h pre-treatment catalase (CAT) activity Pre-treatment at 2 and 4% retained CAT and GSH-Px activities vitro study treated by high glucose Control groups Glutathione (GSH) content Control (5.5 mM glucose) Glutathione peroxidase activity (GSH- Px) Model control (33 mM glucose) 2 cell line (rat adrenal gland ochromocytoma) Aqueous extract at 0.25, 0.5 or 1% ROS level by 2′,7′-Dichloroﬂuorescein diacetate (DCFH-DA) GB pre-treatments ↑GSH content, ↑ GSH-Px activity, concentration- dependently ↓ROS level and ↑ catalase activity Yang et al. (2019) 48 h pre-treatment Glutathione level (GSH) Control groups Glutathione peroxidase activity (GSH- Px) Normal control CAT activity assay Model control (H2O2 stimulation) vitro study of H2O2 induced injury e C57BL/6 mice (8 mice/group) 0.25 or 0.5% G. bicolor aqueous extract diet Hepatic GSH or GSSG content 0.25% or 0.5% GB ↑GSH content, ↓ GSSG, ↓ROS levels, maintained GSH-Px, GR, and catalase activities Yin et al. (2017) vivo study using chronic ethanol onsumption-induced hepatic injury ice model Glutathione peroxidase activity (GSH- Px) Oral route Glutathione reductase (GR) and catalase 6 weeks Control groups Hepatic ROS level using DCFH-DA Normal control Liquid diet group (without ethanol) Ethanol diet group male Balb/cA mice (10 mice/group) 0.25, 0.5, 1% GB diet ROS level by DCFH-DA GB at 3 doses Pai et al. (2019) Oral route Glutathione level (GSH) 8 weeks ↑GSH content, ↑GPX, ↑GR, and ↑ catalase activities↓ROS level in heart and kidney Glutathione peroxidase activity (GSH- Px) Control groups Glutathione reductase (GR) Normal control (standard mouse basal diet) CAT activity assay Diabetic model control (40 mg/kg BW streptozotocin via i.p. injection for 5 days) vivo streptozotocin-induced type diabetic mice model male imprinting control region mice mice/group) Diets with 1.2% and 4.8% GD Glutathione peroxidase activity (GSH- Px) 1.2% GD ↑↑T-SOD levels, ↓↓ Xu et al. (2015) Oral route MDA level 4.8% GD Total superoxide dismutase activity (SOD) ↑↑GSH-Px level, ↑T-SOD level, ↓ MDA level Lipid peroxidation level by MDA level assay vivo study using high-fat diet and reptozotocin (STZ) induced type 2 abetic mice Daily for 4 weeks Control groups Normal control (normal diet) Diabetic model control (high-fat diet (18% lard, 20% sugar, 3% egg yolk, 59% basal diet) and 100 mg/kg STZ) (Continued on following page) Solvents Used for Extraction Carcinogen group (azoxymethane (AOM) subcutaneous injection +10% tween 20 oral administration) Reference group (AOM, ﬂuorouracil intraperitoneal injection) rague-dawley rats (6 rats/ 250 and 500 mg/kg body weight Plasma malondialdehyde (MDA) level using high-performance liquid chromatography (HPLC) ↓MDA level in treatment groups 250 and 500 mg/kg GP compared to postmenopausal control group at month 3 and month 6 Nazri et al. (2019) ↑SOD, GSH-Px and CAT enzyme activities in treated rats (250 and 500 mg/kg) compared to postmenopausal control group. GP maintained SOD activity from month 3 to month 6 Oral route Daily for 24 weeks dy using postmenopausal th cholesterol diet with repeatedly heated Superoxide dismutase (SOD) activity Glutathione peroxidase (GSH-Px) activity Control groups Sham group (rats received basal diet) Postmenopausal (PM) group (rats subjected to ovariectomy, received 2% cholesterol diet fortiﬁed with ﬁve-time heated palm oil (5HPO) catalase (CAT) activity Positive control (10 mg/kg atorvastatin) (Continued on following page) Nazri et (2019) wing page roup. G om mo ed on fo e sprague-dawley rats (6 rats/ study using postmenopausal d with cholesterol diet d i h dl h d carbon -induced s e-dawley rats (6 carcinogen- cer rats atedly heated g y Cell line/Animal study model Concentration/dose, control groups Parameter measured and technique used Findings Reference male C57BL/6 J mice (8 mice/ up) 500 and 1,000 mg/kg body weight Hepatic malondialdehyde (MDA) level ↓↓MDA level in treatment groups 500 and 1,000 mg/kg GP. Liu Y. et al. (2019) Oral route Glutathione peroxidase (GSH-Px) activity vivo study using non-alcoholic eatohepatitis (NASH) mice ↑↑GSH-Px, CAT and HO-1 activities in treated mice (500 and 1,000 mg/kg) Daily for 6 weeks catalase (CAT) activity Control groups Heme oxygenase 1 (HO-1) activity Normal control (methionine- and choline-sufﬁcient (MCS) diet) Model control (methionine- and choline-deﬁcient (MCD) diet) man umbilical vein endothelial cells Aqueous or ethanol extract at 1, 2 or 4% (v/v) ROS production level by 2′,7′- dichloroﬂuorescein (DCF) content Pre-treatments with aqueous or ethanol extract dose-dependently ↓ ROS level and preserved GSH content Chao et al. ge) (2016) 1 h pre-treatment Control groups y H2O2 CAT activity assay ↓↓↓ROS production in H2O2-treated cells, even at a concentration of 0.78 μM Normal control Model control (0.3 mM H2O2) ↑↑↑H2O2-induced decrease in CAT activity at doses of 25 and 50 μM s (8 rats/ 100 mg/kg, 250 mg/kg, 500 mg/kg body weight CAT activity assay 250 and 500 mg/kg GF treatment ↑↑ activities of CAT, ↑↑SOD, ↑↑GSH and ↓↓lipid peroxidation in rat liver Ma et al. (2017) Total superoxide dismutase activity (SOD) Oral route pellet- odel Once daily for 7 days Glutathione level (GSH) Lipid peroxidation level Control groups Normal control Model control (0.5% carboxymethylcellulose, 1 ml/kg) Standard drug (4 mg/kg indomethacin) ↑↑indicates signiﬁcantly induce (p < 0.001), ↓indicates signiﬁcantly inhibit (p < 0.05), ↓↓indicates signiﬁcantly inhibit (p < 0.01), and ↓↓↓indicates ge) OD level, ↓ ued on following MDA level 4.8% xide dismutase activity ↑↑GSH-Px leve MDA level ation level by MDA level GS de ca ) e activity (GSH- G content 0.2 GS GS e activity (GSH- (GR) and ng DCFH-DA A GB ) ↑G ca an e activity (GSH- (GR) e activity (GSH 1 2 ROS level by diacetate (DC Glutathione le Glutathione p Px) CAT activity a act Hepatic GSH Glutathione p Px) Glutathione re catalase H i ROS y for 4 weeks ol groups mal control (normal di betic model control (h % lard, 20% sugar, 3% % basal diet) and 100 sing high-fat diet and (STZ) induced type 2 (rat adrena cytoma) dy of H2O2 i 6 mice (8 m dy using chr on-induced l male Balb/cA mice (10 n vivo streptozotocin- diabetic mice model male imprinting contro i / ) a. Concentration/dose, control groups Parameter measured and technique used Findings Reference up) Diets with 1%, 5% and 10% GD Hepatic glutathione peroxidase activity (GSH-Px) 1% GD: ↑GSH-Px level, ↑T-SOD level Dong et al., (2019) at diet and ced type 2 Oral route 5% GD: ↑GSH-Px level, ↑↑T-SOD level, ↓8-OHdG level Daily for 4 weeks Hepatic total superoxide dismutase activity (SOD) Hepatic lipid peroxidation level by MDA level assay 10% GD: ↑↑GSH-Px level, ↑T-SOD level, ↓MDA level, ↓8-OHdG level Control groups Normal control (normal chow) Hepatic 8-hydroxy-2′- deoxyguanosine (8-OHdG) level using ELISA Model control (high-fat diet (18% lard, 20% sugar, 3% egg yolk and 59% basal diet) and 100 mg/kg STZ) utrophils Extract: 6.25–100 μg/ml ROS production level by luminol method GS extract exhibited inhibitory activity upon activation by PMA (IC50 1.41 ± 0.63 μg/ml) and zymosan (IC50 2.63 ± 0.89 μg/ml) Yuandani et al. (2017) Compounds: 3.125–50 μg/ml Control groups Negative control (without sample) 8,8′-(ethene-1,2-diyl)-dinaphtalene- 1,4,5-triol revealed ROS inhibitory upon activation by PMA (IC50 0.13 μM) and zymosan (IC50 0.05 μM) opsonized Positive control (acetylsalicylic acid) Rutin inhibited ROS activated by PMA (IC50 0.08 μM) and zymosan (IC50 0.13 μM) 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100 μM Intracellular ROS production by DCF content Compound 6 (3,5-dicaffeoylquinic acid ethyl ester) exhibited a more potent cytoprotective effect thus selected for further evaluation Yu et al. y level usin minol g/kg CAT activit Total supe (SOD) Glutathione Lipid perox kg) methacin) cantly induce (p < cantly induce (p < oup) at die ced t utrop opso by H2 ts (8 n pell odel ↑↑in al roup) -fat d uced neutro y ops by H rats (8 on pe mode ↑↑↑in Gynura Antioxidant and Anti-inﬂammatory Effects Tan et al. ethanol extract was used in this study for successive bioassays by considering ethanol as a safer solvent for health product application. production (Yin et al., 2017; Pai et al., 2019), superoxide dismutase (SOD) activity (Shwter et al., 2014; Xu et al., 2015; Ma et al., 2017; Dong et al., 2019; Nazri et al., 2019), heme oxygenase 1 (HO-1) (Liu Y. et al., 2019), 8-hydroxy-2′-deoxyguanosine (8-OHdG) level (Dong et al., 2019), and GSH-related parameters, including GSH- Px activity (Xu et al., 2015; Yin et al., 2017; Dong et al., 2019; Liu Y. et al., 2019; Nazri et al., 2019; Pai et al., 2019), GSH-S-transferase (GST) activity (Shwter et al., 2014), GSH reductase (GR) activity (Yin et al., 2017; Pai et al., 2019), GSH disulﬁde (GSSG) content (Yin et al., 2017), and GSH content (Ma et al., 2017; Yin et al., 2017; Pai et al., 2019). Eleven studies tested the antioxidant or anti-inﬂammatory activities of the ethanol extract of G. procumbens, G. bicolor, G. pseudochina and G. nepalensis in human keratinocytes (Kim et al., 2011; Sukadeetad et al., 2018), human dermal ﬁbroblasts (Kim et al., 2011), human endothelial cells (Chao et al., 2015), murine macrophages (Chandradevan et al., 2020; Liu M. et al., 2019; Ning et al., 2019), ear-inﬂamed mice (Iskander et al., 2002; Rahman et al., 2018), rat colon cancer model (Shwter et al., 2014), parasite-infected mice (Wong et al., 2015), and hypercholesterolemic rats (Nazri et al., 2019). Liu M. et al. (2019) simultaneously studied the anti-inﬂammatory effect of the ethanol extract and its fractions, including the petroleum ether fraction, ethyl acetate fraction, n-butanol fraction, and water fraction. Yu et al. (2016) studied the antioxidant and anti-inﬂammatory effects of nine caffeoylquinic acid analogs isolated from the ethanol extract on cardiomyoblasts. The optimal extraction of G. divaricata was determined with 45% ethanol for 30 min at 90°C, where the increase in the extraction temperature (from 40 to 100°C) led to a signiﬁcant elevation of the TFC, TPC, and FRSA (Wan et al., 2011). Anti-inﬂammatory Parameters Overall, in vitro anti-inﬂammatory studies on Gynura species focused on the secretion or expression of pro-inﬂammatory mediators, including interleukin-1 (IL-1) (Siriwatanametanon et al., 2010; Yuandani et al., 2017; Yang et al., 2019), IL-6 (Siriwatanametanon et al., 2010; Kim et al., 2011; Chao et al., 2015; Liu M. et al., 2019; Yang et al., 2019), IL-8 (Kim et al., 2011; Sukadeetad et al., 2018), tumor necrosis factor alpha (TNF-α) (Siriwatanametanon et al., 2010; Chao et al., 2015; Yuandani et al., 2017; Liu M. et al., 2019; Yang et al., 2019), prostaglandin E2 (PGE2) (Siriwatanametanon et al., 2010; Wu et al., 2013; Chao et al., 2015), cyclooxygenase-2 (COX-2) (Wu et al., 2013; Chao et al., 2015), inducible nitric oxide synthase (iNOS) (Wu et al., 2013; Ning et al., 2019), nitric oxide (NO) (Wu et al., 2013; Yuandani et al., 2017; Liu M. et al., 2019; Ning et al., 2019; Chandradevan et al., 2020), lactate dehydrogenase (LDH) activity, mitochondrial membrane potential (Δψm) (Yu et al., 2016; Yang et al., 2019), matrix metalloproteinase-1 and 9 (MMP-1 and MMP-9) (Kim et al., 2011), and inﬁltration of inﬂammatory cells (Huang et al., 2019). Moreover, in vitro studies included investigations on the anti-inﬂammatory effect of the Gynura species on the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway (Liu Y. et al., 2019), nuclear factor kappa B (NF-κB) signaling pathway (Siriwatanametanon et al., 2010; Siriwatanametanon and Heinrich, 2011; Wu et al., 2013; Sukadeetad et al., 2018; Yang et al., 2019), and mitogen-activated protein kinases (MAPK) signaling pathway, including c-Jun N-terminal kinase (JNK) (Yu et al., 2016; Liu Y. et al., 2019), p38 (Yu et al., 2016; Yang et al., 2019), and extracellular signal-regulated kinase (ERK) (Yu et al., 2016). Similarly, the in vivo studies also measured the parameters of IL-1 (Seow et al., 2014; Ma et al., 2017; Yin et al., 2017; Pai et al., 2019), IL-6 (Yin et al., 2017; Pai et al., 2019), and TNF-α (Seow et al., 2014; Wong et al., 2015; Ma et al., 2017; Yin et al., 2017; Dong et al., 2019; Pai et al., 2019). The other anti- inﬂammatory effect parameters of in vivo studies were interferon-γ (IFN-γ), IL-10, phosphorylation of glycogen synthase kinase-3 (GSK3β (Wong et al., 2015; Dong et al., 2019), LDH, alanine aminotransferase (GPT), c-reactive protein (CRP) (Ma et al., 2017), Nrf2 (Liu Y. Another study on the extraction method optimization of G. bicolor showed that 40% ethanol, 40°C, 30 min sonication time, and 50:1 liquid-to-solid ratio were the optimal conditions for a higher extraction yield of TPC (Qiu X. L. et al., 2018). Anti-inﬂammatory Parameters et al., 2019), peroxisome proliferator-activated receptor gamma (PPARγ) (Dong et al., 2019; Liu Y. et al., 2019), COX-2 (Huang et al., 2019), inﬂamed-ear thickness (Iskander et al., 2002; Rahman et al., 2018; Huang et al., 2019), cotton pellet granuloma (Seow et al., 2014; Ma et al., 2017), paw edema (Rahman et al., 2018; Huang et al., 2019), signaling pathways of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) (Xu et al., 2015; Dong et al., 2019), JNK (Liu Y. et al., 2019), p38 (Pai et al., Five studies used the aqueous extract of G. procumbens and G. bicolor to determine their antioxidant or anti-inﬂammatory activities in human endothelial cells (Chao et al., 2015), murine adrenal gland pheochromocytoma (Yang et al., 2019), murine hepatocytes (Liu Y. et al., 2019), diabetic mice (Pai et al., 2019), and liver-injured mice (Yin et al., 2017; Liu Y. et al., 2019). The ethyl acetate extract of G. formosana was studied for its antioxidant and anti-inﬂammatory activities in granuloma model rats (Ma et al., 2017). Meanwhile, the ether extract of G. bicolor was studied for its anti-inﬂammatory activity in murine macrophages (Wu et al., 2013). G. divaricata was lyophilized into powder and tested in diabetic mice (Xu et al., 2015; Dong et al., 2019). The study by Huang et al. (2019) determined the anti-inﬂammatory effect of G. procumbens essential oil and its active ingredients in murine macrophage and ear-inﬂamed mice. One randomized controlled study on patients with moderate plaque psoriasis was conducted to study the anti-inﬂammatory effect of G. pseudochina ointment from an ethanol extract with a ratio of one extract to 10 vehicles (Rerknimitr et al., 2016). Antioxidant Parameters In general, ROS production level was the major parameter measured in vitro antioxidant studies (Kim et al., 2011; Chao et al., 2015; Yu et al., 2016; Yuandani et al., 2017; Liu Y. et al., 2019; Yang et al., 2019). The other in vitro parameters included glutathione (GSH) content and GSH peroxidase (GSH-Px) or catalase (CAT) activity (Chao et al., 2015; Yu et al., 2016; Yang et al., 2019). The main focus of in vivo antioxidant studies of Gynura species was lipid peroxidation (Akowuah et al., 2012; Shwter et al., 2014; Xu et al., 2015; Ma et al., 2017; Dong et al., 2019; Liu Y. et al., 2019; Nazri et al., 2019). The other in vivo parameters measured were plasma total antioxidant status (TAS) (Akowuah et al., 2012), CAT activity (Ma et al., 2017; Liu Y. et al., 2019; Nazri et al., 2019; Pai et al., 2019), ROS November 2020 \| Volume 11 \| Article 504624 Frontiers in Pharmacology \| www.frontiersin.org 12 Gynura Antioxidant and Anti-inﬂammatory Effects Tan et al. product (Ayala et al., 2014). G. procumbens, G. divaricata, and G. formosana exhibited inhibition of lipid peroxidation by lowering the MDA level in carbon tetrachloride-induced oxidative stress rats (Akowuah et al., 2012), carcinogen-induced colon cancer rats (Shwter et al., 2014), high-fat-diet and streptozotocin (STZ)- induced diabetic mice (Xu et al., 2015; Dong et al., 2019), cotton pellet-induced granuloma rats (Ma et al., 2017), postmenopausal rats fed with cholesterol diet enriched with repeatedly heated palm oil (Nazri et al., 2019), and non-alcoholic steatohepatitis mice (Liu Y. et al., 2019). The reduction in the MDA level of G. procumbens extract-treated rats was comparable with treatments of anticancer drug, 5-ﬂuorouracil (Shwter et al., 2014), and lipid-lowering drug atorvastatin (Nazri et al., 2019). It is worth noticing that Xu et al. (2015) and Dong et al. (2019) both studied on G. divaricata using identical treatment methods and diabetic mice models. The results of the study by Xu et al. (2015) showed a signiﬁcant inhibitory effect of G. divaricata lyophilized powder on lipid peroxidation at treatment doses as low as 1.2% of diet. However, only 10% lyophilized powder diet was able to exert signiﬁcant inhibition of lipid peroxidation in the study by Dong et al. (2019). Gynura Inhibits Reactive Oxygen Species Gynura Inhibits Reactive Oxygen Species At physiological concentrations, ROS is involved in many cellular activities, including gene transcription, signaling transduction, and immune response. However, overproduction of ROS can result in oxidative damage to biomolecules, including lipids, proteins, and DNA, which is the underlying cause of various diseases (Liu et al., 2018). Inhibition of ROS production using G. procumbens extracts was determined in human HaCaT keratinocytes at 50 μg/ml (inhibitory effect comparable to 200 μg/ml vitamin C) (Kim et al., 2011) and in murine hepatocytes at 80 and 160 μg/ml (Liu Y. et al., 2019); that of G. bicolor in human umbilical vein endothelial cell (huvec) at 1–4% v/v (Chao et al., 2015), in adrenal gland pheochromocytoma in a concentration dependent manner (Yang et al., 2019), and in liver-injured mice at 0.25 and 0.5% diet (Yin et al., 2017); and that of G. segetum (IC50 for zymosan 2.63 ± 0.89 μg/ml; IC50 for PMA 1.41 ± 0.63 μg/ml) in polymorphonuclear cells (PMNs) neutrophils. The compound isolated from G. segetum extract, 8,8′-(ethene-1,2-diyl)- dinaphtalene-1,4,5-triol, possessed ROS inhibitory effect upon the activation by PMA (IC50 0.13 μM) and zymosan (IC50 0.05 μM). At the same time, rutin, which was also isolated from G. segetum extract, inhibited the ROS activated by PMA (IC50 0.08 μM) and zymosan (IC50 0.13 μM). Both isolated compounds from G. segetum showed a higher antioxidant effect than the positive control aspirin (Yuandani et al., 2017). One of the compounds isolated from G. nepalensis extract, 3,5-dicaffeoylquinic acid ethyl ester, showed an inhibitory effect on intracellular ROS production in cardiomyoblasts at a concentration of 0.78 μM and above (Yu et al., 2016). In nuclear and mitochondrial DNA, the interaction of the hydroxyl radical (HO•) with the nucleobases of the DNA strand will produce 8-hydroxy-2′-deoxyguanosine (8-OHdG), a predominant form of a free radical-induced oxidative lesion, which is a critical biomarker of oxidative stress (Valavanidis et al., 2009). G. divaricata diet (5 and 10%) showed the inhibition of the hepatic 8-OHdG level in a diabetic mice model (Dong et al., 2019). G. procumbens also improved the plasma TAS in rats with induced oxidative stress (Akowuah et al., 2012). Antioxidant Parameters This variation between ﬁndings could be due to the phytochemical or bioactivity variations that possibly correlated to intrinsic factors (age of the plant and part of the plant used) and extrinsic factors (geographical climate, nature of soil, season, and processing methods). 2019), and NF-κB (Dong et al., 2019; Pai et al., 2019). The effect of Gynura on NF-κB phosphorylation was also studied in a randomized controlled study (Rerknimitr et al., 2016). Gynura Modulates Enzymatic Antioxidant Production or Activities The antioxidant mechanism of heme oxygenase-1 (HO-1) is found to be associated with an increase in superoxide dismutase (SOD) and CAT (Turkseven et al., 2005). HO-1 is responsible for the oxidative cleavage of heme groups, which generates biliverdin, carbon monoxide, and ferrous iron. Biliverdin is converted to bilirubin, and both of these bile pigments are potent scavengers of singlet oxygen (Stocker et al., 1990). Oral administration of G. procumbens extract was shown to increase HO-1 activity in liver-injured mice model (Liu Y. et al., 2019). SOD is an antioxidant enzyme that is responsible for the catalytical conversion of the superoxide radical (pO2) or singlet oxygen radical (1O2 −) to hydrogen peroxide (H2O2) and molecular oxygen (O2). However, the accumulation of H2O2 causes toxicity to body tissues or cells. Successively, CAT breaks down H2O2 into water and molecular oxygen, thus minimizing free radical- induced damage (Ighodaro and Akinloye, 2018). G. procumbens, G. divaricata, and G. formosana showed an antioxidant effect by inducing in vivo total SOD activity (Shwter et al., 2014; Xu et al., 2015; Ma et al., 2017; Dong et al., 2019; Nazri et al., 2019). The in vitro CAT activity was induced by G. bicolor (Chao et al., 2015; Yang et al., 2019) and a compound isolated from G. nepalensis, which were 25 and 50 μM of 3,5-dicaffeoylquinic acid ethyl ester (Yu et al., 2016). At the same time, in vivo CAT activity was increased by G. procumbens (Liu Y. et al., 2019; Nazri et al., 2019), G. bicolor (Pai et al., 2019), and G. formosana (Ma et al., 2017). Notably, the induction of SOD and CAT by the G. procumbens extract was comparable to the effects of atorvastatin (Nazri et al., 2019). Antioxidant Effects of Genus Gynura y Table 3 shows the list of studies on the antioxidant effects of genus Gynura. Several potential mechanisms for the antioxidant activity of Gynura are suggested as follows: inhibition of ROS, inhibition of lipid peroxidation, modulation of enzymatic antioxidant production or activities, and modulation of GSH- related parameters. Figure 2 illustrates the proposed signaling pathways of antioxidant effects by Gynura species. Gynura Inhibits Prostaglandin E2 and Nitric Oxide Production Prostaglandin E2 (PGE2) is a bioactive lipid that physiologically mediates the regulation of immune responses, blood pressure, gastrointestinal integrity, and fertility. However, the sequential actions of cyclooxygenase-2 (COX-2) catalyze PGE2 synthesis. Consequently, dysregulated PGE2 production has been correlated to a variety of pathological conditions, such as chronic inﬂammation (Legler et al., 2010). Inhibition of COX-2 expression in mice model was exerted by G. procumbens essential oil and its active ingredients. Both G. procumbens essential oil and its active ingredients were showing similar COX-2 inhibitory effects as that of the positive control drug diclofenac diethylamine emulgel (Huang et al., 2019). The use of the G. bicolor extract inhibited PGE2 production and COX- 2 protein expression and activity in activated macrophages (Wu et al., 2013) and endothelial cells (Chao et al., 2015). PGE2 production was also inhibited using the extract of G. pseudochina var. hispida with an IC50 value of 25.23 μg/ml (Siriwatanametanon et al., 2010). Anti-inﬂammatory Effects of Genus Gynura Table 4 shows the studies on the anti-inﬂammatory effects of Gynura. The potential mechanisms for the anti-inﬂammatory activity of Gynura are suggested as follows: modulation of inﬂammatory cytokine production, inhibition of PGE2 and NO production, inhibition of cellular inﬂammatory-related parameters, and inhibition of inﬂammation on animal models. As shown in Figure 2, the several potential anti-inﬂammatory signaling pathways of Gynura are PI3K/Akt, Nrf2, PPARγ, GSK3, NF-κB, and MAPK. Anti-inﬂammatory Effects of Genus Gynura Table 4 shows the studies on the anti-inﬂammatory effects of Gynura. The potential mechanisms for the anti-inﬂammatory activity of Gynura are suggested as follows: modulation of inﬂammatory cytokine production, inhibition of PGE2 and NO production, inhibition of cellular inﬂammatory-related parameters, and inhibition of inﬂammation on animal models. As shown in Figure 2, the several potential anti-inﬂammatory signaling pathways of Gynura are PI3K/Akt, Nrf2, PPARγ, GSK3, NF-κB, and MAPK. Gynura Modulates Inﬂammatory Cytokine Production Cytokines are released by cells for signaling, where critical pro- inﬂammatory cytokines, including IL-1, IL-6, IL-8, IFN-γ, and TNF-α, are involved in the upregulation of inﬂammatory reactions (Turner et al., 2014) and IL-10 is a potent anti-inﬂammatory cytokine (Zhang and An, 2007). G. procumbens inhibited the IL-6 and IL-8 production in UV-induced HaCaT keratinocytes (Kim et al., 2011). G. procumbens extract also dose-dependently suppressed the IL-6 production in macrophages and inhibited the TNF-α level. At the same time, the ethyl acetate fraction of G. Gynura Inhibits Lipid Peroxidation Lipid peroxidation is a reaction of oxygen with unsaturated lipids that successively results in the production of oxidation products, including lipid peroxyl radicals and hydroperoxides. Among a wide variety of oxidation products from lipid peroxidation, malondialdehyde (MDA) appears to be the most mutagenic Gynura Modulates Glutathione-Related Parameters Owing to the absence of CAT in the mitochondria, the reduction of H2O2 and lipid peroxides is carried out by GSH peroxidase November 2020 \| Volume 11 \| Article 504624 Frontiers in Pharmacology \| www.frontiersin.org 13 Gynura Antioxidant and Anti-inﬂammatory Effects Tan et al. hispida showed strong inhibition of the release of IL-6 (IC50 8.14 μg/ml) and TNF-α (IC50 1.49 μg/ml) in monocytes (Siriwatanametanon et al., 2010). The extract of G. pseudochina and its marker compounds of chlorogenic acid, caffeic acid, rutin, and p-coumaric acid also showed signiﬁcant inhibition of IL-8 production in keratinocytes (Sukadeetad et al., 2018). G. segetum extract inhibited the release of TNF-α (IC50 16.20 ± 3.94 μg/ml) and IL-1β (IC50 2.72 ± 1.84 μg/ml) in macrophages. Among the isolated compounds of G. segetum, 4,5,4′-trihydroxychalcone was the most potent sample in inhibiting IL-1β (IC50 6.69 μM), and another isolated compound, rutin, demonstrated the strongest inhibition against TNF-α release in macrophages (IC50 16.96 μM) (Yuandani et al., 2017). The anti-inﬂammatory effects of G. segetum and G. formosana have been demonstrated by the in vivo inhibition of TNF-α and IL-1, where the inhibitory effects of 500 mg/kg of G. segetum and G. formosana extracts were comparable to that of the nonsteroidal anti-inﬂammatory drug indomethacin (Seow et al., 2014; Ma et al., 2017). G. divaricata also suppressed TNF-α production in a diabetic mice model (Dong et al., 2019). (GSH-Px). Therefore, protection of cells against oxidative stress is reinforced by increasing the activity of GSH-Px, which plays a crucial role in inhibiting the lipid peroxidation process (Ighodaro and Akinloye, 2018). GSH-Px activity was induced by G. procumbens (Liu Y. et al., 2019; Nazri et al., 2019), G. bicolor (Chao et al., 2015; Yin et al., 2017; Pai et al., 2019; Yang et al., 2019), and G. divaricata (Xu et al., 2015; Dong et al., 2019). G. procumbens showed higher efﬁcacy in inducing GSH-Px activity as compared to atorvastatin (Nazri et al., 2019). GSH is a crucial low- molecular-weight antioxidant that protects cells from oxidative damage via reduction, conjugation, and interaction with other non-enzymatic antioxidants (Forman et al., 2009). Gynura Inhibits Lipid Peroxidation G. formosana extract diet (250 and 500 mg/kg) was found to increase hepatic GSH level in granuloma rat model (Ma et al., 2017), and G. bicolor extract also showed in vitro and in vivo preservation on GSH content (Chao et al., 2015; Yin et al., 2017; Pai et al., 2019; Yang et al., 2019). GSH reductase (GR) is vital in maintaining the supply of reduced GSH, and GSH disulﬁde (GSSG) indicates the level of oxidized GSH (Chakravarthi et al., 2006). G. bicolor extract (0.25, 0.5, and 1% of diet) maintained GR activity (Yin et al., 2017; Pai et al., 2019) and suppressed the hepatic GSSG content in the liver-injured mice model (Yin et al., 2017). GSH-S-transferase (GST) exerted protection on cellular macromolecules from damage of reactive electrophiles by catalyzing the conjugation of GSH to various endogenous and exogenous electrophilic compounds (Townsend and Tew, 2003). G. procumbens extract diet showed antioxidant potential by increasing in vivo GST activity (Shwter et al., 2014). Gynura Inhibits Prostaglandin E2 and Nitric Oxide Production Gynura Inhibits Prostaglandin E2 and Nitric Oxide Production procumbens showed the best inhibitory effect on IL-6 and TNF-α (Liu M. et al., 2019). In a parasite-infected mice model, G. procumbens reduced the levels of the liver and serum TNF-α and IFN-γ as well as increased IL-10 level (Wong et al., 2015). G. bicolor possessed an anti-inﬂammatory effect by inhibiting the production of IL-1β, IL-6, and TNF-α both in vitro (Chao et al., 2015; Yang et al., 2019) and in vivo (Yin et al., 2017; Pai et al., 2019). G. pseudochina var. hispida methanol extract also caused inhibition of IL-1β production (IC50 2.46 μg/ml). Meanwhile, ethyl acetate extract of G. pseudochina var. Nitric oxide (NO) is a signaling molecule that physiologically exerts an anti-inﬂammatory effect and acts as a pro-inﬂammatory mediator in the overproduction of the enzyme iNOS (Sharma et al., 2007). G. procumbens extract had shown an inhibitory effect on NO production in macrophages (Liu M. et al., 2019; Ning et al., 2019; Chandradevan et al., 2020). This signiﬁcant NO inhibitory effect by 250 μg/ml G procumbens was comparable to the positive control of the non-selective NOS inhibitor Nω-nitro-l-arginine methyl ester hydrochloride (L-NAME) (Ning et al., 2019). G. procumbens fractions (petroleum ether, ethyl acetate, n-butanol, and water) at concentrations of 0.3–0.8 mg/ml also exerted 2–64% inhibition of NO production in macrophages. Among the fractions, the ethyl acetate fraction possessed the highest NO inhibitory effect (Liu M. et al., 2019). The protein expression of iNOS in macrophages was shown to be inhibited by the G. procumbens extract at a concentration of 250 μg/ml (Ning et al., 2019). Similarly, G. bicolor showed concentration dependent November 2020 \| Volume 11 \| Article 504624 Frontiers in Pharmacology \| www.frontiersin.org 14 Gynura Antioxidant and Anti-inﬂammatory Effects Tan et al. FIGURE 2 \| A schematic illustrating the proposed signaling pathways of the antioxidant and anti-inﬂammatory effects of Gynura species. The antioxidant effects of Gynura are manifested by the inhibition of reactive oxygen species (ROS) production, suppression of lipid peroxidation, modulation of enzymatic antioxidant production or activities, and modulation of glutathione-related parameters. The reported anti-inﬂammatory effects of Gynura are modulation of inﬂammatory cytokines and molecule production as well as inhibition of cellular inﬂammation and inﬂammation in an animal model. Gynura Inhibits Prostaglandin E2 and Nitric Oxide Production The potential anti-inﬂammatory signaling pathways of Gynura include the induction of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), nuclear factor erythroid 2-related factor 2 (Nrf2), and peroxisome proliferator-activated receptor gamma (PPARγ) signaling pathways as well as the inhibition of the nuclear factor kappa B (NF-κB), glycogen synthase kinase 3 (GSK3), and mitogen-activated protein kinase (MAPK) signaling pathways. Dark blue arrow lines indicate activation, dark blue perpendicular lines indicate inhibition, green tick indicates induction by Gynura species, and red cross indicates suppression by Gynura species. FIGURE 2 \| A schematic illustrating the proposed signaling pathways of the antioxidant and anti-inﬂammatory effects of Gynura species. The antioxidant effects of Gynura are manifested by the inhibition of reactive oxygen species (ROS) production, suppression of lipid peroxidation, modulation of enzymatic antioxidant production or activities, and modulation of glutathione-related parameters. The reported anti-inﬂammatory effects of Gynura are modulation of inﬂammatory cytokines and molecule production as well as inhibition of cellular inﬂammation and inﬂammation in an animal model. The potential anti-inﬂammatory signaling pathways of Gynura include the induction of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), nuclear factor erythroid 2-related factor 2 (Nrf2), and peroxisome proliferator-activated receptor gamma (PPARγ) signaling pathways as well as the inhibition of the nuclear factor kappa B (NF-κB), glycogen synthase kinase 3 (GSK3), and mitogen-activated protein kinase (MAPK) signaling pathways. Dark blue arrow lines indicate activation, dark blue perpendicular lines indicate inhibition, green tick indicates induction by Gynura species, and red cross indicates suppression by Gynura species. responses (Yue and Yao, 2016). In a concentration dependent manner, Δψm was increased, and LDH activity was inhibited by G. bicolor extract (Yang et al., 2019) and the isolated compound from the G. nepalensis, 3,5-dicaffeoylquinic acid ethyl ester (Yu et al., 2016). The induction of Δψm by the isolated compound of G. nepalensis was more effective than that of carbonyl cyanide m-chlorophenyl hydrazone, a chemical inhibitor of oxidative phosphorylation (Yu et al., 2016). G. formosana also suppressed plasma inﬂammatory biomarker (LDH, GPT, and CRP) activities in the treated group, where these inhibitory effects were comparable to indomethacin (Ma et al., 2017). The essential oil of G. procumbens and its active ingredient, limonene, showed an inhibitory effect on inﬂammatory cell inﬁltration (Huang et al., 2019). suppression on iNOS protein expressions and NO production (30% decrease with 120 μg/ml extract) in macrophages (Wu et al., 2013). G. Gynura Inhibits Prostaglandin E2 and Nitric Oxide Production segetum extract also inhibited NO production in macrophages, with IC50 0.16 ± 0.03 μg/ml. Meanwhile, one of the compounds isolated from G. segetum extract, 8,8′-(ethene-1,2- diyl)-dinaphtalene-1,4,5-triol, depicted the strongest NO inhibitory activity with an IC50 value of 0.15 μM (Yuandani et al., 2017). Gynura Inhibits Cellular Inﬂammatory-Related Parameters Matrix metalloproteinases (MMPs) act as crucial regulatory enzymes in both pro- and anti-inﬂammatory pathways through the actions of cytokine or chemokine activation and antagonism (Manicone and McGuire, 2008). G. procumbens dose-dependently inhibited MMP-1 and MMP-9 expression in human dermal ﬁbroblasts (HDFs), where the inhibition of the MMP-1 expression by the G. procumbens extract was even more effective than that of the positive control drug retinoic acid (Kim et al., 2011). Mitochondrial membrane potential (Δψm), the energy provider to generate ATP, is produced when free energy is used to pump protons out of the mitochondrial matrix via oxidative phosphorylation. Under a stress condition, Δψm can be reduced and can possibly lead to mitochondrial dysfunction in inﬂammatory Gynura Inhibits Inﬂammation in Animal Models Gynura Inhibits Inﬂammation in Animal Models Tissue swelling (edema) is one of the cardinal signs of inﬂammation, where the increased ﬂuid ﬁltration is further enhanced by the arteriolar vasodilator action of the inﬂammatory mediators (Amelang et al., 1981). G. nepalensis extract diet (Rahman et al., 2018), topical administration of G. procumbens extract (Iskander et al., 2002), G. procumbens essential oil, and the active ingredients mixture from the G. procumbens essential oil (Huang et al., 2019) showed anti- November 2020 \| Volume 11 \| Article 504624 Frontiers in Pharmacology \| www.frontiersin.org 15 mal ﬁbroblasts (HDFs) 1, 10, 20 μg/ml (48 h) MMP-1 expression using western blotting udy stimulated by UV Control groups: MMP-9 expression using Zymography GP extract dose-dependently inhibited MMP-1 and MMP-9 expression in UV-B irradiated HDFs. Normal control Model control (UV 40 mJ/cm2) Positive control (10 μM Retinoic acid) macrophages GPEO: 0.003, 0.01, 0.03 μg/ml Inhibition on inﬂammatory cell inﬁltrates using migration assay GPEO ↓↓↓LPS-induced cell migration. Limonene, but not α-pinene, 3-carene, or their components mixture, ↓↓↓cell migration. Huang et al. (2019) udy stimulated by LPS Active ingredients: concentration not stated Treatment time not stated Control groups: Normal control Model control (LPS) udy stimulated by LPS 0.4, 0.6, 0.8 mg/ml TNF-α and IL-6 production level using ELISA GP extract dose-dependently ↓IL-6. GP extract at 0.8 mg/ml ↓TNF-α. Ethyl acetate fraction showed the best inhibitory effect on IL-6 and TNF- α. Liu M. et al. (2019) 24 h incubation with LPS Control groups: Normal control Model control (1 μg/ml LPS) macrophages 3.9, 15.63, 62.5 and 250 μg/ml Nitric oxide production level using Griess assay Pre-treatment of 250 μg/ml GP: Ning et al. (2019) udy stimulated by LPS 1 h pre-treatment iNOS protein expression using western blotting ↓NO production (dose dependent) Control groups: ↓iNOS protein expression. Normal control Vehicle control (0.1% DMSO) Model control (1 μg/ml LPS) l liver cell line NCTC- 80 and 160 μg/ml (24 h) Nrf2 and p-JNK protein expressions using western blotting 80 μg/ml GP treatment ↑Nrf2 protein level, ↓p-JNK Liu Y. et al. (2019) udy transfected with Ad- Control groups: 160 μg/ml GP treatment ↑Nrf2 protein levels, ↓↓p-JNK Normal control (Ad-shCtrl) Model control (pre-treated with Ad- shCFLAR for 24 h) macrophages 15.63, 31.25, 62.5, 125, 250, 500 μg/ ml Nitric oxide production level using Griess assay GP extract showed anti-inﬂammatory activity by inhibiting NO production Chandradevan et al. o Mo 0.75 To 30 wa Cont Mo ea Sta (hy enzyme iNOS, CO and p65 immunob v) NF-κB D Nuclear electroph (EMSA Control groups: Vehicle control gr methanol) Model control (1 Gynura Inhibits Inﬂammation in Animal Models (2020) udy stimulated by LPS Control groups: Normal control Model control (IFN-γ + LPS) e mice (5 mice/group) 0.75 mg/20 μl Ear thickness (anti-inﬂammatory activity) Original organic crude extract ↓↓↓ croton oil-induced ear inﬂammation. Iskander et al. (2002) udy using croton oil- ear inﬂammation mice Topical application 30 min pre-treatment, ear thickness was measured at 24 h Control groups: Model control (20 μl acetone at left ear + 0.1 mg/20 μl/ear croton oil) Standard anti-inﬂammatory agent (hydrocortisone) (Continued on following page) 02) age) 02) page) c crude e ced ear in ntinued on following nd COX-2 prot ns (concentrati t) protein (47% de GB) p65 protein lev with 30, 60, 12 ation of NF-κB nuclei nding activity o otein by 30 60 κBα ng ↓iNO expr depe ay ↓p-I 120 ↓nu decr ↓tra cytos ↓DN l GB f2 and p-JNK protein expressio ng western blotting RNA expression of PPARγ usin T-qPCR ric oxide production level using ess assay GE2 production using competitiv COX-2 e immunoh multispec on of % grou kg body weig itoneally trea y pre-infectio groups: mal control el control (B. ion) O: 0.433, 0.8 of all 3 ingr cal administr groups: ated contro el control (to me oil + 20 ermal inject alin) ve control (d l i ice (8 mice/ non-alcoho SH) mic ages on/dose, control roups Parameter measured and technique used Findings Reference nol extract at 1, 2 or IL-6 and TNF-α production using cytoscreen immunoassay Pre-treatments with aqueous or ethanol extract of GB at 2 and 4% Chao et al. (2015) ment Prostaglandin E (PGE2) production ↓IL-6, TNF-α formation Cyclooxygenase (COX)-2 activity ↓PGE2 formation M glucose) ↓COX-2 activity. (33 mM glucose) at 0.25, 0.5 or 1% LDH activity ↓LDH activity in concentration dependent manner Yang et al. (2019) ment Mitochondrial membrane potential (Δψm) using ﬂuorescent dye Rh123 ↑Δψm in concentration dependent manner IL-6, IL-1β and TNF-α production level by cytoscreen assay ↓IL-6, IL-1β and TNF-α production level NF-κB and p38 mRNA expression using RT-PCR Test concentrations (0.25, 0.5 and 1%) ↓NF-κB mRNA expression (H2O2 stimulation) 0.5 and 1% extract ↓p38 mRNA expression. bicolor aqueous Hepatic levels of IL-1β, IL-6 and TNF- α using cytoscreen immunoassay kits GB dose-dependently ↓hepatic levels of inﬂammatory cytokines of IL- 1β, IL-6 and TNF-α. Yin et al. (2017) up (without ethanol) oup B diet Cardiac or renal level of IL-1β, IL-6 or TNF-α using cytoscreen immunoassay kits GB at 3 doses: Pai et al. (2019) p38 and NF-κB mRNA expression using RT-PCR ↓level of IL-1β, IL-6, and TNF-α in heart and kidney ↓p38 and NF-κB mRNA expression in heart or kidney (basal diet) control (40 mg/kg ocin (in citrate buffer, njection for 5 days) (Continued on following page) cytoscreen immunoassay ethanol extract of GB at 2 and 4% ment Prostaglandin E (PGE2) production ↓IL-6, TNF-α formation Cyclooxygenase (COX)-2 activity ↓PGE2 formation mM glucose) ↓COX-2 activity. (33 mM glucose) at 0.25, 0.5 or 1% LDH activity ↓LDH activity in concentration dependent manner Yang et al. (2019) ment Mitochondrial membrane potential (Δψm) using ﬂuorescent dye Rh123 ↑Δψm in concentration dependent manner IL-6, IL-1β and TNF-α production level by cytoscreen assay ↓IL-6, IL-1β and TNF-α production level ol NF-κB and p38 mRNA expression using RT-PCR Test concentrations (0.25, 0.5 and 1%) ↓NF-κB mRNA expression (H2O2 stimulation) 0.5 and 1% extract ↓p38 mRNA expression. bicolor aqueous Hepatic levels of IL-1β, IL-6 and TNF- α using cytoscreen immunoassay kits GB dose-dependently ↓hepatic levels of inﬂammatory cytokines of IL- 1β, IL-6 and TNF-α. Yin et al. (2017) ol oup (without ethanol) roup B diet Cardiac or renal level of IL-1β, IL-6 or TNF-α using cytoscreen immunoassay kits GB at 3 doses: Pai et al. (2019) p38 and NF-κB mRNA expression using RT-PCR ↓level of IL-1β, IL-6, and TNF-α in heart and kidney ↓p38 and NF-κB mRNA expression in heart or kidney ol (basal diet) el control (40 mg/kg tocin (in citrate buffer, injection for 5 days) (Continued on following page) inhibitory effects, as well as inhibition on release of IL-1, IL-6, TNF-α and PGE2. production level using ELISA and EIA roups: e control (ethanol) ve control (Unstimulated cells) nce group (Parthenolide, ortisone) c concentrations (using the ay) IL-6/luciferase assay (NF-κB assay) Quercetin 3-rutinoside showed the highest NF-κB inhibitory effect. Siriwatanametanon and Heinrich (2011) roups: NF-κB inhibitory activities IC50: e control (ethanol) Quercetin 3-rutinoside: 24.1 ± 0.1 μg/ml ve control (Unstimulated cells) 3,5-di-caffeoylquinic acid: 42.8 ± 0.2 μg/ml nce group (Parthenolide) 4,5-di-caffeoylquinic acid: 49.1 ± 0.1 μg/ml 5-mono-caffeoylquinic acid: 83.0 ± 0.1 μg/ml 375 and 750 μg/ml IL-8 production level using ELISA Extract at both tested concentrations and some concentration of each marker compounds: Sukadeetad et al. (2018) nic acid: 140 and 280 μg/ml RelA and RelB localization by immunoﬂuorescence assay ↓IL-8 production level cid: 30 and 60 μg/ml ↓translocation of RelB S573 into nucleus 0 and 1,500 μg/ml ric acid: 1,400 and 2,800 μg/ roups: l control control (50 ng/ml TNF-α with/ t 0.7% DMSO) e control (50 μg/ml curcumin) f extract and vehicle (1:10) Phosphorylation of NF-κB p65 using immunohistochemistry (skin sections from two patients) Immunohistochemical staining revealed diminution of phosphorylated NF-κB p65 in the lesions treated with the GP ointment. Rerknimitr et al. nce daily fo trol groups: egative cont eference gro domethacin (2019) route phosphatidylinositol 3-kinase (PI3K) y for 4 weeks phosphorylated protein kinase B (p- Akt) ol groups: phosphorylated glycogen synthase kinase 3β (p-GSK3β) 5% GD: ↑PI3K, ↑p-Akt, ↑p-GSK3β, ↑PPARγ, ↓TNF-α mal control (Normal chow) PPARγ control (high-fat diet (18% lard, ugar, 3% egg yolk and 59% diet) and 100 mg/kg STZ) Tumor necrosis factor-α (TNF-α) 10% GD: ↑PI3K, ↑↑p-Akt, ↑p- GSK3β, ↑↑PPARγ, ↓TNF-α, ↓NF-κB Nuclear factor kappa B (NF-κB) t: 6.25–100 μg/ml Nitric oxide production level using Griess assay GS extract showed inhibition on NO production with IC50 0.16 ± 0.03 μg/ml 8,8′-(ethene-1,2-diyl)- dinaphtalene-1,4,5-triol depicted the strongest NO inhibitory activity with IC50 value of 0.15 μM. Yuandani et al. (2017) ounds: 3.125–50 μg/ml IL-1β and TNF-α production level using ELISA GS extract showed IC50 TNF-α 16.20 ± 3.94 μg/ml; IC50 IL-1β 2.72 ± 1.84 μg/ml 4,5,4′- trihydroxychalcone demonstrated the highest inhibition on IL-1β release (IC50 6.69 μM). Rutin was the most potent sample against TNF-α release with IC50 16.96 μM. Seow et al. (2014) (Griess assay) or 12 h pre- tment (ELISA) ol groups: tive control (0.025 μM amethasone) 50 and 500 mg/kg body weight Cotton pellet granuloma assay GS dose-dependently ↓↓↓formation of granuloma tissues (17.1, 39.7, and 47.2% inhibition by 125, 250 and 500 mg/kg GS), ↓↓↓TNF-α and IL-1 levels in circulating pro-inﬂammatory cytokine levels. route TNF-α and IL-1 production level using ELISA e daily for 7 days ol groups: ative control (1% Tween 80) erence group (5 mg/kg methacin) (Continued on following page) ats 125, 250 and 500 mg/kg body weight Cotton pellet granuloma assay GS dose-dependently ↓↓↓formation of granuloma tissues (17.1, 39.7, and 47.2% inhibition by 125, 250 and 500 mg/kg GS), ↓↓↓TNF-α and IL-1 levels in circulating pro-inﬂammatory cytokine levels. tton pellet- t model Oral route TNF-α and IL-1 production level using ELISA Once daily for 7 days Control groups: Negative control (1% Tween 80) Reference group (5 mg/kg indomethacin) (Continued on following page) age) Yuandani et al. (2 nued on following dinaphtalene-1,4,5-triol depicted the strongest NO inhibitory activity with IC50 value of 0.15 μM. GS extract showed IC50 TNF-α 16.20 ± 3.94 μg/ml; IC50 IL-1β 2.72 ± 1.84 μg/ml 4,5,4′- trihydroxychalcone demonstrated the highest inhibition on IL-1β release (IC50 6.69 μM). Rutin was the most potent sample against TNF-α release with IC50 16.96 μM. E 0) at diet yolk, kg D W pr ph echnique Akt, PI3K, essions us p-Akt, PI3K ressions us (2016) ily for 4 weeks 0.1% triamcinolone cream (Continued on following page) on release of IL-1, IL-6, TNF-α and PGE2. l groups: tive control (ethanol) ative control (Unstimulated cells) rence group (Parthenolide, ocortisone) xic concentrations (using the ssay) IL-6/luciferase assay (NF-κB assay) Quercetin 3-rutinoside showed the highest NF-κB inhibitory effect. Siriwatanametanon and Heinrich (2011) l groups: NF-κB inhibitory activities IC50: tive control (ethanol) Quercetin 3-rutinoside: 24.1 ± 0.1 μg/ml ative control (Unstimulated cells) 3,5-di-caffeoylquinic acid: 42.8 ± 0.2 μg/ml rence group (Parthenolide) 4,5-di-caffeoylquinic acid: 49.1 ± 0.1 μg/ml 5-mono-caffeoylquinic acid: 83.0 ± 0.1 μg/ml : 375 and 750 μg/ml IL-8 production level using ELISA Extract at both tested concentrations and some concentration of each marker compounds: Sukadeetad et al. (2018) genic acid: 140 and 280 μg/ml RelA and RelB localization by immunoﬂuorescence assay ↓IL-8 production level acid: 30 and 60 μg/ml ↓translocation of RelB S573 into nucleus 750 and 1,500 μg/ml maric acid: 1,400 and 2,800 μg/ l groups: mal control el control (50 ng/ml TNF-α with/ out 0.7% DMSO) tive control (50 μg/ml curcumin) e of extract and vehicle (1:10) Phosphorylation of NF-κB p65 using immunohistochemistry (skin sections from two patients) Immunohistochemical staining revealed diminution of phosphorylated NF-κB p65 in the lesions treated with the GP ointment. Rerknimitr et al. (2016) daily for 4 weeks l: 0.1% triamcinolone cream (Continued on following page) ncentration/dose, control groups Parameter measured and technique used Findings Reference with 1.2 and 4.8% GD Pancreatic Akt, PI3K, and PDK-1 mRNA expressions using qPCR 1.2% GD: Xu et al. (2015) route Pancreatic p-Akt, PI3K, and PDK-1 protein expressions using western blotting ↑Akt mRNA, ↑p-Akt protein expression, ↑PI3K mRNA and protein expression, ↑PDK-1 mRNA and protein expression y for 4 weeks ol groups: 4.8% GD: mal control (Normal diet) ↑↑Akt mRNA, ↑↑p-Akt protein expression, ↑↑PI3K mRNA and protein expression, ↑↑PDK-1 mRNA, ↑PDK-1 protein expression. betic model control (high-fat diet % lard, 20% sugar, 3% egg yolk, % basal diet) and 100 mg/kg ) ets with 1, 5 and 10% GD Western blotting to determine hepatic protein expression of 1% GD: ↑p-GSK3β, ↑PPARγ Dong et al. GS dose-dependently ↓↓↓formation of granuloma tissues (17.1, 39.7, and g 1,4,5 triol depicted the inhibitory activity with 0.15 μM. howed IC50 TNF-α μg/ml; IC50 IL-1β μg/ml 4,5,4′- cone demonstrated hibition on IL-1β release μM). Rutin was the most e against TNF-α release 6.96 μM. endently ↓↓↓formation tissues (17.1, 39.7, and ein expression, protein express % GD: Akt mRNA, ↑↑p ession, ↑↑PI3K ein expression, DK-1 protein ex GD: ↑p-GSK3 5% GD: ↑PI3K, ↑p ↑PPARγ, ↓TNF-α 10% GD: ↑PI3K, GSK3β, ↑↑PPARγ GS extract showe production with IC 0.03 μg/ml 8,8′-(et dinaphtalene-1 4 5 with IC50 16.96 125, 250 and 500 mg/kg body weight Cotton pellet granuloma assay GS dose-depende of granuloma tissue 47.2% inhibition b 500 mg/kg GS), ↓ levels in circulating cytokine levels. n pellet- model Oral route TNF-α and IL-1 production level using ELISA Once daily for 7 days Control groups: Negative control (1% Tween 80) Reference group (5 mg/kg indomethacin) α production level anuloma assay production level us prote glyco SK3β) facto appa ductio prote glyco K3β) facto appa ductio at diet yolk, kg D W pr ph p A p k chow) P (18% lard, nd 59% STZ) T N N G /ml IL u h pre- p A p k chow) P (18% lard, nd 59% STZ) T N N G /ml IL u h pre- M ear cells Compounds: 3.125–50 μg/ml LPS 3 h (Griess assay) or 12 h pre- treatment (ELISA) Control groups: Positive control (0.025 μM Dexamethasone) 125, 250 and 500 mg/kg body weig n pellet- Oral route ntrol groups: Normal contro Diabetic mode 18% lard, 20 9% basal die STZ) Diets with 1 Oral route Daily for 4 we ntrol groups: Normal contro del control (h % sugar, 3% sal diet) and ract: 6.25–10 aily for 4 we ntrol groups: ormal contr del control (h % sugar, 3% al diet) and ract: 6.25–10 dose, control ps Parameter measured and technique used Findings Reference 5, 25, 50, 100 μM LDH production level Compound 6 (3,5-dicaffeoylquinic acid ethyl ester) exhibited a more potent cytoprotective effect thus selected for further evaluation. Yu et al. (2016) Mitochondrial membrane potential (Δψm) ↓LDH leakage at 6.25, 12.5, 25, 50 and 100 μM compound 6. Phosphorylation of ERK, JNK, and p38 ↑↑↑Δψm in cells cultured with compound 6 (6.25, 12.5, 25.0 μM). ↓Phosphorylation of JNK and ERK by 12.5 and 25.0 μM compound 6 3 mM H2O2) arbonyl cyanide drazone (CCCP) body weight Xylene-induced ear edema test 250 and 500 mg/kg GN extract ↓ xylene-induced ear edema and carrageenan-induced models of inﬂammation. Rahman et al. (2018) Carrageenan-induced paw edema test n illed water + 20 μl ection of 0.1 ml 00 mg/kg ) /kg, 500 mg/kg Cotton pellet granuloma assay GF dose-dependently ↓granuloma formation. Ma et al. (2017) LDH, GPT, CRP, TNF-α and IL-1β production level using ELISA 100, 250, and 500 mg/kg GF ↓levels of plasma inﬂammatory biomarkers (LDH, GPT and CRP) activities. ays GF dose-dependently ↓plasma pro- inﬂammatory cytokines (TNF-α and IL-1β). % ulose, 1 ml/kg) mg/kg e (p < 0.001), ↓indicates signiﬁcantly inhibit (p < 0.05), ↓↓indicates signiﬁcantly inhibit (p < 0.01), and ↓↓↓indicates signiﬁcantly acid ethyl ester) exhibited a more potent cytoprotective effect thus selected for further evaluation. Mitochondrial membrane potential (Δψm) ↓LDH leakage at 6.25, 12.5, 25, 50 and 100 μM compound 6. Phosphorylation of ERK, JNK, and p38 ↑↑↑Δψm in cells cultured with compound 6 (6.25, 12.5, 25.0 μM). ↓Phosphorylation of JNK and ERK by 12.5 and 25.0 μM compound 6 mM H2O2) bonyl cyanide azone (CCCP) ody weight Xylene-induced ear edema test 250 and 500 mg/kg GN extract ↓ xylene-induced ear edema and carrageenan-induced models of inﬂammation. Rahman et al. (2018) Carrageenan-induced paw edema test ed water + 20 μl ction of 0.1 ml 0 mg/kg g, 500 mg/kg Cotton pellet granuloma assay GF dose-dependently ↓granuloma formation. Ma et al. (2017) LDH, GPT, CRP, TNF-α and IL-1β production level using ELISA 100, 250, and 500 mg/kg GF ↓levels of plasma inﬂammatory biomarkers (LDH, GPT and CRP) activities. s GF dose-dependently ↓plasma pro- inﬂammatory cytokines (TNF-α and IL-1β). ose, 1 ml/kg) g/kg p < 0.001), ↓indicates signiﬁcantly inhibit (p < 0.05), ↓↓indicates signiﬁcantly inhibit (p < 0.01), and ↓↓↓indicates signiﬁcantly DH leakage 100 μM c Δψm in ce pound 6 osphoryla 2.5 and 2 oups 12.5, 25 ent 0.3 mM l (Carbo lhydraz /kg bod ction distilled + injectio an) l (100 m um) mg/kg, 7 days 0.5% cellulose (4 mg/k uce (p < Gynura Antioxidant and Anti-inﬂammatory Effects Tan et al. model signiﬁcantly induced the mRNA and protein expression of Akt and PI3K (Xu et al., 2015; Dong et al., 2019). Similarly, a mice model with G. Anti-inﬂammatory Signaling Pathways of Gynura y g g y y Mitogen-activated protein kinases (MAPK) consist of three signaling pathways: ERK, JNK, and p38 MAPK, which mediate fundamental cellular processes by regulating immunomodulatory cytokine expression (Kaminska, 2005). G. bicolor extract possessed an anti-inﬂammatory effect by inhibiting p38 mRNA expression both in vitro (Yang et al., 2019) and in vivo (Pai et al., 2019). The isolated compound from G. nepalensis extract, 3,5-dicaffeoylquinic acid ethyl ester, exhibited inhibitory effects on the phosphorylation of JNK and ERK in cardiomyoblasts (Yu et al., 2016). Similarly, G. procumbens treatment also inhibited the protein expression of p-JNK in murine hepatocytes and in a mice model (Liu Y. et al., 2019). NF-κB represents a family of inducible transcription factors that activate the transcription of various pro-inﬂammatory genes (Liu et al., 2017). The NF-κB signaling pathway had been proposed as a potential mechanism of the anti-inﬂammatory effect of G. bicolor through the inhibition of the p-IκBα protein, nucleic p65 protein levels, translocation of NF-κB from the cytosol to the nuclei, and DNA-binding activity of the NF-κB nuclear protein (Wu et al., 2013). To complement the study by Wu et al. (2013), two studies demonstrated that the G. bicolor aqueous extract was able to inhibit NF-κB mRNA expression in both in vitro (Yang et al., 2019) and in vivo studies (Pai et al., 2019). Meanwhile, G. pseudochina var. hispida extract caused the inhibition of NF-κB activation with IC50 41.96 μg/ml in HeLa cells (Siriwatanametanon et al., 2010). Successively, quercetin 3- rutinoside, which was one of the isolated compounds from G. pseudochina var. hispida extract, showed a strong NF-κB inhibitory effect with IC50 24.1 ± 0.1 μg/ml (Siriwatanametanon and Heinrich, 2011). G. pseudochina extract and its marker compounds of chlorogenic acid, caffeic acid, rutin, and p- coumaric acid also inhibited RelB S573 (protein transcription factors of NF-κB family) translocation into the nucleus of keratinocytes (Sukadeetad et al., 2018). Moreover, G. divaricata lyophilized powder at 10% of diet inhibited the NF-κB signaling pathway (Dong et al., 2019). In a randomized controlled study, NF- κB phosphorylation in the lesions treated with G. pseudochina ointment was inhibited (Rerknimitr et al., 2016). divaricata lyophilized powder in its diet also exhibited signiﬁcant induction of the mRNA and protein expression of PDK-1 (Xu et al., 2015). By Akt-mediated phosphorylation, the PI3K/Akt signaling pathway was able to inactivate GSK3 (Salazar et al., 2006), where GSK3 has been determined as one of the mechanisms that phosphorylate and degrade Nrf2. In turn, Nrf2, a transcription factor, will be upregulated and will ultimately lead to the production of downstream cytoprotective protein expression (Cuadrado et al., 2018). GSK3 acts as the central regulator of the inﬂammatory response to bacterial infections and other insults. Hence, inactivation of GSK3 by the phosphorylation of GSK3β has been proposed as a potential therapeutic target in the control of bacterial-driven inﬂammatory diseases (Wang et al., 2014). The anti-inﬂammatory effect of G. procumbens was correlated to the increased phosphorylation of liver GSK3β (Ser9), which inhibited the activities of GSK3 (Wong et al., 2015). G. divaricata lyophilized powder diet increased GSK3β phosphorylation in a diabetic mice model (Dong et al., 2019). G. procumbens treatment showed anti-inﬂammatory potential through the induction of the Nrf2 protein level in murine hepatocytes and in a mice model (Liu Y. et al., 2019). Akt activation is also essential for the transcriptional activation of peroxisome proliferator-activated receptor-γ (PPARγ) (Kim et al., 2010), a nuclear receptor that inhibits the expression of inﬂammatory cytokines and directs the differentiation of immune cells toward anti-inﬂammatory phenotypes (Tyagi et al., 2011). G. procumbens extract in diet signiﬁcantly induced in vivo PPARγ mRNA expression (Liu Y. et al., 2019), and G. divaricata lyophilized powder diet increased in vivo hepatic protein expression of PPARγ (Dong et al., 2019). inﬂammatory effects by signiﬁcantly reducing ear thickness and paw edema in inﬂammatory mice model. Granulomatous inﬂammation is a special variety of chronic inﬂammation in which cells of the mononuclear phagocyte system are aggregated into well-demarcated focal lesions called granulomas (Williams and Williams, 1983). The anti-inﬂammatory effect of G. segetum and G. formosana had been demonstrated by signiﬁcant inhibition of granuloma tissue formation in a rat model (Seow et al., 2014; Ma et al., 2017). Frontiers in Pharmacology \| www.frontiersin.org Interplay Between Physiological, Biochemical and Immunological Aspects The antioxidant and anti-inﬂammatory effects of Gynura species are corroborated to each other. As aforementioned, inﬂammation and oxidative stress are highly interdependent pathophysiological events. Hence the antioxidant effects of Gynura species has supported anti-inﬂammatory effects by these plants. In general, Gynura species depict anti-inﬂammatory effects by inhibiting inﬂammatory signaling pathways, cellular pathogenicity, inﬂammatory biomolecules secretion, and clinical manifestation of inﬂammatory diseases. Granulomatous inﬂammation is the end result of a prolonged complex interplay among causal agents, mononuclear phagocytes activity, circulating immune complexes, and a vast array of biological mediators (Zumla and James, 1996). Inﬂammatory cell inﬁltration is the critical process in granuloma formation while tissue swelling is one of the possible associated clinical manifestation. The pathogenesis of granulomatous inﬂammation is manipulated by the vital player of macrophages along with secretion of cytokines and chemokines by immune cells through stimulation of inﬂammatory signaling pathways (Facco et al., 2007). Not only granulomatous inﬂammation, the signaling pathways, biomolecules and cellular responses also contribute Upon cellular induction, phosphoinositide 3-kinases (PI3K) generate lipid products that recruit cytosolic protein kinase B (Akt) to cellular membranes for Akt activation. Phosphoinositide-dependent kinase-1 (PDK-1) is a serine/ threonine kinase that facilitates Akt phosphorylation. The activated Akt detaches from the plasma membrane and translocates through the cytosol to the nucleus for further downstream reactions (Vanhaesebroeck and Alessi, 2000). G. divaricata lyophilized powder in the diet of a diabetic mice November 2020 \| Volume 11 \| Article 504624 Frontiers in Pharmacology \| www.frontiersin.org 22 Gynura Antioxidant and Anti-inﬂammatory Effects Tan et al. TABLE 5 \| Phytochemicals of Gynura species. Species Identiﬁed phytochemicals Gynura procumbens (Lour.) Merr. 15,16-Dihydroxy-9Z, 12Z-octadecadienoic acid (PubChem CID: 16061068) Akowuah et a Kaewseejan a et al. (2018), et al. (2019), L et al. Frontiers in Pharmacology \| www.frontiersin.org (Continued on following page) Interplay Between Physiological, Biochemical and Immunological Aspects (2020) 3,4-Dicaffeoylquinic acid (Isochlorogenic acid B) (PubChem CID: 5281780) 3,5-Dicaffeoylquinic acid (Isochlorogenic acid A) (PubChem CID: 6474310) 3,5-O-Dicaffeoylquinic acid (PubChem CID: 13604688) 3-O-Methyl gallic acid sulfate (PubChem CID: not found) 3-Carene (PubChem CID: 26049) 4,5-Dicaffeoylquinic acid (Isochlorogenic acids C) (PubChem CID: 6474309) 4-O-Methyl gallic acid sulfate (PubChem CID: not found) 5-O-(E)-Caffeoyl-galactaric acid (PubChem CID: not found) Apigenin (PubChem CID: 5280443) Caffeic acid (PubChem CID: 689043) Chlorogenic acid (PubChem CID: 1794427) Choline (PubChem CID: 305) Citric acid (PubChem CID: 311) Cynarine (PubChem CID: 5281769) Dicaffeoylquinic acids (PubChem CID: 6474310) Eriocitrin (PubChem CID: 83489) Ferulic acid (PubChem CID: 445858) Feruloylquinic acid (PubChem CID: 10133609) Gallic acid (PubChem CID: 370) Genkwanin isomer (PubChem CID: 5281617) Isobioquercetin (PubChem CID: not found) Kaempferol (PubChem CID: 5280863) Kaempferol 3-O-glucoside (Astragalin) (PubChem CID: 5282102) Kaempferol 3-O-rhamnosyl-(1→6)-glucoside (PubChem CID: not found) Kaempferol-3-O-rutinoside (Nicotiﬂorin) (PubChem CID: 5318767) Limonene (PubChem CID: 22311) Malic acid (PubChem CID: 525) Myricetin (PubChem CID: 5281672) Neochlorogenic acid (PubChem CID: 5280633) Oxooctadecanoic acid (PubChem CID: 439332) p-Coumaric acid (PubChem CID: 637542) p-Coumaroylquinic acid (PubChem CID: 6441280) p-Hydroxybenzoic acid (PubChem CID: 135) Phenylalanine (PubChem CID: 6140) Protocatechuic acid (PubChem CID: 72) Quercetin (PubChem CID: 5280343) Quercetin 3-O-rhamnosyl-(1→2)-galactoside (PubChem CID: 44259099) Quercetin 3-O-rhamnosyl-(1→6)-glucoside (PubChem CID: not found) Quercetin 3-O-rutinoside (Rutin) (PubChem CID: 5280805) Sinapic acid (PubChem CID: 637775) Syringic acid (PubChem CID: 10742) Trimethyl gallic acid glucuronide (PubChem CID: not found) Vanillic acid (PubChem CID: 8468) α-Pinene (PubChem CID: 6654) (Continued on following page) Interplay Between Physiological, Biochemical and Immunological Aspects (2020) 3,4-Dicaffeoylquinic acid (Isochlorogenic acid B) (PubChem CID: 5281780) 3,5-Dicaffeoylquinic acid (Isochlorogenic acid A) (PubChem CID: 6474310) 3,5-O-Dicaffeoylquinic acid (PubChem CID: 13604688) 3-O-Methyl gallic acid sulfate (PubChem CID: not found) 3-Carene (PubChem CID: 26049) 4,5-Dicaffeoylquinic acid (Isochlorogenic acids C) (PubChem CID: 6474309) 4-O-Methyl gallic acid sulfate (PubChem CID: not found) 5-O-(E)-Caffeoyl-galactaric acid (PubChem CID: not found) Apigenin (PubChem CID: 5280443) Caffeic acid (PubChem CID: 689043) Chlorogenic acid (PubChem CID: 1794427) Choline (PubChem CID: 305) Citric acid (PubChem CID: 311) Cynarine (PubChem CID: 5281769) Dicaffeoylquinic acids (PubChem CID: 6474310) Eriocitrin (PubChem CID: 83489) Ferulic acid (PubChem CID: 445858) Feruloylquinic acid (PubChem CID: 10133609) Gallic acid (PubChem CID: 370) Genkwanin isomer (PubChem CID: 5281617) Isobioquercetin (PubChem CID: not found) Kaempferol (PubChem CID: 5280863) Kaempferol 3-O-glucoside (Astragalin) (PubChem CID: 5282102) Kaempferol 3-O-rhamnosyl-(1→6)-glucoside (PubChem CID: not found) Kaempferol-3-O-rutinoside (Nicotiﬂorin) (PubChem CID: 5318767) Limonene (PubChem CID: 22311) Malic acid (PubChem CID: 525) Myricetin (PubChem CID: 5281672) Neochlorogenic acid (PubChem CID: 5280633) Oxooctadecanoic acid (PubChem CID: 439332) p-Coumaric acid (PubChem CID: 637542) p-Coumaroylquinic acid (PubChem CID: 6441280) p-Hydroxybenzoic acid (PubChem CID: 135) Phenylalanine (PubChem CID: 6140) Protocatechuic acid (PubChem CID: 72) Quercetin (PubChem CID: 5280343) Quercetin 3-O-rhamnosyl-(1→2)-galactoside (PubChem CID: 44259099) Quercetin 3-O-rhamnosyl-(1→6)-glucoside (PubChem CID: not found) Quercetin 3-O-rutinoside (Rutin) (PubChem CID: 5280805) Sinapic acid (PubChem CID: 637775) Syringic acid (PubChem CID: 10742) Trimethyl gallic acid glucuronide (PubChem CID: not found) Vanillic acid (PubChem CID: 8468) α-Pinene (PubChem CID: 6654) TABLE 5 \| Phytochemicals of Gynura species. cals of Gynura species. Identiﬁed phytochemicals References our.) Merr. 15,16-Dihydroxy-9Z, 12Z-octadecadienoic acid (PubChem CID: 16061068) Akowuah et al. (2002), Rosidah et al. (2008), Kim et al. (2011) Kaewseejan and Siriamornpun (2015), Murugesu et al. (2017), et al. (2018), Murugaiyah et al. (2018), Nazri et al. (2019), Huan et al. (2019), Liu M. et al. (2019), Liu Y. et al. (2019), Chandradeva et al. November 2020 \| Volume 11 \| Article 504624 Gynura pseudochina (L.) DC. References Species Species Identiﬁed phytochemicals 15,16-Dihydroxy-9Z, 12Z-octadecadienoic acid (PubChem CID: 16061068) 3,4-Dicaffeoylquinic acid (Isochlorogenic acid B) (PubChem CID: 5281780) 3,5-Dicaffeoylquinic acid (Isochlorogenic acid A) (PubChem CID: 6474310) 3,5-O-Dicaffeoylquinic acid (PubChem CID: 13604688) 3-O-Methyl gallic acid sulfate (PubChem CID: not found) 3-Carene (PubChem CID: 26049) 4,5-Dicaffeoylquinic acid (Isochlorogenic acids C) (PubChem CID: 6474309) 4-O-Methyl gallic acid sulfate (PubChem CID: not found) 5-O-(E)-Caffeoyl-galactaric acid (PubChem CID: not found) Apigenin (PubChem CID: 5280443) Caffeic acid (PubChem CID: 689043) Chlorogenic acid (PubChem CID: 1794427) Choline (PubChem CID: 305) Citric acid (PubChem CID: 311) Cynarine (PubChem CID: 5281769) Dicaffeoylquinic acids (PubChem CID: 6474310) Eriocitrin (PubChem CID: 83489) Ferulic acid (PubChem CID: 445858) Feruloylquinic acid (PubChem CID: 10133609) Gallic acid (PubChem CID: 370) Genkwanin isomer (PubChem CID: 5281617) Isobioquercetin (PubChem CID: not found) Kaempferol (PubChem CID: 5280863) Kaempferol 3-O-glucoside (Astragalin) (PubChem CID: 5282102) Kaempferol 3-O-rhamnosyl-(1→6)-glucoside (PubChem CID: not found) Kaempferol-3-O-rutinoside (Nicotiﬂorin) (PubChem CID: 5318767) Limonene (PubChem CID: 22311) Malic acid (PubChem CID: 525) Myricetin (PubChem CID: 5281672) Neochlorogenic acid (PubChem CID: 5280633) Oxooctadecanoic acid (PubChem CID: 439332) p-Coumaric acid (PubChem CID: 637542) p-Coumaroylquinic acid (PubChem CID: 6441280) p-Hydroxybenzoic acid (PubChem CID: 135) Phenylalanine (PubChem CID: 6140) Protocatechuic acid (PubChem CID: 72) Quercetin (PubChem CID: 5280343) Quercetin 3-O-rhamnosyl-(1→2)-galactoside (PubChem CID: 44259099) Quercetin 3-O-rhamnosyl-(1→6)-glucoside (PubChem CID: not found) Quercetin 3-O-rutinoside (Rutin) (PubChem CID: 5280805) Sinapic acid (PubChem CID: 637775) Syringic acid (PubChem CID: 10742) Trimethyl gallic acid glucuronide (PubChem CID: not found) Vanillic acid (PubChem CID: 8468) α-Pinene (PubChem CID: 6654) Gynura procumbens (Lour.) Merr. Feruloylquinic acid (PubChem CID: 10133609) Isobioquercetin (PubChem CID: not found) Kaempferol 3-O-glucoside (Astragalin) (PubChem CID: 5282102) Kaempferol 3-O-rhamnosyl-(1→6)-glucoside (PubChem CID: not Kaempferol-3-O-rutinoside (Nicotiﬂorin) (PubChem CID: Limonene (PubChem CID: 22311) Neochlorogenic acid (PubChem CID: 5280633) Oxooctadecanoic acid (PubChem CID: 439332) p-Coumaric acid (PubChem CID: 637542) p-Coumaroylquinic acid (PubChem CID: 6441280) p-Hydroxybenzoic acid (PubChem CID: 135) Phenylalanine (PubChem CID: 6140) Protocatechuic acid (PubChem CID: 72) Protocatechuic acid (PubChem CID: 72) Quercetin (PubChem CID: 5280343) Quercetin (PubChem CID: 5280343) Quercetin 3-O-rhamnosyl-(1→2)-galactoside (PubChem CID: Quercetin 3-O-rutinoside (Rutin) (PubChem CID: 5280805) Sinapic acid (PubChem CID: 637775) Syringic acid (PubChem CID: 10742) Trimethyl gallic acid glucuronide (PubChem CID: not found) Vanillic acid (PubChem CID: 8468) α-Pinene (PubChem CID: 6654) (Continued on following page) November 2020 \| Volume 11 \| Article 504624 Frontiers in Pharmacology \| www.frontiersin.org Frontiers in Pharmacology \| www.frontiersin.org 23 Gynura Antioxidant and Anti-inﬂammatory Effects Tan et al. TABLE 5 \| (Continued) Phytochemicals of Gynura species. Gynura pseudochina (L.) DC. (Continued on following page) Siriwatanametanon and Heinrich (2011), Ferlinahayati et al. (2017), Sukadeetad et al. (2018) Frontiers in Pharmacology \| www.frontiersin.org References Species Identiﬁed phytochemicals References Gynura bicolor (Roxb. ex Willd.) DC. 3,5-Di-O-caffeoylquinic acid (Isochlorogenic acid A) (PubChem CID: 6474310) Lu et al. (2012), Wu et al. (2015), Qiu X. L. et al. (2018) 3-O-Feruloylquinic acid (PubChem CID: 9799386) 3-O-p-Coumaroylquinic acid (PubChem CID: 9945785) 4,5-Di-O-Caffeoylquinic acid (Isochlorogenic acid C) (PubChem CID: 6474309) 5-O-Caffeoylquinic acid (Neochlorogenic Acid) (PubChem CID: 5280633) 5-O-p-Coumaroylquinic acid (PubChem CID: 9945785) Anthocyanin (PubChem CID: 145858) Caffeoyl glucose (PubChem CID: 129715972) Citric acid (PubChem CID: 311) Dihydro-phellopterin (PubChem CID: not found) Gallic acid (PubChem CID: 370) Geniposide (PubChem CID: 107848) Guanosine (PubChem CID: 135398635) Isobavachalcone (PubChem CID: 5281255) Kaempferol-3-O-caffeoylate (PubChem CID: not found) Kaempferol-3-O-glucoside (Astragalin) (PubChem CID: 5282102) Malic acid (PubChem CID: 525) Phenylalanine (PubChem CID: 6140) Protocatechuate-O-glucoside (PubChem CID: not found) Quercetin (PubChem CID: 5280343) Quercetin-3-acetylhexose (PubChem CID: not found) Quercetin-3-O-galactoside (PubChem CID: 5281643) Quercetin-3-O-rutinoside (Rutin) (PubChem CID: 5280805) Tryptophan (PubChem CID: 6305) Uridine (PubChem CID: 6029) β-Carotene (PubChem CID: 5280489) Gynura pseudochina (L.) DC. (+)-Tephropurpurin (PubChem CID: 10047971) Siriwatanametanon and Heinrich (2011), Ferlinahayati et al. (2017), Sukadeetad et al. (2018) 1-(9Z-octadecenoyl)-sn-glycero-2,3-cyclic phosphate (PubChem CID: 52922109) 1,3,8-Trihydroxy-4-methyl-2,7-diprenylxanthone (PubChem CID: 67261902) 2-(2,4-Dihydroxyphenyl)-5-hydroxy-8-methyl-8-(4-methyl-3- penten-1-yl)-2,3-dihydro-4H,8H-pyrano[2,3-f]chromen-4-one (PubChem CID: not found) 3,4-Dihydroxycinnamoyl-(Z)-2-(3,4-Dihydroxyphenyl) Ethanol (PubChem CID: 14353342) 3,5-Dicaffeoyl quinic acid (Isochlorogenic acid A) (PubChem CID: 6474310) 3-O-Caffeoyl-1-O-methylquinic acid (PubChem CID: 131752768) 4,5-Dicaffeoyl quinic acid (PubChem CID: 13887346) 5-Hydroxy-2′-methoxy-6,7-methylenedioxyisoﬂavone (PubChem CID: 5491929) 5-Caffeoyl quinic acid (Chlorogenic acid) (PubChem CID: 1794427) Caffeic acid (PubChem CID: 689043) Isochlorogenic acid B (PubChem CID: 5281780) Isochlorogenic acid C (PubChem CID: 6474309) Kaempferol rutinoside (Nicotiﬂorin) (PubChem CID: 5318767) Quercetin (PubChem CID: 5280343) Quercetin 3-rutinoside (Rutin) (PubChem CID: 5280805) Stigmasterol (PubChem CID: 5280794) β-Sitosterol (PubChem CID: 222284) (Continued on following page) Identiﬁed phytochemicals References . ex Willd.) DC. 3,5-Di-O-caffeoylquinic acid (Isochlorogenic acid A) (PubChem CID: 6474310) Lu et al. (2012), Wu et al. (2015), Qiu X. L. et al. November 2020 \| Volume 11 \| Article 504624 References (2018) 3-O-Feruloylquinic acid (PubChem CID: 9799386) 3-O-p-Coumaroylquinic acid (PubChem CID: 9945785) 4,5-Di-O-Caffeoylquinic acid (Isochlorogenic acid C) (PubChem CID: 6474309) 5-O-Caffeoylquinic acid (Neochlorogenic Acid) (PubChem CID: 5280633) 5-O-p-Coumaroylquinic acid (PubChem CID: 9945785) Anthocyanin (PubChem CID: 145858) Caffeoyl glucose (PubChem CID: 129715972) Citric acid (PubChem CID: 311) Dihydro-phellopterin (PubChem CID: not found) Gallic acid (PubChem CID: 370) Geniposide (PubChem CID: 107848) Guanosine (PubChem CID: 135398635) Isobavachalcone (PubChem CID: 5281255) Kaempferol-3-O-caffeoylate (PubChem CID: not found) Kaempferol-3-O-glucoside (Astragalin) (PubChem CID: 5282102) Malic acid (PubChem CID: 525) Phenylalanine (PubChem CID: 6140) Protocatechuate-O-glucoside (PubChem CID: not found) Quercetin (PubChem CID: 5280343) Quercetin-3-acetylhexose (PubChem CID: not found) Quercetin-3-O-galactoside (PubChem CID: 5281643) Quercetin-3-O-rutinoside (Rutin) (PubChem CID: 5280805) Tryptophan (PubChem CID: 6305) Uridine (PubChem CID: 6029) β-Carotene (PubChem CID: 5280489) (L.) DC. (+)-Tephropurpurin (PubChem CID: 10047971) Siriwatanametanon and Heinrich (2011), Ferlinahayati et al. (2017 Sukadeetad et al. (2018) 1-(9Z-octadecenoyl)-sn-glycero-2,3-cyclic phosphate (PubChem CID: 52922109) 1,3,8-Trihydroxy-4-methyl-2,7-diprenylxanthone (PubChem CID: 67261902) 2-(2,4-Dihydroxyphenyl)-5-hydroxy-8-methyl-8-(4-methyl-3- penten-1-yl)-2,3-dihydro-4H,8H-pyrano[2,3-f]chromen-4-one (PubChem CID: not found) 3,4-Dihydroxycinnamoyl-(Z)-2-(3,4-Dihydroxyphenyl) Ethanol (PubChem CID: 14353342) 3,5-Dicaffeoyl quinic acid (Isochlorogenic acid A) (PubChem CID: 6474310) 3-O-Caffeoyl-1-O-methylquinic acid (PubChem CID: 131752768) 4,5-Dicaffeoyl quinic acid (PubChem CID: 13887346) 5-Hydroxy-2′-methoxy-6,7-methylenedioxyisoﬂavone (PubChem CID: 5491929) 5-Caffeoyl quinic acid (Chlorogenic acid) (PubChem CID: 1794427) Caffeic acid (PubChem CID: 689043) Isochlorogenic acid B (PubChem CID: 5281780) Isochlorogenic acid C (PubChem CID: 6474309) Kaempferol rutinoside (Nicotiﬂorin) (PubChem CID: 5318767) Quercetin (PubChem CID: 5280343) Quercetin 3-rutinoside (Rutin) (PubChem CID: 5280805) Stigmasterol (PubChem CID: 5280794) β-Sitosterol (PubChem CID: 222284) (Continued on following page) Identiﬁed phytochemicals Stigmasterol (PubChem CID: 5280794) β-Sitosterol (PubChem CID: 222284) (Continued on following page) (Continued on following page) November 2020 \| Volume 11 \| Article 504624 Frontiers in Pharmacology \| www.frontiersin.org Frontiers in Pharmacology \| www.frontiersin.org 24 Gynura Antioxidant and Anti-inﬂammatory Effects Tan et al. TABLE 5 \| (Continued) Phytochemicals of Gynura species. Species Identiﬁed phytochemicals References Gynura divaricata (L.) DC. 3,4-Dicaffeoylquinic acid (Isochlorogenic acid B) (PubChem CID: 5281780) Jiangseubchatveera et al. (2015), Dong et al. (2019) 3,5-Dicaffeoylquinic acid (Isochlorogenic acid A) (PubChem CID: 6474310) 4,5-Dicaffeoylquinic acid (Isochlorogenic acid C) (PubChem CID: 6474309) 3-Caffeoylquinic acid (Chlorogenic acid) (PubChem CID: 1794427) Cubenol (PubChem CID: 519857) Spathulenol (PubChem CID: 92231) Gynura segetum (Lour.) Merr. Rutin (PubChem CID: 5280805) Yuandani et al. (2017) Gallic acid (PubChem CID: 370) 4,5,4′-Trihydroxychalcone (PubChem CID: 468135) 8,8’-(Ethene-1,2-diyl)-dinaphtalene-1,4,5-triol (PubChem CID: not found) Gynura nepalensis DC. 3,4-Dicaffeoylquinic acid methyl ester (PubChem CID: not found) Yu et al. (2016), Aktar et al. References (2019) 3,5-Dicaffeoylquinic acid ethyl ester (PubChem CID: not found) 3,5-Dicaffeoylquinic acid methyl ester (PubChem CID: 10075681) 3-O-cis-p-Coumaroylquinic acid (PubChem CID: 9945785) 4,5-Dicaffeoylquinic acid methyl ester (PubChem CID: not found) Chlorogenic acid (PubChem CID: 1794427) Isochlorogenic acid A (PubChem CID: 6474310) Isochlorogenic acid B (PubChem CID: 5281780) Isochlorogenic acid C (PubChem CID: 6474309) Saponins (PubChem CID: 6540709) Tannins (PubChem CID: 250395) TABLE 5 \| (Continued) Phytochemicals of Gynura species. Identiﬁed phytochemicals to pathogenesis of various diseases. Thus the plants in genus Gynura are having high potential to be explored for pharmacological evidence of different ailments. only one non-systematic randomized controlled trial that has been carried out. Rerknimitr et al. (2016) investigated the efﬁcacy of G. pseudochina DC. var. hispida Thv. ointment in treating chronic plaque psoriasis in a randomized controlled trial. Frontiers in Pharmacology \| www.frontiersin.org REFERENCES and anti-inﬂammatory activities of Gynura procumbens and Cleome gynandra. Plant Foods Hum. Nutr. 75, 243–251. doi:10.1007/s11130-020-00805-3 Chao, C., Liu, W., Wu, J., and Yin, M. (2015). Phytochemical proﬁle, antioxidative and anti-inﬂammatory potentials of Gynura bicolor DC. J. Sci. Food Agric. 95 (5), 1088–1093. doi:10.1002/jsfa.6902 Akowuah, G. A., Ahmad, M., and Fei, M. Y., (2012). Effects of Gynura procumbens leaf extracts on plasma lipid peroxidation and total antioxidant status in CCl4- treated rats. Nat. Prod. J. 2 (4), 247–251. doi:10.2174/2210315511202040247 Chen, J., Lü, H., Fang, L. X., Li, W. L., Verschaeve, L., Wang, Z. T., et al. (2017). Detection and toxicity evaluation of pyrrolizidine alkaloids in medicinal plants Gynura bicolor and Gynura divaricata collected from different Chinese locations. Chem. Biodivers. 14 (2), e1600221. doi:10.1002/cbdv. 201600221 Akowuah, G. A., Mariam, A., and Chin, J. H. (2009). The effect of extraction temperature on total phenols and antioxidant activity of Gynura procumbens leaf. Phcog. Mag. 4 (17), 81–85. Akowuah, G. A., Sadikun, A., and Mariam, A. (2002). Flavonoid identiﬁcation and hypoglycaemic studies of the butanol fraction from Gynura procumbens. Pharmaceut. Biol. 40 (6), 405–410. doi:10.1076/phbi.40.6.405.8440 Chen, J., Mangelinckx, S., Lü, H., Wang, Z. T., Li, W. L., and De Kimpe, N. (2015). Proﬁling and elucidation of the phenolic compounds in the aerial parts of Gynura bicolor and G. divaricata collected from different Chinese origins. Chem. Biodivers. 12 (1), 96–115. doi:10.1002/cbdv.201400134 Aktar, A., Haﬁz Hassan, S. M., Parvin, T., Akhlas, M. B., Khatun, F., Islam, M. T., et al. (2019). Further phytochemical screening; non-clinical evaluation of toxic and anti-inﬂammatory effects of crude aqueous extract of Gynura nepalensis. Pharmacologyonline 1, 136–153. Available at: https://pharmacologyonline.silae. it/ﬁles/archives/2019/vol1/PhOL_2019_1_A015_Anzuman.pdf Chen, Z., Huo, J. R., Yang, L., and Zhu, H. Y. (2011). Effect of ligustrazine on mice model of hepatic veno-occlusive disease induced by Gynura segetum. J. Gastroenterol. Hepatol. 26 (6), 1016–1021. doi:10.1111/j.1440-1746.2011. 06661.x Algariri, K., Atangwho, I. J., Meng, K. Y., Asmawi, M. Z., Sadikun, A., and Murugaiyah, V. (2014). Antihyperglycaemic and toxicological evaluations of extract and fractions of Gynura procumbens leaves. Trop. Life Sci. Res. 25 (1), 75–93. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4156475/ Cuadrado, A., Kügler, S., and Lastres-Becker, I. (2018). Pharmacological targeting of GSK-3 and NRF2 provides neuroprotection in a preclinical model of tauopathy. Redox Biol. 14, 522–534. doi:10.1016/j.redox.2017.10.010 Davies, F. G. (1980). The genus Gynura (compositae) in India, Sri Lanka and the Seychelles. Kew Bull. 35 (2), 363. doi:10.2307/4114583 Amelang, E., Prasad, C. M., Raymond, R. FUNDING We would like to thank the Ministry of Higher Education for FRGS grant FRGS/1/2019/SKK06/UKM/02/1. FUTURE PERSPECTIVE The bioavailability of bioactive molecules from genus Gynura also has limited investigation. On the basis of the study by Wu et al. (2015), the use of G. bicolor extract showed improvement on in vivo iron absorption and storage protein, which might be related to its rich phytoactive ingredients. Although the traditional uses of genus Gynura are supported by scientiﬁc evidence, the processing, and application or consumption methods by the public may alter the phytochemical proﬁle, thus leading to pharmacological effect variations. Herbal combination is possible to provide better effects or beneﬁts in developing therapeutic drugs. The study by Sari et al. (2015) showed that the combination of Andrographis paniculata (Burm. f.) Ness and G. procumbens could be a potential candidate for the development of an antidiabetic agent. The optimum therapeutic effect could be attributed to the combination of the potent hypoglycemic effect of A. paniculata and the potent antioxidant effect of G. procumbens. Hence, further studies on the therapeutic effects of the combinations of Gynura species with other medicinal plants are potentially producing more effective disease remedies. However, the potential issues of herb–herb and herb–drug interactions should be given due consideration, and further studies are needed to ensure no adverse interactions in polypharmacy and polyherbacy conditions. Previous studies on Gynura species have indicated that several Gynura species possessed strong antioxidant and anti-inﬂammatory effects that are fundamental to various therapeutic purposes. Hence, Gynura species are potential continual source of new and useful bioactive compounds. Identiﬁcation of the bioactive compounds in the plants contributing to the bioactivities, along with their mechanisms of action responsible for the pharmacological activities, is needed. Table 5 shows the list of phytochemicals identiﬁed in Gynura species. There is a need to investigate whether the crude extracts or isolated pure compounds are more effective. Synergistic effects of multiple active compounds in the extract may lead to a stronger pharmacological effect than that achievable by a single compound. At this stage, limited clinical studies on the antioxidant and anti-inﬂammatory effects of genus Gynura have been conducted. Human clinical trials with clearly deﬁned symptomology to evaluate the therapeutic value of genus Gynura are necessary because animal experiments cannot be a substitute for clinical trials in the evaluation of therapeutic efﬁcacy. To the best of our knowledge, there is November 2020 \| Volume 11 \| Article 504624 Frontiers in Pharmacology \| www.frontiersin.org 25 Gynura Antioxidant and Anti-inﬂammatory Effects Tan et al. CONCLUSION sources of Gynura research. TJN conducted the literature search, extracted the data, and wrote the ﬁrst draft. ZJ and NMF oversaw the research project, including checking the research work, reviewing, and interpreting the results. SMS and FB provided methodological advice on the project (literature search, screening, and selection). All authors are involved in reviewing and approval of the ﬁnal manuscript. The extracts and phytochemicals of several Gynura species, particularly G. procumbens, G. bicolor, G segetum, G. divaricata, G. formosana, G. nepalensis, and G. pseudochina, have been reported to exhibit strong antioxidant and anti-inﬂammatory effects. However, in vitro and in vivo studies and clinical studies that have been carried out on the species are by no means free of methodological ﬂaws. All 27 studies selected in this systematic review depicted risk of bias at different extent. The lack of randomization, lack of blinding, and unclear methodology explanation are some prevalent limitations. Further preclinical studies, including toxicity and pharmacokinetic studies on Gynura extracts and their bioactive compounds, are necessary before they can be subjected to clinical studies. Genus Gynura has high potential to be developed into medicinal agents for prophylactic supplements of diseases related to oxidative stress or inﬂammation. AUTHOR CONTRIBUTIONS The authors would like to thank Ernieda Md Hatah for her advice on the issues raised during the review process. The authors would like to thank Enago (www.enago.com) for the English language review. TJN, ZJ, and NMF designed the study. 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Mitochondrial dysfunction in inﬂammatory responses and cellular senescence: pathogenesis and pharmacological targets for chronic lung diseases. Br. J. Pharmacol. 173 (15), 2305–2318. doi:10.1111/bph.13518 Zahra, A. A., Kadir, F. A., Mahmood, A. Frontiers in Pharmacology \| www.frontiersin.org November 2020 \| Volume 11 \| Article 504624 REFERENCES A., Al hadi, A. A., Suzy, S. M., Sabri, S. Z., et al. (2011). Acute toxicity study and wound healing potential of Gynura procumbens leaf extract in rats. J. Med. Plants Res. 5 (12), 2551–2558. Copyright © 2020 Tan, Mohd Safﬁan, Buang, Jubri, Jantan, Husain and Mohd Fauzi. 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. 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https://openalex.org/W4318706061	https://www.researchsquare.com/article/rs-1844844/latest.pdf	English	null	Expression of hormone receptors is associated with specific immunological profiles of the breast cancer microenvironment	Breast cancer research	2,023	cc-by	9,933	Toru Hanamura Tokai University School of Medicine Shigehisa Kitano The Cancer Institute Hospital of JFCR Hiroshi Kagamu Saitama Medical University International Medical Center M kik Y hit Makiko Yamashita The Cancer Institute Hospital of JFCR Mayako Terao Tokai University School of Medicine Takuho Okamura Tokai University School of Medicine Nobue Kumaki Tokai University School of Medicine Katsuto Hozumi Tokai University School of Medicine Takayuki Iwamoto Okayama University Hospital Research Article Keywords: breast cancer, estrogen receptor, progesterone receptor, androgen receptor, tumor immunity, microenvironment, im-mune cell composition Posted Date: July 14th, 2022 Posted Date: July 14th, 2022 Page 1/23 DOI: https://doi.org/10.21203/rs.3.rs-1844844/v1 Conclusion Hormone receptor expression correlates with specific immunological profiles in the breast cancer microenvironment both at the gene and protein expression levels. Results Expressions of ESR1, PGR, and AR were negatively correlated with overall immune composition. Expressions of ER and AR, but not that of PgR, were inversely associated with hTIL and hPD-L1 expression. FCM analysis showed that the expressions of ER and AR, but not that of PgR, were associated with decreased total leukocyte infiltration. Both CIBERSORTx and FCM analysis showed that ER expression was associated with reduced infiltration of macrophages and CD4 + T cells, and that of AR with reduced macrophage infiltration. DOI: https://doi.org/10.21203/rs.3.rs-1844844/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: Competing interest reported. Hiroshi Kagamu has an advisory role in ImmuniT Research Inc, received honoraria from AstraZeneca K.K., Ono Pharmaceutical Co. Ltd., and Bristol-Myers Squibb Co. Ltd. Shigehisa Kitano received honoraria from Ono Pharmaceutical Co., Bristol-Myers Squibb Co., Ltd., AstraZeneca K.K., Chugai Pharmaceutical Co., Ltd., Pfizer Japan Inc., and MSD Co. Ltd, received research fund-ing from Astellas Pharma Inc., Gilead Sciences Inc., Eisai Co., Ltd., Regeneron Additional Declarations: Competing interest reported. Hiroshi Kagamu has an advisory role in ImmuniT Research Inc, received honoraria from AstraZeneca K.K., Ono Pharmaceutical Co. Ltd., and Bristol-Myers Squibb Co. Ltd. Shigehisa Kitano received honoraria from Ono Pharmaceutical Co., Bristol-Myers Squibb Co., Ltd., AstraZeneca K.K., Chugai Pharmaceutical Co., Ltd., Pfizer Japan Inc., and MSD Co. Ltd, received research fund-ing from Astellas Pharma Inc., Gilead Sciences Inc., Eisai Co., Ltd., Regeneron Pharmaceuticals Inc., Boehringer Ingelheim GmbH, Daiichi Sankyo Co., Ltd., Ono Pharmaceutical Co., Takara Bio Inc., PACT Pharma Inc., Chugai Pharmaceutical Co., Ltd., and MSD Co., Ltd. Naoki Niikura received honoraria from AstraZeneca K.K., Daiichi Sankyo Co. Ltd., Pfizer Japan Inc., Eisai Co. Ltd., and Nippon Kayaku Co. Ltd. Sasagu Kurozumi has received honoraria from Eli Lilly and Company, Daiichi Sankyo co. ltd, Taiho Pharmaceutical co. ltd, MSD K.K., AstraZeneca K.K., Chugai Pharmaceutical, Ltd., Dinow Inc., Eisai Co., Ltd. and Novartis Japan. The funders 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 decision to publish the results. Version of Record: A version of this preprint was published at Breast Cancer Research on January 31st, 2023. See the published version at https://doi.org/10.1186/s13058-023-01606-7. Page 2/23 Methods Using publicly available gene expression profile datasets, METABRIC and SCAN-B, the associations between the expressions of hormone receptors and the immune cell compositions in breast cancer tissue, estimated by CIBERSORTx algorithm, were analyzed. We histologically evaluated tumor infiltrating lymphocytes (hTIL), PD-L1 (hPD-L1) expression, and the infiltration of 11 types of immune cells by flow cytometry (FCM) for 45 breast cancer tissue samples. The relationships between them and the expressions of ER, PgR, and AR of tumor tissues, evaluated immunohistochemically, were analyzed. Background Elucidating the unique immunoregulatory mechanisms in breast cancer microenvironment may help develop new therapeutic strategies. Some studies have suggested that hormone receptors also have immune regulatory functions, but their mechanisms are not fully understood. In this study, we have comprehensively analyzed the relationship between the expressions of estrogen (ER), progesterone (PgR), and androgen receptors (AR), and the immunological profile in breast cancer. 1. Background Breast cancer is the most commonly occurring cancer in women. Despite recent advances in multimodal treatment, advanced and recurrent cases are challenging to cure [1], and the urgent need to develop innovative treatment strategies has to be addressed. Owing to the development of immune checkpoint inhibitors (ICIs) that reinvigorate the adaptive immune response in the tumor microenvironment and the successful application of these ICIs in various neoplasms, tumor immunology has recently gained Page 3/23 renewed interest across multiple cancers [2, 3]. ICIs have also been applied for breast cancer treatment, but their effectiveness is limited, likely due to the immunosuppressive tumor microenvironment [4–9]. Elucidating the unique immunomodulatory mechanisms of the breast cancer microenvironment will provide significant insights into the development of new therapeutic strategies. Hormone dependency is one of the prominent biological features of breast cancer. Approximately 70% of breast cancer cells express the estrogen receptor (ER), resulting in ER-dependent growth of breast cancer [1]. Therefore, treatment strategies that inhibit ER function are frequently used as postoperative adjuvant therapy for patients with early-stage breast cancer and as systemic therapy for metastatic cases [1]. The progesterone receptor (PgR) is clinically considered a complementary marker of hormone dependency in breast cancer [1, 10] because it is driven partly, but not exclusively, by ER-mediated transcriptional events [11, 12]. PgR is a binding partner and major modifier of ER-mediated processes, suggesting its additional role in breast cancer other than its identification as an ER activity marker [13, 14]. The androgen receptor (AR) is a nuclear transcription factor with a diverse range of biological actions, mainly in the development and maintenance of the male reproductive system [15]. It is widely expressed in all breast cancer subtypes to varying extents, with approximately 60–80% of the cases being AR-positive [16–18]. Furthermore, according to The Human Protein Atlas, AR expression, at both gene and protein levels, is second-highest in breast cancer, after prostate cancer, among various malignancies [19]. Although the function of AR in breast cancer depends on the tumor subtype, treatment, and other factors, it is suggested to have a tumor- promoting role [20–22], thereby attracting attention as a new therapeutic target for breast cancer treatment [23, 24]. Some recent studies have suggested that sex steroid hormones and their receptor signaling have immune regulatory functions. In vitro studies have shown that estrogen can expand the regulatory T-cell fraction and reduce the function of antigen-presenting cells [25, 26]. 2.1 Gene expression profile datasets Two publicly available gene expression profile datasets of patients with breast cancer used in this study were the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) [40, 41] cohort (n = 1904) and the Sweden Cancerome Analysis Network-Breast (SCAN-B) [42, 43] cohort (n = 3273). Gene expression data of METABRIC and SCAN-B, generated by microarray and RNA-sequencing, were downloaded from the cBioPortal: https://www.cbioportal.org/ (accessed on 20/2/2019) and Gene Expression Omnibus: https://www.ncbi.nlm.nih.gov/geo/ (accessed on 16/6/2019), respectively. The inclusion criteria and clinicopathological information for each cohort have been provided in the original papers. The METABRIC cohort included patients with primary invasive breast cancer, including < 1% metastatic breast cancer. However, information on the use of preoperative chemotherapy was not provided. On the contrary, the SCAN-B cohort consisted of patients with non-metastatic primary invasive breast cancer and included the cases of preoperative chemotherapy. Both cohorts included all breast cancer subtypes, and all samples were obtained from primary lesions. 1. Background In addition, estrogen can promote immune tolerance by interfering with human leukocyte antigen-II expression in ER-positive breast cancer cell lines [27]. These findings suggest that estrogen signaling in the tumor microenvironment regulates anti-tumor immunity [2]. In agreement with this, hormone receptor-positive breast cancer is characterized by low infiltration of tumor-infiltrating lymphocytes (TILs) and minimal response to ICIs [5, 9, 28–32]. To the best of our knowledge, the immunological function of PgR in breast cancer has not yet been reported. However, limited studies have shown a relationship between PgR expression and tumor immunity in breast cancer; PgR expression is inversely associated with programmed death-ligand 1 (PD-L1) expression in epithelial cells or the stroma and the infiltration of CD8 + T and CD20 + B cells [9, 33, 34]. Further, immune regulatory functions of AR signals have been demonstrated via in vivo models of various autoimmune diseases and some malignancies [35]. Moreover, in breast cancer, AR expression is inversely correlated with immune cell infiltration and cytotoxic immune activity, suggesting an immunosuppressive effect of AR signals [36–38]. Despite fragmentary evidence on sex steroid hormone signals and tumor immunity, the interactions between hormone receptors and immune cells are not well understood because of the complexity of the immune milieu in the breast cancer microenvironment and limited reports on systematic evaluation of immune cell composition in breast cancer tissue [39]. In this study, we systematically analyzed the Page 4/23 Page 4/23 relationship between the expression of sex steroid hormone receptors such as ER, PgR, and AR and the immunological profiles of breast cancer tissues. Our results demonstrated that hormone receptor expression, at both gene and protein levels, correlates with specific immunological profiles of the breast cancer microenvironment, strongly suggesting their direct or indirect immunomodulatory role. 2.2 CIBERSORTx Immune cell composition in breast cancer tissue was determined from gene expression profiles using a bioinformatics algorithm, CIBERSORTx: https://cibersortx.stanford.edu/ (accessed on 25/2/2022) [44]. Briefly, “LM22” representing the profiling of 22 functionally defined human immune cell types [45] was applied as a signature matrix file. The non-log transformed gene expression data from METABRIC and SCAN-B were applied to the mixture file. The program was run in the absolute mode with 100 permutations. A B-mode batch correction was applied, and quantile normalization was set as disabled. Absolute scores representing the overall immune content and absolute abundance of each immune cell fraction in the mixture were produced by the algorithm. Cases with a CIBERSORTx p-value < 0.05 were filtered and selected for subsequent analysis. Absolute scores and absolute abundance of each cell type in a mixture were used for correlation analysis with the expression values of the indicated genes that were log2 transformed. Immune cell composition in breast cancer tissue was determined from gene expression profiles using a bioinformatics algorithm, CIBERSORTx: https://cibersortx.stanford.edu/ (accessed on 25/2/2022) [44]. Briefly, “LM22” representing the profiling of 22 functionally defined human immune cell types [45] was applied as a signature matrix file. The non-log transformed gene expression data from METABRIC and SCAN-B were applied to the mixture file. The program was run in the absolute mode with 100 2.3 Gene set enrichment analysis (GSEA) Page 5/23 The gene expression values in the METABRIC dataset were log2 transformed before performing GSEA [46, 47]. Hallmark gene set collections (50 gene sets) representing specific well-defined biological states or processes were obtained from MsigDB v7.1: https://www.gsea-msigdb.org/gsea/msigdb/ (accessed on 13/5/2020) and applied to GSEA using GSEA software v4.0: https://www.gsea-msigdb.org/gsea/msigdb/ (accessed on 21/11/2019). While performing GSEA, the permutations were set at 1000 with phenotype as the permutation type. Expression values of the indicated genes were used as phenotype labels, and Pearson’s correlation was set as the metric for ranking genes. Thresholds for nominal p-value and false discovery rate (FDR) q-value were set at < 0.05 and < 0.25, respectively. According to user guidelines, the SCAN-B dataset was not applied to GSEA because of the incompatible normalization method used for this analysis. the permutation type. Expression values of the indicated genes were used as phenotype labels, and Pearson’s correlation was set as the metric for ranking genes. Thresholds for nominal p-value and false discovery rate (FDR) q-value were set at < 0.05 and < 0.25, respectively. According to user guidelines, the SCAN-B dataset was not applied to GSEA because of the incompatible normalization method used for this analysis. 2.4 Patients In a previous study, immune cell composition of breast cancer tissue was evaluated using flow cytometry (FCM) and an association between histologically assessed expression of TIL and PD-L1 and the immunological profile of the tumor microenvironment was reported [39]. The inclusion criteria and patient characteristics have also been described previously [39], and this dataset was used for further analyses. Briefly, 47 breast cancer samples were obtained, regardless of clinicopathological factors or treatment histories, except for patients with distant metastases or complete clinical responses to neoadjuvant chemotherapy. None of the patients had received irradiation or endocrine therapy before surgery. Clinicopathological data were collected by reviewing the case records. Two cases were excluded from the analysis, and the reasons are mentioned in section 2.6. 2.5 Histological evaluation of hormone receptors and tumor immunity-related biomarkers Rabbit monoclonal antibodies for ER (SP1) and PgR (1E2) were purchased from Ventana Medical Systems Japan Inc. (Tokyo, Japan) and for AR (AR27) from Leica Biosystems Inc. (Wetzlar, Germany). The immunohistochemistry (IHC) staining was performed using the VENTANA BenchMark ULTRA automated IHC device (Roche Diagnostics, Basel, Switzerland) for ER and PgR and BOND-III automated IHC device (Leica Biosystems Inc.; Wetzlar, Germany) for AR. The antigen–antibody complex was visualized using diaminobenzidine and counterstained with hematoxylin. The nuclear staining of ER, PgR, and AR in carcinoma cells was counted, and the percentage of immunoreactive cells was determined. ER and PgR were determined as positive when nuclear staining-positive cells were ≥ 10%. [48]. However, the accepted cutoff value for AR expression is not known; since the median value of nuclear staining-positive cells for AR was 60% in the present study, AR was considered positive at ≥ 60%. We diverted the data from histological analysis of the expression of TIL and PD-L1 from our previous study, referred to as hTIL and hPD-L1, respectively [39]. According to the International TILs Working Group guidelines [49], the percentages of TILs in stromal tissue sections stained with hematoxylin and eosin (H&E) were evaluated and categorized into three grades: low (0–10%), intermediate (10–40%), and high (40–90%). PD-L1 expression was assessed by IHC using an anti-PD-L1 antibody (SP142; Spring Bioscience, Pleasanton, CA, USA). Tumors with ≥ 1% immune cells showing cytoplasmic and/or membrane PD-L1 staining were determined to be PD-L1 positive [50]. A previous study report accounted that in a case, hTIL and hPD-L1 could not be evaluated because the tumor tissue was insufficient for analysis. 2.6 TIL preparation/ FCM analysis Page 6/23 FCM data from breast cancer tissue samples were obtained from a previously reported study, which contains the detailed method [39]. To perform the analysis, fresh breast cancer tissues were mechanically dissociated and filtered using a 70-micron cell strainer. From the filtered cell suspension, mononuclear cell components were separated by density-gradient centrifugation and subjected to FCM analysis. Samples stained with an antibody cocktail were detected using LSR II Fortessa with the fluorescence-activated cell sorting Diva software (BD Biosciences). All analyses were performed using the FlowJo software v10.6.1 (BD Biosciences). The list of antibodies used, gating strategy, and definitions of immune cell fractions have been described in the previous report [39]. 2.5 Histological evaluation of hormone receptors and tumor immunity-related biomarkers According to the staining profile of the surface antigen, evaluated by FCM, the cells were classified as follows: leukocytes, total T cells (total T), CD4 + T cells (CD4 + T), CD8 + T cells (CD8 + T), B cells (B), monocytes/macrophages (Mo/Mφ), non-classical monocytes (CD16 + Mo), myeloid-derived suppressor cells (MDSCs), dendritic cells (DCs), myeloid dendritic cells (mDCs), natural killer (NK) cells, minor NK cells, and natural killer T cells (NKT). As mentioned in section 2.4, two cases were excluded because they had a low number of living cells (count < 1000) in the FCM analysis of tumor tissue. Leukocyte density was defined as the count of total CD45 + cells per weight of tumor tissue (count/g), as described in a previous paper [51]. Similarly, we determined the count of each immune cell fraction per weight of the tissue fragment (count/g). 2.7 Statistical analyses GraphPad Prism ver. 9.1.0 software was used for statistical analyses and graph preparation. The gene expression and FCM data were tested using the D'Agostino–Pearson normality test, which showed non- normal distribution in almost all the datasets. Correlation analyses between groups were performed using Spearman’s rank correlation coefficient. \|r-value\| > 0.3 and a significant p-value was defined as a positive or negative correlation [52, 53]. The Mann–Whitney U test and Fisher’s exact test were used to compare continuous and categorical variables between the two unpaired groups, respectively. The p-value < 0.05 was defined as statistically significant. In the previous study [39], the FCM data contained outliers. Here, although all analyses were performed without omitting outliers, we identified the outliers using the robust regression and outlier removal method, excluded them, and performed all statistical analyses to ensure the reliability of our analyses. Statistics with omitted outliers are shown in each figure along with the original data. 3.3 Association of hormone receptor status with leukocyte density and tumor-infiltrating immune cells in breast cancer tissue The relationship between each hormone receptor status, density of leukocytes, and each immune cell fraction in breast cancer tissue was analyzed. ER positivity was associated with decreased leukocyte density and reduced infiltration of total T, CD4 + T, Mo/Mφ, MDSC, DC, and mDC in breast cancer tissue (Fig. 3a–m). PgR positivity was associated with decreased infiltration of DC but not with leukocyte density or other immune cell fractions (Fig. 4a–m). AR positivity was associated with decreased leukocyte density and infiltration of CD4 + T, Mo/Mφ, CD16 + Mo, MDSC, DC, mDC, and minor NK cells (Fig. 5a–m). These results suggest a strong association of the expression of ER and AR with decreased infiltration of leukocytes and specific immune cell fractions such as CD4 + T and Mo/Mφ into breast cancer tissue. 3.1 Gene expression levels of hormone receptors in breast cancer tissue Gene expression of estrogen receptor 1 (ESR1), PGR, and AR negatively correlated with the absolute score in the METABRIC cohort (r>-0.3, p < 0.05); however, the negative correlation was weak for PGR in the SCAN- B cohort (r = -0.284, p < 0.05) (Fig. 1a–f). ESR1 expression levels inversely correlated with Mφ M1 and M0, CD4 + T (memory activated), CD8 + T, and CD4 + T (memory resting) in at least one dataset (r<-3, p < 0.05) (Fig. 1g). Likewise, PGR expression levels inversely correlated with Mφ M1 and M0 and CD4 + T (memory Page 7/23 Page 7/23 activated) in at least one dataset (r<-3, p < 0.05) (Fig. 1h). Similarly, AR expression levels inversely correlated with Mφ M0 and M1 in at least one dataset (r<-3, p < 0.05) (Fig. 1i). These results suggest that the expression of hormone receptors ESR1, PGR, and AR is inversely associated with the total immune content and the infiltration of specific immune cell fractions in the tumor tissue. 3.2 Correlation of histologically assessed TIL and expression of PD-L1 with protein expression of ER and AR 3.2 Correlation of histologically assessed TIL and expression of PD-L1 with protein expression of ER and AR AR The status of ER, PgR, AR, PD-L1 (hPD-L1), and TIL (hTIL) in breast cancer tissue was histologically evaluated by IHC and H&E staining using the samples obtained at our facility from 45 patients with breast cancer. A summary of clinicopathological findings according to hormone receptor status is shown in Additional file: Supplementary Tables S1–S3. The correlation between hormone receptor expression and immune-related biomarkers was further analyzed at the protein level. Expression of both hTIL and hPD-L1 showed a negative correlation with the protein expression of ER and AR, but not with that of PgR, probably due to the small number of PgR-positive cases (Fig. 2a–f). The status of ER, PgR, AR, PD-L1 (hPD-L1), and TIL (hTIL) in breast cancer tissue was histologically evaluated by IHC and H&E staining using the samples obtained at our facility from 45 patients with breast cancer. A summary of clinicopathological findings according to hormone receptor status is shown in Additional file: Supplementary Tables S1–S3. The correlation between hormone receptor expression and immune-related biomarkers was further analyzed at the protein level. 3.1 Gene expression levels of hormone receptors in breast cancer tissue Expression of both hTIL and hPD-L1 showed a negative correlation with the protein expression of ER and AR, but not with that of PgR, probably due to the small number of PgR-positive cases (Fig. 2a–f). 4. Discussion This study systematically analyzed the relationship between hormone receptor expression (at both gene and protein levels) and the immunological profile of breast cancer tissues, including multiple immune cell fractions. Expression of ER and AR in breast cancer tissues was associated with a decreased infiltration of immune cells into the tumor microenvironment. More specifically, ER expression was associated with decreased infiltration of macrophages and CD4 + T cells, while AR expression with reduced macrophage infiltration. These results were consistent at the gene and protein expression levels. The present analysis showed that gene expression of hormone receptors was inversely correlated to total immune cell infiltration into the tumor microenvironment (Fig. 1a–f). This was further verified by performing GSEA on the METABRIC dataset, which analyzed the relationship between the expression levels of hormone receptors and hallmark gene set collection (50 gene sets) representing specific well- defined biological states or processes. Expression levels of ESR1, PGR, and AR showed a significant Page 8/23 Page 8/23 positive correlation with gene sets representing estrogen response, such as estrogen response early and estrogen response late (Additional file: Figure S1a–c). On the other hand, ESR1 and AR expression levels showed a significant negative correlation with multiple gene sets representing immunological processes such as inflammatory response, allograft rejection, complement, and interferon-gamma response (Additional file: Figure S1d, f). Likewise, a significant negative correlation was observed between PgR expression level and gene sets of inflammatory response, allograft rejection, and interferon-gamma response (Additional file: Figure S1e). These findings suggest that the gene expression levels of hormone receptors, such as ESR1, PGR, and AR, correlate with immunosuppressive phenotypes of breast cancer. Several preclinical studies have suggested that ER signaling may suppress the immune response of breast cancer [2, 25–27], and ER-positive breast cancers have shown reduced TIL infiltration and minimal response to ICI [5, 9, 28–32]. This is consistent with the present findings, suggesting an immunomodulatory function of ER. Although numerous studies have analyzed the relationship between a single immune cell lineage and hormone receptor expression [32, 54–57], few reports have systematically investigated the multiple immune cell composition of breast cancer tissues [51, 58–60]. The present study reports the first systematic analysis demonstrating that ER expression is preferentially associated with reduced infiltration of macrophages and CD4 + T cells (Fig. 1g, 3c, 3f). 4. Discussion Thus, ER positivity and AR positivity may reflect decreased immune response in the breast cancer microenvironment. Findings of this study suggest that the hormone receptor signals may primarily affect specific immune cell lineages. In particular, the expression of ER and AR showed a negative correlation not only with total immune content in the tumor microenvironment but also with certain immune cell subsets such as macrophages and CD4 + T cells. This indicates that specific immune cell lineages may be primary targets of the immunoregulatory function of the hormone receptor signals. Our next goal is to verify this hypothesis using an in-vitro or in-vivo analysis model. In our previous study [39], a relatively small number of samples were used for FCM analysis, and compared to those of the general breast cancer cohort, the clinicopathological characteristics of tumors were biased with larger invasive tumor sizes, more ER-negative cases, and higher Ki67 cases. Similarly, in this study, the small sample size prevented us from performing subgroup analyses based on the tumor subtype. Therefore, the inclusion of a greater number of samples and more detailed analyses are required in future studies. 5. Conclusions In the present study, the gene expression levels of hormone receptors correlated with immunosuppressive phenotypes of breast cancer. The expression level of ER and AR proteins was associated with decreased tumor-infiltrating immune cells and decreased PD-L1 expression. These data suggest that hormone receptor signaling may suppress tumor immunity through a specific mechanism. Additionally, our data showed that certain immune cell lineages might get more strongly affected by hormonal signals than others, providing useful suggestions for further analysis of the effects of hormonal signals on tumor immunity. 4. Discussion This result is consistent with earlier reports that show a negative correlation between ER expression and intratumoral infiltration of macrophages and CD4 + T cell [54–57]. Further, an inverse correlation between ER expression and CD8 + T cells has been reported previously [32, 54]; however, in our analysis, this correlation was weaker than that for CD4 + T cells, both at the gene and protein levels (Fig. 1g and 3). In addition, PGR expression showed a weaker correlation with total immune content than ESR1 and AR, as estimated by CIBERSORTx. Moreover, the correlation between PgR status and hTIL and hPD-L1 was insignificant at the protein level, probably due to the small number of PgR-positive samples. To validate these findings, a greater number of samples are required to be analyzed. AR signaling is suggested to have immunomodulatory functions in some malignancies, including breast cancer [35–38]; however, its immunological role in breast cancer has not been fully validated. To the best of our knowledge, a single study has investigated the relationship between immune cell composition and AR expression in breast cancer using the CIBERSORT algorithm [38]. Tumors with high AR expression were reported to be associated with pro-cancer regulatory T cells, and those with low AR expression were associated with anti-cancer immune cells, such as CD4, CD8, gamma delta T cells, and memory B cells in ER-positive breast cancer. In our study, CIBERSORTx was run in absolute mode and included all subtypes in the analysis; therefore, a simple comparison was not possible. High AR-expressing tumors showed reduced macrophage infiltration into the tumor microenvironment; this finding was consistent with CIBERSORTx and FCM analysis results. In the present study, expressions of ER and AR inversely correlated with hPD-L1 expression (Fig. 2b, f). We analyzed the PD-L1 expression in each immune cell fraction by FCM analysis, similar to our previous study [39]. According to hormone receptor status, we found that ER positivity was associated with decreased PD-L1 expression in Mo/Mφ and mDC (Additional file: Figure S2d, h), PgR positivity with Page 9/23 Page 9/23 decreased PD-L1 expression in MDSCs and NK cells (Additional file: Figure S3f, j), and AR positivity with decreased PD-L1 expression in CD8 + T and Mo/Mφ cells (Additional file: Figure S4b, d). PD-L1 expression reflects ongoing (or active) immune responses in addition to immunosuppression via the PD-1/PD-L1 pathway [60–62]. Abbreviations ER: estrogen receptor PgR: progesterone receptors AR: androgen receptor hTIL: histologically evaluated tumor infiltrating lymphocytes hPD-L1: histologically evaluated programmed death-ligand 1 FCM: flow cytometry Page 10/23 ICIs: immune checkpoint inhibitors Declarations Ethics Approval and Consent to Participate: This study was conducted at Tokai University Hospital and approved by the Ethics Committee, which conforms to the provisions of the Declaration of Helsinki (Accepted project No. 16R-279 and 20R-049). Patients were enrolled from May 2015 to April 2019, and written informed consent was obtained from all the patients to publish this paper. Consent for publication: Not applicable Consent for publication: Not applicable Availability of data and materials: The datasets generated and/or analyzed during the current study are available upon reasonable request from the corresponding author. Competing interests: Hiroshi Kagamu has an advisory role in ImmuniT Research Inc, received honoraria from AstraZeneca K.K., Ono Pharmaceutical Co. Ltd., and Bristol-Myers Squibb Co. Ltd. Shigehisa Kitano received honoraria from Ono Pharmaceutical Co., Bristol-Myers Squibb Co., Ltd., AstraZeneca K.K., Chugai Pharmaceutical Co., Ltd., Pfizer Japan Inc., and MSD Co. Ltd, received research fund-ing from Astellas Pharma Inc., Gilead Sciences Inc., Eisai Co., Ltd., Regeneron Pharmaceuticals Inc., Boehringer Ingelheim GmbH, Daiichi Sankyo Co., Ltd., Ono Pharmaceutical Co., Takara Bio Inc., PACT Pharma Inc., Chugai Pharmaceutical Co., Ltd., and MSD Co., Ltd. Naoki Niikura received honoraria from AstraZeneca K.K., Page 11/23 Page 11/23 Daiichi Sankyo Co. Ltd., Pfizer Japan Inc., Eisai Co. Ltd., and Nippon Kayaku Co. Ltd. Sasagu Kurozumi has received honoraria from Eli Lilly and Company, Daiichi Sankyo co. ltd, Taiho Pharmaceutical co. ltd, MSD K.K., AstraZeneca K.K., Chugai Pharmaceutical, Ltd., Dinow Inc., Eisai Co., Ltd. and Novartis Japan. The funders 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 decision to publish the results. Funding: This research was funded by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (Project No.18K16266 to NN, and No.20K22856 to TH), and the 2021 Tokai University School of Medicine Research Aid (to TH). This work was also supported by Chugai Pharmaceutical Co. Ltd. Authors' contributions: TH analyzed and interpreted the data and wrote the manuscript. MT, BT, TO, and NN collected clinical samples from the participants. 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Cancer 12:252-264. doi: 10.1038/nrc3239 61. Pardoll DM (2012) The blockade of immune checkpoints in cancer immunotherapy. Nature reviews. Cancer 12:252-264. doi: 10.1038/nrc3239 62. References Cimino-Mathews A, Thompson E, Taube JM, Ye X, Lu Y, Meeker A, Xu H, Sharma R, Lecksell K, Cornish TC, Cuka N, Argani P, Emens LA (2016) PD-L1 (B7-H1) expression and the immune tumor microenvironment in primary and metastatic breast carcinomas. Human pathology 47:52-63. doi: 10.1016/j.humpath.2015.09.003 Figures Figures Page 18/23 Page 18/23 Figure 1 Association between gene expression levels of hormone receptors and immunological profile of the breast cancer tissue. (a–f) Scatterplots showing a correlation between the gene expression levels of estrogen receptor 1 (ESR1), progesterone receptor (PGR), and androgen receptor (AR), and absolute score estimated by CIBERSORTx. (g–i) Graphs showing the correlation coefficient (r-value) between indicated gene expression and the absolute amount of various immune cell fractions estimated by CIBERSORTx. Immune Association between gene expression levels of hormone receptors and immunological profile of the breast cancer tissue. (a–f) Scatterplots showing a correlation between the gene expression levels of estrogen receptor 1 (ESR1), progesterone receptor (PGR), and androgen receptor (AR), and absolute score estimated by CIBERSORTx. (g–i) Graphs showing the correlation coefficient (r-value) between indicated gene expression and the absolute amount of various immune cell fractions estimated by CIBERSORTx. Immune Page 19/23 Page 19/23 cell fraction data showing consistently significant p-values in the METABRIC and SCAN-B datasets have been displayed in ascending order of r-value cell fraction data showing consistently significant p-values in the METABRIC and SCAN-B datasets have been displayed in ascending order of r-value been displayed in ascending order of r-value Figure 2 Histological assessment of the expression of tumor-infiltrating lymphocytes (TILs) and programmed death-ligand 1(PD-L1) based on protein expression status of estrogen receptor (ER) and androgen receptor (AR). (a–f) Graphs showing the number of patients with positive or negative immune-related markers according to hormone receptor status. Fisher’s exact test was used to compare categorical variables between two groups; actual p-values are shown in the figure Figure 2 Histological assessment of the expression of tumor-infiltrating lymphocytes (TILs) and programmed death-ligand 1(PD-L1) based on protein expression status of estrogen receptor (ER) and androgen receptor (AR). (a–f) Graphs showing the number of patients with positive or negative immune-related markers according to hormone receptor status. Fisher’s exact test was used to compare categorical variables between two groups; actual p-values are shown in the figure Figure 2 Histological assessment of the expression of tumor-infiltrating lymphocytes (TILs) and programmed death-ligand 1(PD-L1) based on protein expression status of estrogen receptor (ER) and androgen receptor (AR). (a–f) Graphs showing the number of patients with positive or negative immune-related markers according to hormone receptor status. Fisher’s exact test was used to compare categorical variables between two groups; actual p-values are shown in the figure Page 20/23 Page 20/23 Figure 3 Association between estrogen receptor (ER) status and subsets of tumor-infiltrating immune cells. (a) Total leukocyte density (count/g) in breast cancer tissue according to ER status (b m) Count of each Figure 3 Association between estrogen receptor (ER) status and subsets of tumor-infiltrating immune cells. (a) Total leukocyte density (count/g) in breast cancer tissue according to ER status. (b–m) Count of each immune cell fraction per unit weight of the tissue (count/g) according to ER status. A tabulated summary of the statistics values and recalculated statistics values excluding outliers is shown on the upper and lower left sides of the figure, respectively. ns p>0.05 (not significant); * p<0.05; p<0.01; * p<0.001 Figure 3 Association between estrogen receptor (ER) status and subsets of tumor-infiltrating immune cells. (a) Total leukocyte density (count/g) in breast cancer tissue according to ER status. (b–m) Count of each immune cell fraction per unit weight of the tissue (count/g) according to ER status. A tabulated summary of the statistics values and recalculated statistics values excluding outliers is shown on the upper and lower left sides of the figure, respectively. ns p>0.05 (not significant); * p<0.05; p<0.01; * p<0.001 Page 21/23 Figure 4 Association between estrogen receptor (ER) status and subsets of tumor-infiltrating immune cells. (a) Total leukocyte density (count/g) in breast cancer tissue according to ER status. (b–m) Count of each immune cell fraction per unit weight of the tissue (count/g) according to ER status. A tabulated summary of the statistics values and recalculated statistics values excluding outliers is shown on the upper and lower left sides of the figure, respectively. ns p>0.05 (not significant); * p<0.05; p<0.01; * p<0.001 Figure 5 Association between androgen receptor (AR) status and subsets of tumor-infiltrating immune cells. (a) Total leukocyte density (count/g) in breast cancer tissue according to AR status. (b–m) Count of each immune cell fraction per unit weight of the tissue (count/g) according to AR status. A tabulated summary of the statistics values and recalculated statistics values excluding outliers is shown on the upper and lower left sides of the figure, respectively. ns p>0.05 (not significant); * p<0.05; p<0.01; * p<0.001 Association between androgen receptor (AR) status and subsets of tumor-infiltrating immune cells. (a) Total leukocyte density (count/g) in breast cancer tissue according to AR status. (b–m) Count of each immune cell fraction per unit weight of the tissue (count/g) according to AR status. A tabulated summary of the statistics values and recalculated statistics values excluding outliers is shown on the upper and lower left sides of the figure, respectively. ns p>0.05 (not significant); * p<0.05; p<0.01; * p<0.001 Association between androgen receptor (AR) status and subsets of tumor-infiltrating immune cells. (a) Total leukocyte density (count/g) in breast cancer tissue according to AR status. (b–m) Count of each immune cell fraction per unit weight of the tissue (count/g) according to AR status. A tabulated summary of the statistics values and recalculated statistics values excluding outliers is shown on the upper and lower left sides of the figure, respectively. ns p>0.05 (not significant); * p<0.05; p<0.01; * p<0.001 Figure 4 Association between estrogen receptor (ER) status and subsets of tumor-infiltrating immune cells. (a) Total leukocyte density (count/g) in breast cancer tissue according to ER status. (b–m) Count of each immune cell fraction per unit weight of the tissue (count/g) according to ER status. A tabulated summary of the statistics values and recalculated statistics values excluding outliers is shown on the upper and lower left sides of the figure, respectively. ns p>0.05 (not significant); * p<0.05; p<0.01; * p<0.001 Association between estrogen receptor (ER) status and subsets of tumor-infiltrating immune cells. (a) Total leukocyte density (count/g) in breast cancer tissue according to ER status. (b–m) Count of each immune cell fraction per unit weight of the tissue (count/g) according to ER status. A tabulated summary of the statistics values and recalculated statistics values excluding outliers is shown on the upper and lower left sides of the figure, respectively. ns p>0.05 (not significant); * p<0.05; p<0.01; * p<0.001 Page 22/23 Figure 5 Association between androgen receptor (AR) status and subsets of tumor-infiltrating immune cells. (a) Total leukocyte density (count/g) in breast cancer tissue according to AR status. (b–m) Count of each immune cell fraction per unit weight of the tissue (count/g) according to AR status. A tabulated summary of the statistics values and recalculated statistics values excluding outliers is shown on the upper and lower left sides of the figure, respectively. ns p>0.05 (not significant); * p<0.05; p<0.01; * p<0.001 S l t Fil Additionalfile.docx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Additionalfile.docx Page 23/23 Page 23/23 Page 23/23
W1881138695.txt	https://inria.hal.science/hal-01054967/document	en		User-Centric Privacy Preservation in Data-Sharing Applications	Lecture notes in computer science	2,010	cc-by	3,264	User-Centric Privacy Preservation in Data-Sharing Applications Feng Gao, Jingsha He, Shufen Peng To cite this version: Feng Gao, Jingsha He, Shufen Peng. User-Centric Privacy Preservation in Data-Sharing Applications. IFIP International Conference on Network and Parallel Computing (NPC), Sep 2010, Zhengzhou, China. pp.423-431, �10.1007/978-3-642-15672-4_35�. �hal-01054967� HAL Id: hal-01054967 https://inria.hal.science/hal-01054967 Submitted on 11 Aug 2014 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. 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. Distributed under a Creative Commons Attribution 4.0 International License User-Centric Privacy Preservation in Data-Sharing Applications Feng Gao1, 1 Jingsha He2, and Shufen Peng1 College of Computer Science and Technology, Beijing University of Technology Beijing China 2 School of Software Engineering, Beijing University of Technology Beijing China maple0371@emails.bjut.edu.cn jhe@bjut.edu.cn Abstract. During data sharing process across people, users do not want the information that contains privacy to be shared with everyone else; some people may only want to share it with someone they are familiar with. To solve this issue of privacy preservation in data-sharing applications, we propose a novel user-centric method. Our main contributions include the followings. (1) Users can select key words or characters for their own privacy information, which is a user-centric way to protect privacy. (2) During the process of data sharing, data substitution can be used to ensure privacy preservation as well as high successful rate of data sharing. (3) Our method can be used in different data-sharing applications in a flexible way. Simulation results show that our method can achieve our privacy preservation goal. Keywords: user-centric, privacy preservation, data sharing, trust, P2P network, online social network 1 Introduction The evolution of peer-to-peer (P2P) networks has triggered large scale distributed applications. The main application domain is data sharing across a very large number of highly autonomous participants. The popular scenarios of data sharing in the peer-to-peer (P2P) networks focus on massive file sharing. Advanced scenarios such as online communities (e.g., medical communities) need to share private or sensitive data frequently. The other typical data-sharing application is online social networks, which is immensely popular, claiming over 200 million users. In online social networks, the information that users share may include privacy, such as personal information or private photographs that should not be misused [2]. Privacy is a concept combining law, sociology and psychology. The dimension of privacy refers to user’s culture, education level, preference and so on. Therefore, the definition, the sensitive degree and the range of privacy information is diversiform. According to the multiplicity of privacy understandings, it should be important to research the privacy preservation mechanism that is user-centric way. To solve the issue of privacy preservation in data-sharing applications, we propose a novel user-centric method in this paper that includes following contributions. Firstly, user can select key words or characters by themselves for their own privacy information which means a user-centric way to protect privacy. Secondly, if the data under sharing contains too much privacy according to user’s privacy policy and trust model, data substitution can be used to ensure privacy preservation. The substitution also brings higher successful rate of data sharing. Thirdly, our method can be applied in different data-sharing applications in a flexible way. The rest of this paper is organized as follows. In section 2, we introduce some related work. In section 3, we explain our user-centric privacy preservation method that includes formally description and the execution model. In section 4, we perform some simulations. The results show that our method can achieve our privacy preservation goal. Finally, we conclude this paper in Section 5. 2 Relate Work Privacy preservation is an important issue in data-sharing applications. Jawad et al. [1] propose a P2P data privacy model which combines the Hippocratic principles and the trust notions to support P2P systems. Di Crescenzo and Lipton [2] describe an evolving access control mechanism in social networking to provide non-trivial quantifiable guarantees for formally specified requirements of utility and privacy. Wei Q and Yansheng L [3] introduce a practical solution to defend against background knowledge attacks with considering the privacy disclosure in social network data publishing. Kobsa A. and Teltzrow M. [4] proposed a user interface design approach in which the privacy practice of a website was explicated in a contextualized manner. These methods for privacy preservation in data-sharing applications focus on certain computing application, none of them can offer privacy preservation in all scenarios for data sharing. Selective partial encryption offers protection for compressed image data [5], audio data and video data. Although this method can protect privacy by encrypting partial data which the user does not want to be shared, the receiver can recognize that the sender does not trust him from the partial encryption data. Therefore, this method is unsuitable for data sharing with privacy preservation. 3 User-Centric Privacy Preservation Method 3.1 Formally Description In this section, we describe the concepts and expressions used in our privacy preservation method. Data object We consider the user’s data as objects. These objects make up to a logical database. Let D1 , D 2 ,.... D m ∈ D be the user’s data objects (they can be image, text, file and so on), {D1, D2 ,.... D m} is the user’s logical database, and D is the space of data object. Privacy range The range of privacy information is defined by the customized setting and the default setting. The customized setting means the user can choose the key K words and the characters of their privacy information. The set {k / k ∈ (k1 , k 2 ,.... k n )} and the set C {c / c ∈ (c1 , c2 ,.... co)} denote the choice of the user’s key words and characters for their privacy information respectively. The default setting means that makes all the general acknowledged privacy information into privacy range. Privacy policy ontology We abstract the user’s privacy policy as privacy policy ontology and its attribute as trust evidence with constraint which need to be satisfied in semantic way ([6]). Privacy information ontology According to the customized setting, the default setting and privacy policy ontology, privacy information ontology created in semantic way ([6]) correspondingly. Privacy information ontology describes the needed trust evidences when disclosing privacy. Privacy mapping function P The mapping function P : D → {0,1} denotes the user data D whether contains privacy based on privacy information ontology. Let d1 , d 2 ,.... d r ∈ {0,1} express the value of function P where d j = P( D j )( j = 1,2,...r ) . And d j = 1 means the user’s data Dj involves privacy while d j = 0 means Dj does not involve privacy. Privacy entropy We describe the entity’s trust that should be achieved when disclosing a piece of privacy as T a , the trust that the entity has already been achieved before the privacy disclosure as T b .Conditional probability P = prob(T a / T b) denotes the probability of achieving T a under condition T b . Set T e {e1, e 2 ,...e s} denotes the needed trust evidences when disclosing one piece of privacy information. Conditional probability P i = prob(T ai / T bi ) represents the i th trust evidence’s conditional probability when disclosing the privacy information. Let p1, p 2 ,.. p s denote the original values of conditional probability p i , preprocess Pi by formula 1. Pi = pi p1 + p 2 + .... p s (1) We use H to denote privacy entropy which means privacy information quantity: s H = −k ( ∑ P i log P i ) i =1 s ( ∑ P i = 1 , k = p1 + p 2 + .... + p s ) (2) i =1 Trust mapping function T The mapping function T : D → (a, b) denotes that for certain trust, the interval of privacy entropy can be afforded in data sharing. This mapping function related to the user’s trust model and privacy policy. Substitution data object Let D1 ' , D2 ' ,.... Dt '∈ D be the substitution data objects (they can be image, text, file and so on), {D1 ' , D 2 ' ,.... Dt '} as logical substitution database, and D' 3.2 is the substitute data object space. Privacy Preservation Method The architecture of our user-centric privacy preservation method in data sharing is illustrated in Fig.1. And this method includes privacy setting module and privacy processing module. Privacy setting module, in this module the users can choose their own key words ( K {k / k ∈ (k1 , k 2 ,.... k n)} ) and characters ( C {c / c ∈ (c1 , c2 ,....co)} ) for their privacy with the default setting which makes the all general acknowledged privacy information into privacy range. Privacy processing module, this module process the user’s sharing data by four steps. Step1 Scan the information that the user want to share by using privacy mapping function P, for the sharing data set Ds ( D1 , D 2 ,.... Dt )(t ≤ m) , if d l = P( Dl ) = 1(l = 1,2,...m) , record d l to set d ' d '∈ {d k ' / d k ' = d j , d j = P( D j ) = 1}, (k ∈ (1, w), j ∈ (1, k )) . Step2 For each D j that P( D j ) = d j , (d j ∈ d ' ) , compute H ( D j ) , then let H ' records the sum of the H ( D j ) based on formula (2). n w H ' = ∑ H ( D j ) ( H ( D j ) = −k ( ∑ P i log P i ) j =1 i =1 ) (3) Step3 Divide the trust interval to define that for the certain trust how much privacy information can be shared. The total of interval can be set by the user or default as a certain constant. Denote there is u trust interval T , each interval controls v terms privacy information Q , for t i ∈ T , q j ∈ Q , t i controls q j privacy information. For trust interval t i , the lower bound of mapping function T : D → (a, b) says ai compute by formula (4). ai = u v 1 (i − 1) ∑ ∑ H (q j )(i = 1,2,....u ) u i =1 j =1 (4) And the upper bound says bi compute by formula (5). bi = u v 1 ⋅ i ⋅ ∑ ∑ H (q j )(i = 1,2,....u ) u i =1 j =1 (5) So the interval (ai , bi) denotes the privacy entropy that the i th trust interval can be afforded in data sharing. Step4 For the sharing data set, if H '∈ (ai , bi) , share the data with the entity. If H '∉ (ai , bi) , substitute some sharing data from substitution database, until the H ' satisfied H '∈ (ai , bi ) , then share that new data set. Our user-centric privacy preservation method in data-sharing applications includes 9 steps as follows. 1. The user input the data that he wants to share to privacy preservation model; 2. Creates privacy policy ontology according to privacy policy and based on it also with the user setting and the default setting, privacy setting module creates the privacy ontology; 3. Scan input data set by using privacy mapping function P then record the information which involved privacy; 4. Compute the entropy of privacy information; 5. Divide the trust interval; 6. According to entity’s trust, judge the privacy entropy whether in the interval or not. If privacy entropy is in it, share the data set, otherwise do step 7; 7.Substitute some data from substitution data base; 8. Do 4 until the privacy entropy is in relevant interval; 9. Output the new data set which means privacy preserved. Privacy preserving module Privacy setting module Privacy policy Share data set Privacy policy ontology User set Default set Privacy mapping Privacy information ontology Privacy processing module Privacy entropy Substitution database Trust interval Trust model New share data set with privacy preservation Fig. 1. Architecture of Privacy Preservation Model 4 Simulations and Analysis We have performed evaluation of our method using the .NET Framework 2.0 platform and implemented a simulator written in C#. The experiment was carried out on a laptop computer with an Intel Pentium (R) Core(TM) 2 Duo 1.83 GHz CPU and 1G bytes of memory. In our simulation, we choose the parameters as follows. The data set {D1 , D2 ,.... Dm}(m = 500) is the user’s logical database, there is u (u = 5) trust interval T , each interval controls v(v = 10) terms privacy information Q . The space of privacy information is 50 terms and the data set {D1 ' , D 2 ' ,.... Dt '}(t = 15) is logical substitution database. Privacy entropy uses the data form our previous work [7], the sum of privacy information entropy is between 0 and 3.5 in our randomly selecting data simulations. In case 1, we select 10 terms data from 500 randomly by using our privacy entropy computing method but without substitution method to describe the situation of data sharing. We run our simulation 50 rounds, the x-axis records the time of round, and y-axis denotes the trust interval. There are 5 trust intervals which can afford privacy entropy [0,0.7) [0.7,1.4) [1.4,2.1) [2.1,2.8) [2.8,3.5] respectively. In our randomly selection, if privacy entropy of sharing data is in arbitrary interval, record as one successful data sharing. The result of case 1 is illustrated in Fig.2. The coordinate ( x, y) means the yth simulation involved x privacy. Take point (40,0.7) as example, it means the th 40 simulation data sharing scenario is successful if entity’s trust can afford privacy entropy 0.7. That is to say, if the entity’s trust is in the certain trust interval which can afford privacy entropy between 0.7 and 1.4, this entity can share data with user. And the entity whose trust is in the certain trust interval which can afford privacy entropy [1.4,2.1) , [2.1,2.8) and [2.8,3.5] respectively also can share data with user. So our simulations show the situation of once data selecting, each trust interval that entity’s trust in whether can share this selected data or not. Then we do case 2 that select 20 terms data from 500 randomly and show result in Fig.3. We can see that the points which mean successful data sharing in Fig.3 is universal lower than the points in Fig.2. That is to say in case 1 the demand of trust to share selected data is more strictly. In case 3 and case 4, we select 10 data terms and 20 data terms respectively from the user data space randomly with using our substitution method to describe the situation of data sharing. Fig.4 show the result of case 3 and Fig.5 illustrates the result of case 4 respectively. We denote “successful rate of data sharing” as successful data sharing to all of data sharing scenarios. We then record the successful rate of data sharing and illustrate in table 1. We can see that without the substitution method, successful rate of data sharing is 37.2% and 34.8% respectively according to our simulation scenario. That is clear that in process of data sharing with privacy considering the success rate is low. With substitution method, the successful rate of user data sharing is close to 54.4% and 54.8% respectively. That accounts for our method is effective in data sharing with privacy preservation. Also we can learn that the more data selection for share the smaller successful rate by comparing case 1 to case 2. Fig.6 illustrates information quantity of data sharing compare case 1 to case 3. By using substitute method, the information quantity is less than the data sharing without substitute method which means privacy enhanced. Fig. 2. The situation of data sharing selecting 10 from 500 (without substitution) Fig. 3. The situation of data sharing selecting 20 from 500 (without substitution) Fig. 4. The situation of data sharing selecting 10 from 500 (with substitution) Fig. 5. The situation of data sharing selecting 20 from 500 (with substitution) Fig. 6. Information quantities compare case 1 to case 3 Table 1. Successful Rate of Data Sharing Simulation Case Successful Rate of Data Sharing Case 1 37.2% Case 2 34.8% Case 3 54.4% Case 4 54.8% 5 Conclusions and Future Work Privacy preservation is an important issue for data sharing in applications such as P2Pnetworking, online social networking and so on. Privacy is a concept combining all kinds of elements, and the dimension of privacy is diversiform. To solve the privacy preservation problem in data sharing, we propose a novel user-centric method in this paper. In our method, the users can select key words and characters for their privacy information, which means user-centric way to protect privacy. Then during the process of data sharing, if the sharing data contains too much privacy according to user’s privacy policy and trust model, our data substitution method can be used to ensure privacy preservation and as well as high successful rate for data sharing. And our method can be used in different data sharing applications in a flexible way. We did five simulation cases and simulation results show that our method can achieve our privacy preservation goal. In the future, we will analyze the complexity and cost of our approach; refine our method to balance privacy preservation and its additional cost. We need further improve our method with real implementation and applications. Acknowledge The work in this paper has been supported by research funding from Beijing Education Commission (Grant No. KM201010005027). References 1. Jawad, M., Serrano-Alvarado, P., and Valduriez, P.: Protecting Data Privacy in Structured P2P Networks, Data Management in Grid and Peer-to-Peer Systems, Linz, Austria. (2009). 2. Di Crescenzo, G., and Lipton, Richard J.: Social Network Privacy via Evolving Access Control, Lecture Notes in Computer Science (including sub series Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics), v 5682 LNCS, p 551-560, (2009). 3. Wei, Q., and Yansheng, L.: Preservation of Privacy in Publishing Social Network Data, International Symposium on Electronic Commerce and Security (2008). 4. Kobsa, A., and Teltzrow, M.: Contextualized Communication of Privacy Practices and Personalization Benefits: Impacts on Users’ Data Sharing and Purchase Behavior, Lecture Notes in Computer Science, v 3424, p 329-343, Springer Verlag, (2005). 5. Spinsante, S. and Gambi, E.: Selective Encryption for Efficient and Secure Transmission of Compressed Space Images, International Workshop on Satellite and Space Communications (IWSSC), 9-11 , Tuscany, Italy, Sept. (2009). 6. Gao, F., He, J., Peng, S., Wu, X. and Liu, L.: An Approach for Privacy Protection Based-on Ontology, The 2nd International Conference on Networks Security, Wireless Communications and Trusted Computing, Wuhan, China, April 24-25, 2010. 7. Gao, F., He, J., Peng, S., and Wu, X.: A Quantify Metric for Privacy Protection Based on Information Theory, 3rd International Symposium on Intelligent Information Technology and Security Informatics (IITSI 2010), Jinggangshan, China April 2-4, 2010.
https://openalex.org/W1975722505	https://edoc.hu-berlin.de/bitstream/18452/9960/1/282ycnD78FHjA.pdf	English	null	The Folk Heritage Collections in Crisis conference	Library hi tech	2,001	public-domain	2,363	Library Hi Tech Volume 19 . Number 1 . 2001 . pp. 7±9 # MCB University Press . ISSN 0737-8831 The variety of historical evidence is nearly infinite. Everything that man says or writes, everything that he makes, everything he touches can or ought to teach us about him (Bloch, 1953, p. 66). Even if they [family papers] have not been lost, eaten by rats, or scattered by the caprice of sale or inheritance through the attics of three or four houses on different estates, there is nothing to oblige their present owners to let you see them (Bloch, 1953, p. 73). The variety of historical evidence is nearly infinite. Everything that man says or writes, everything that he makes, everything he touches can or ought to teach us about him (Bloch, 1953, p. 66). Even if they [family papers] have not been lost, eaten by rats, or scattered by the caprice of sale or inheritance through the attics of three or four houses on different estates, there is nothing to oblige their present owners to let you see them (Bloch, 1953, p. 73). Intellectual property, Preservation, Archives Intellectual property, Preservation, Archives Intellectual property, Preservation, Archives The Folk Heritage Collections in Crisis conference Editorial Michael Seadle The ``Folk Heritage Collections in Crisis'' conference that took place in December 2000 at the Library of Congress addressed precisely the problems that Bloch raised half a century earlier: everything having to do with a culture is of interest, and even seemingly robust records are vulnerable to destruction or concealment. No one doubted the first point, and no one had a perfect solution to the second. Perfect solutions take too long anyway. Finding ways to take imperfect but immediate action had to suffice as a common goal. The author Michael Seadle is Editor of Library Hi Tech. He is also Digital Services and Copyright Librarian at Michigan State University, East Lansing, Michigan, USA. E-mail: seadle@mail.lib.msu.edu A sense of urgency pervaded the conference. Paper records have a relatively long lifespan, even when printed on acidic media. But analog sound and video recordings, even on quality media, begin deteriorating immediately. Some recordings from the 1970s suffer from the ``sticky shed'' syndrome and might at best survive one more use, after a restorative baking. Other physical artifacts are also vulnerable, not necessarily because of deterioration, but because many libraries and archives have inadequate provision for storing and describing non-text objects. Even worse: many folk collections remain in private hands or unofficial collections. The death or retirement of a single person might send hundreds of unique items into the trash. Digital preservation elements of the intellectual infrastructure: those shared terms and ideas without which discourse cannot proceed. At a recent interview at Michigan State University, an excellent job candidate answered a question about digital preservation by saying that she assumed people did not accept digitization as preservation. In this conference, the opposite was true. Janet Gertz of Columbia said that the question is not whether we do digital, but how. And one person infamous for his opposition to digital preservation of sound merely encouraged archives not to discard the analog versions after digitization. As a digital advocate, I felt a milestone had been passed. But the standards for digital preservation received relatively little attention. This was partly because other groups were working on those standards. Research Libraries Group (RLG) is preparing a magnetic media manual, and the Library of Congress (LC) is establishing standards for its own massive multimedia digitization project. It was also partly because issues like sampling rates and file formats are easier to solve than the choices of which items to preserve first, and where to find the financial resources to make that possible. Is a unique copy of linguistic interviews of Yiddish speakers in now-nonexistent eastern European villages more important to preserve than an anthropologist's interview with tribal elders who are now having second thoughts about how widely they should share secret knowledge? It is an unanswerable question, but in a world of limited resources, someone or some institution may well have to decide. At a recent interview at Michigan State University, an excellent job candidate answered a question about digital preservation by saying that she assumed people did not accept digitization as preservation. In this conference, the opposite was true. Janet Gertz of Columbia said that the question is not whether we do digital, but how. And one person infamous for his opposition to digital preservation of sound merely encouraged archives not to discard the analog versions after digitization. As a digital advocate, I felt a milestone had been passed. Abstract The Folk Heritage Collections in Crisis conference pursued three themes: access, preservation, and intellectual property. The 100 invited participants were mainly middle-aged, established, upper-echelon professionals. What is interesting is how much agreement there was on issues like digital preservation and the use of encoded archival description. Participants saw the US copyright law as a barrier which neither upheld the rights of people of non-European origin, nor facilitated the free movement of information. The conference pursued three themes: (1) access; (2) preservation; and (3) intellectual property. I participated in the last of these, and in the plenary sessions. More information about the action items and the conference in general will be available from the Council on Library and Information Resources[1], which is publishing a white paper on the meeting in March. Rather than repeat those action items, I want to talk about some of the significant areas which are not action-oriented, but which represent 7 The Folk Heritage Collections in Crisis conference Michael Seadle Library Hi Tech Volume 19 . Number 1 . 2001 . 7±9 Library Hi Tech Volume 19 . Number 1 . 2001 . 7±9 Conference demography To understand the discourse, it is essential to know who participated. Roughly four communities had representation: (1) folklorists; (2) librarians/archivists; (3) technologists; and (4) lawyers. But the standards for digital preservation received relatively little attention. This was partly because other groups were working on those standards. Research Libraries Group (RLG) is preparing a magnetic media manual, and the Library of Congress (LC) is establishing standards for its own massive multimedia digitization project. It was also partly because issues like sampling rates and file formats are easier to solve than the choices of which items to preserve first, and where to find the financial resources to make that possible. Is a unique copy of linguistic interviews of Yiddish speakers in now-nonexistent eastern European villages more important to preserve than an anthropologist's interview with tribal elders who are now having second thoughts about how widely they should share secret knowledge? It is an unanswerable question, but in a world of limited resources, someone or some institution may well have to decide. The folklorists were mainly people who went out into the field, did interviews, recorded them on various media, and have a long-term relationship with particular cultural communities. Those communities included native Americans and urban immigrants; they involved multiple languages and a wide range of technology. The librarians and archivists were mainly degreed professionals from established institutions, though at least one major private collector attended as well. The technologists were more diverse. Some had special expertise in making materials available digitally. Others were experts in sound and sound reformatting. The lawyers overwhelmingly worked with copyright issues, sometimes for rights holders, sometimes for organizations wanting access. The majority of participants were over 40, perhaps even over 50. Women outnumbered men, perhaps because librarians outnumbered technologists. Although pinstriped suits were not greatly in evidence (admittedly I wore one), and ties essentially vanished for the Saturday session (I wore turtlenecks both days), the men's clothing was consistently more conservative than what I see on staff in my library. Even those few jeans wearers balanced their informality with a suit jacket. Female participants followed similar patterns: the only actual color in the room came from two red sweaters and one red scarf. Copyright Most people in the room saw the US copyright law as a barrier which neither upheld the rights of people of non-European origin, nor facilitated the free movement of information that is key to the intellectual and educational enterprises. Yet they recognized that they had to deal with it, with its quirks and unclarities, and with the lawyers who knew how to deal with it. They did not like it, and had no collective urge to learn enough to exploit those loopholes and options that do exist. Nor did they take comfort in the excellent suggestion that a wealth of public domain materials remains to be exploited. While such concerns surfaced again and again, and clearly represented the prime intellectual property concern for a significant group of the ethnographic community, the debate focused ultimately on US law. One participant argued that ``bits want to be free''. He meant that digital objects should be shared regardless of the law. Others argued for changing US copyright law to limit ownership rights and increase access. One man with a rich southern accent insisted that this could be done, and that he knew the right committee chairman to make it happen. His conviction and self-assurance were persuasive, yet most of us did not believe him. Memories of past lobbying failures were too vivid to erase. The access issue that received little discussion because of widespread agreement was metadata. Most people seemed to prefer encoded archival description (EAD). EAD has flaws. It does not offer enough detail on copyright and usage restrictions, though it has tags for both. And it does not offer enough detail for describing the nature of sound files. These weaknesses can be remedied with a few modifications. Most people in the room saw the US copyright law as a barrier which neither upheld the rights of people of non-European origin, nor facilitated the free movement of information that is key to the intellectual and educational enterprises. Yet they recognized that they had to deal with it, with its quirks and unclarities, and with the lawyers who knew how to deal with it. They did not like it, and had no collective urge to learn enough to exploit those loopholes and options that do exist. Nor did they take comfort in the excellent suggestion that a wealth of public domain materials remains to be exploited. Conclusion A conference like this is not an end-point, but part of a process of discourse about how libraries and archives handle the increasingly diverse kinds of materials that scholars find interesting. A follow-on discussion will take place at the American Library Association in Washington in January 2001. No one expects quick solutions to the problems. What is interesting is how much agreement there is on issues like digital preservation and the use of EAD. I do not wish to exaggerate. Both are still controversial in the larger library and archival world, and many problems remain to be solved. But progress has been made. Peggy Bolger of the Library of Congress especially deserves thanks for organizing the conference and for providing an intellectual framework for further work. Note 1 http://www.clir.org/ 1 http://www.clir.org/ Bloch, M. (1953), The Historian's Craft, translated by Peter Putnam, Vintage, New York, NY. ACCESS Conference participants recognized that access is difficult enough for materials in established archives that belong to well-known institutions. No one knew how much unique oral material sits in private collections in attics and basements. Art Silverman of National Public Radio talked about his experiences with the Lost and Found Sound program, where people would call in with odd and invaluable items, such as the 78 rpm recording of a man who talked about having heard Abraham Lincoln give the Gettysburg address. Only one avowed private Copyright Tony Seeger, Professor of Ethnomusicology at UCLA, put copyright in the context of the web of agreements that govern objects in all archives. This helped participants step back from viewing copyright as only a part of the US law, and to consider how non-Eurocentric societies think about the ownership of their words, music, art, and artifacts. Although no single answer was possible, even for native American societies, one theme emerged strongly: ownership matters. Often the ownership rights belong to no individual, and sometimes custom limits access rights explicitly The point is that the 100 invited participants were mainly middle-aged, established, upper- echelon professionals. Virtually everyone knew at least some other participants. They were conference-going veterans whose social and intellectual backgrounds had a lot in common. 8 Library Hi Tech Volume 19 . Number 1 . 2001 . 7±9 The Folk Heritage Collections in Crisis conference Michael Seadle The Folk Heritage Collections in Crisis conference Michael Seadle Volume 19 . Number 1 . 2001 . 7±9 to an elite. An argument was made that a tribe should have a say in how its intellectual property is used. The counter argument was that such communities are too ill defined: who really belongs and which faction should control such decision making? collector had been invited to the conference. His collection is unusually large and notable. Other secret collectors came, too. I admit to having a secret stash of oral history tapes in my desk drawer. Probably others at the conference could make similar confessions. Why do we not donate them to institutions that could improve access? Sloth? Privacy concerns? The stupidity of the questions we asked? The problem embraces these issues and more. such decision making? While such concerns surfaced again and again, and clearly represented the prime intellectual property concern for a significant group of the ethnographic community, the debate focused ultimately on US law. One participant argued that ``bits want to be free''. He meant that digital objects should be shared regardless of the law. Others argued for changing US copyright law to limit ownership rights and increase access. One man with a rich southern accent insisted that this could be done, and that he knew the right committee chairman to make it happen. His conviction and self-assurance were persuasive, yet most of us did not believe him. Memories of past lobbying failures were too vivid to erase. Reference Bloch, M. (1953), The Historian's Craft, translated by Peter Putnam, Vintage, New York, NY. 9
https://openalex.org/W4235793241	http://vital.lib.tsu.ru/vital/access/services/Download/koha:000720383/SOURCE1	Russian	null	ОЦК→ГПУ→ОЦК ПРЕВРАЩЕНИЯ КАК МЕХАНИЗМЫ СВЕРХПЛАСТИЧНОСТИ И ФОРМИРОВАНИЯ НАНОКРИСТАЛЛИЧЕСКИХ СТРУКТУР В СПЛАВАХ НА ОСНОВЕ НИКЕЛИДА ТИТАНА И ВАНАДИЯ	Международная конференция "Физическая мезомеханика. Материалы с многоуровневой иерархически организованной структурой и интеллектуальные производственные технологии"	2,021	cc-by	721	МЕЖДУНАРОДНАЯ КОНФЕРЕНЦИЯ «Физическая мезомеханика. Материалы с многоуровневой иерархически организованной структурой и интеллектуальные производственные технологии» 6–10 сентября 2021 г. Томск, Россия Д Д 1,2Тюменцев А.Н., 1,2Дитенберг И.А., 1,2Суханов И.И. 1Институт физики прочности и материаловедения СО РАН, Томск 2Томский государственный университет, Томск Изучены закономерности структурных превращений в условиях сверхвысокой технологической пластичности и формирования наноструктурных состояний при больших пластических деформациях сплавов на основе никелида титана и ванадия прокаткой и кручением на наковальнях Бриджмена при комнатной температуре. В сплаве TiNi(Fe,Mo) в процессе пластической деформации кручением под давлением при величинах истинной логарифмической деформации е ≈ 5-6 получено нанокристаллическое (аморфно-кристаллическое) структурное состояние с размерами нанозерен (d ≈ 10-15 Å) в несколько межатомных расстояний (рис. 1). Это предельная (максимально возможная) степень наноструктурирования кристалла, когда более половины атомов границ нанозерен и их тройных стыков находится в аморфном состоянии. Механизмами формирования таких состояний являются В2→В19′→В2 превращения путем их многократного повторения в процессе повышения степени деформации и квазивязкий механизм переориентации потоками неравновесных точечных дефектов в полях высоких локальных градиентов (моментов) напряжений. Эти механизмы отражают реализацию качественно разных мод дисторсии. Первый осуществляется с участием единственной моды деформации – однородной деформации мартенситного превращения типа деформации Бейна. Второй обеспечивает повороты кристаллической решетки, отражающие антисимметричную часть тензора дисторсии в стесненных условиях деформации или полях локальных градиентов (моментов) напряжений. а б Рис. 1. Картины микродифракции наноструктурных состояний с размерами нанозерен d ≈ 40 (а) и (10-15) Å (б) после деформации сплава TiNi(Fe, Mo) на наковальнях Бриджмена при величинах истинной логарифмической деформации е ≈ 3 (а) и 6 (б) 40 -1 1 А <110> 15 -1 1 А B2 <200> B2 <112> B2 10 -1 1 А б <110> 15 -1 1 А B2 <200> B2 <112> B2 10 -1 1 А а 40 -1 1 А б а Рис. 1. Картины микродифракции наноструктурных состояний с размерами нанозерен d ≈ 40 (а) и (10-15) Å (б) после деформации сплава TiNi(Fe, Mo) на наковальнях Бриджмена при величинах истинной логарифмической деформации е ≈ 3 (а) и 6 (б) Основными факторами формирования указанных выше предельных аморфно- кристаллических структур в никелиде титана являются: Основными факторами формирования указанных выше предельных аморфно кристаллических структур в никелиде титана являются: 1. Низкая дислокационная активность в В2 фазе TiNi сплавов, определяющая высокий уровень деформирующих напряжений и связанных с ними неравновесных дислокационно- дисклинационных субструктур – источников высоких локальных градиентов (моментов) наномасштабного уровня, определяющих характерные размеры микрообъемов, способных быть зародышами новых кристаллитов, и определяющих размеры субмикро- или нанозерен и субзерен. 1. Низкая дислокационная активность в В2 фазе TiNi сплавов, определяющая высокий уровень деформирующих напряжений и связанных с ними неравновесных дислокационно- дисклинационных субструктур – источников высоких локальных градиентов (моментов) наномасштабного уровня, определяющих характерные размеры микрообъемов, способных быть зародышами новых кристаллитов, и определяющих размеры субмикро- или нанозерен и субзерен. DOI: 10.17223/978-5-907442-03-0-2021-040 ОЦК→ГПУ→ОЦК ПРЕВРАЩЕНИЯ КАК МЕХАНИЗМЫ СВЕРХПЛАСТИЧНОСТИ И ФОРМИРОВАНИЯ НАНОКРИСТАЛЛИЧЕСКИХ СТРУКТУР В СПЛАВАХ НА ОСНОВЕ НИКЕЛИДА ТИТАНА И ВАНАДИЯ 1,2Тюменцев А.Н., 1,2Дитенберг И.А., 1,2Суханов И.И. 1Институт физики прочности и материаловедения СО РАН, Томск 2Томский государственный университет Томск Д Д 1,2Тюменцев А.Н., 1,2Дитенберг И.А., 1,2Суханов И.И. 1Институт физики прочности и материаловедения СО РАН, Томс 2Томский государственный университет, Томск Работа выполнена в рамках государственного задания ИФПМ СО РАН, номер FWRW-2021-0008. Д Д 1,2Тюменцев А.Н., 1,2Дитенберг И.А., 1,2Суханов И.И. 1Институт физики прочности и материаловедения СО РАН, Томск 2Томский государственный университет, Томск 2. Снижение в зонах В2→В19`→В2 превращений интенсивности диффузионных механизмов релаксации микроструктуры (например, диффузионного роста нанозерен) 2. Снижение в зонах В2→В19`→В2 превращений интенсивности диффузионных механизмов релаксации микроструктуры (например, диффузионного роста нанозерен) 69 вследствие уменьшения в этих зонах запасенной энергии деформации при высокой плотности низкоэнергетических двойниковых границ специального типа. Пластическая деформация путем многократной последовательности ОЦК→ГПУ→ОЦК превращений в указанном выше TiNi сплаве и малоактивируемом ванадиевом сплаве V-4Ti- 4Cr обеспечивает возможность их сверхвысокой технологической пластичности – способности к неограниченным (е > 7) степеням деформации при комнатной температуре без каких-либо промежуточных отжигов. Эта возможность, в отличие от способности к измельчению зеренной структуры, слабо зависит от характеристик прочности или стабильности кристаллической решетки. Важными условиями ее реализации являются высокая эффективность процессов релаксации неравновесных динамических структур, формирующихся в процессе многократного повторения этих превращений, и геометрии деформирующих напряжений, обеспечивающей высокие компоненты сжатия в направлениях типа <110> в процессе как прямого (ОЦК→ГПУ), так и обратного (ГПУ→ОЦК) превращений. Обсуждаются атомные механизмы деформации и переориентации кристалла в зонах ОЦК→ГПУ→ОЦК превращений в условиях одновременной реализации мартенситной и квазивязкой моды деформации в зависимости от структурного состояния, свойств материала и условий (температура, скорость и др.) деформации. Работа выполнена в рамках государственного задания ИФПМ СО РАН, номер FWRW-2021-0008. 70
https://openalex.org/W4382394664	https://infoscience.epfl.ch/record/304132/files/document.pdf	English	null	United States politicians’ tone became more negative with 2016 primary campaigns	Scientific reports	2,023	cc-by	12,908	United States politicians’ tone became more negative with 2016 primary campaigns OPEN Jonathan Külz 1, Andreas Spitz 2, Ahmad Abu‑Akel 3, Stephan Günnemann 1 & Robert West 4 There is a widespread belief that the tone of political debate in the US has become more negative recently, in particular when Donald Trump entered politics. At the same time, there is disagreement as to whether Trump changed or merely continued previous trends. To date, data-driven evidence regarding these questions is scarce, partly due to the difficulty of obtaining a comprehensive, longitudinal record of politicians’ utterances. Here we apply psycholinguistic tools to a novel, comprehensive corpus of 24 million quotes from online news attributed to 18,627 US politicians in order to analyze how the tone of US politicians’ language as reported in online media evolved between 2008 and 2020. We show that, whereas the frequency of negative emotion words had decreased continuously during Obama’s tenure, it suddenly and lastingly increased with the 2016 primary campaigns, by 1.6 pre-campaign standard deviations, or 8% of the pre-campaign mean, in a pattern that emerges across parties. The effect size drops by 40% when omitting Trump’s quotes, and by 50% when averaging over speakers rather than quotes, implying that prominent speakers, and Trump in particular, have disproportionately, though not exclusively, contributed to the rise in negative language. This work provides the first large-scale data-driven evidence of a drastic shift toward a more negative political tone following Trump’s campaign start as a catalyst. The findings have important implications for the debate about the state of US politics. A vast majority of Americans—85% in a representative survey by the Pew Research Center1—have the impression that “the tone and nature of political debate in the United States has become more negative in recent years”. Many see a cause in Donald Trump, who a majority (55%) think “has changed the tone and nature of political debate [...] for the worse”, whereas only 24% think he “has changed it for the better”1. The purpose of the present article is to investigate whether these subjective impressions reflect the true state of US political discourse.hhf g jl The answer to this question comes with tangible societal implications: The use of negative affective language in a political debate can worsen the audience’s image of the attacker and of the attackee, while inducing a gen- eralized negative mood2. www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| (2023) 13:10495 www.nature.com/scientificreports/ to analyze political leaders’ psychological attributes such as certainty and analytical thinking22; to quantify the turbulence of Trump’s presidency23; or to measure the effect of linguistic features on the success of US presidential candidates24 and on public approval of US Congress25. A combination of political discourse analysis and psycho- logical measurement tools has further been applied to obtain insights into the personality traits and sentiments of politicians in general26,27, as well Donald Trump in particular28–31. g One limiting factor in the above-cited works is the representativeness and completeness of the underlying data, since subtle social or political behavior may only reveal itself in sufficiently big and rigorously processed data32,33. On the one hand, Congressional records20,21,25 and transcripts of public speeches22,24 record scarce events and do not mirror political discourse as perceived by the average American, whose subjective impression of growing negativity we aim to compare with objective measurements of politicians’ language. On the other hand, news text, despite being a better proxy for the average American’s exposure to political discourse, for the most part does not capture politicians’ utterances directly, but largely reports events (laced with occasional direct quotes) and frequently focuses on a commentator’s perspective. Additionally, most of the above-cited linguistic analyses are not longitudinal in nature, but focus on specific points in time. i Transcending these shortcomings, we take a novel approach leveraging Quotebank34, a recently released corpus of nearly a quarter-billion (235 million) unique quotes extracted from 127 million online news articles published by a comprehensive set of online news sources over the course of nearly 12 years (September 2008 to April 2020) and automatically attributed to the speakers who likely uttered them by a machine learning algo- rithm. By focusing on US politicians, we derived a subset of 24 million quotes by 18,627 speakers, enriched with biographic information from the Wikidata knowledge base35. (Details about data in “Materials and methods”.) As no comparable dataset of speaker-attributed quotes was available before, Quotebank enables us to analyze the tone of US politicians’ public language (as seen through the lens of online news media) at a level of representa- tiveness and completeness that was previously impossible, without confounding politicians’ direct utterances with the surrounding news text. www.nature.com/scientificreports/ g In order to quantify the prevalence of negative language over time, we use established psycholinguistic tools36 to score each quote with respect to its emotional content, aggregate quotes by month, and work with the result- ing time series. Anchored in the average American’s subjective perception that Donald Trump has changed the tone and nature of political debate (see above), we hypothesized the start of his primary campaign in June 2015 as an incision point and fitted linear regression models37 with a discontinuity in June 2015 to the time series, as illustrated in Fig. 1. Figure 1. Quantifying the evolution of negative language in US politics (2008–2020). (a) The black points show the fraction of negative emotion words, averaged monthly over all quotes from all 18,627 quoted politicians. The red vs. blue background shows the quote share of Trump vs. Obama (if Trump had T quotes and Obama had O quotes in a given month, the respective red bar covers a fraction T/(T + O) of the full y-range). Whereas the frequency of negative emotion words had decreased continuously during the first 6.5 years of Obama’s tenure, it suddenly and lastingly increased in June 2015, when Trump’s primary campaign started and his quote share began to surpass Obama’s. (b) Regression analysis: The black points again show the fraction of negative emotion words, but now as z-scores (i.e., after subtracting the pre-campaign mean and dividing by the pre-campaign standard deviation). In red, we plot regression lines for the periods before and after June 2015. The coefficients of the ordinary least squares regression model yt = α0 + β0 t + α it + β it t + εt (where t is the number of months since June 2015, and it indicates whether t ≥0 ; cf. Eq. (1)) quantify the slopes of both lines, as well as the sudden increase of α = 1.6 pre-campaign standard deviations coinciding with the discontinuity in June 2015 ( t = 0 ), as visualized. Figure 1. Quantifying the evolution of negative language in US politics (2008–2020). (a) The black points show the fraction of negative emotion words, averaged monthly over all quotes from all 18,627 quoted politicians. The red vs. blue background shows the quote share of Trump vs. United States politicians’ tone became more negative with 2016 primary campaigns OPEN Furthermore, a negative tone of coverage can hurt the level of support and perceived legitimacy of political institutions3, resulting in a decrease of trust in political processes4. As politics impacts nearly every aspect of our personal lives5, changes in political climate can directly affect not only politics itself, but also the well-being of all citizens. g Although longitudinally conducted voter surveys, notably the American National Election Studies, have shown that negative affect towards members of the other party6–8, polarization9, and partisan voting10 have steadily increased over the last decades, such survey-based studies do not directly measure the tone of politi- cal debate—a linguistic phenomenon—and thus cannot answer whether Americans’ impression of increased negativity is accurate. In contrast, we analyze US politicians’ language as conveyed through the media directly, in an objective, data-driven manner, asking: First, is it true that US politicians’ tone has become more negative in recent years? Second, if so, did Donald Trump’s entering the political arena bring about an abrupt shift11,12, or did it merely continue a previously existing trend13,14? y y g Our methodology (for details, see “Materials and methods”) draws on a long history of research on the language of politics and its function in democracies15–18. For instance, prior work has used records of spoken and written political language to establish the prevalence of negative language among political extremists19; to quantify growing partisanship and polarization20, as well as displayed happiness21, among US Congress members; 1Department of Computer Science, Technical University of Munich, Munich, Germany. 2Department of Computer and Information Science, University of Konstanz, Konstanz, Germany. 3School of Psychological Sciences, University of Haifa, Haifa, Israel. 4School of Computer and Communication Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland. email: robert.west@epfl.ch \| https://doi.org/10.1038/s41598-023-36839-1 Scientific Reports \| (2023) 13:10495 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ The results (cf. Fig. 1) provide clear evidence of a sudden shift coinciding with the hypothesized discontinuity at the start of Trump’s primary campaign in June 2015, when the overall political tone became abruptly more negative. The effect was large and highly significant: the frequency of negative emotion words soared by 1.6 pre-campaign standard deviations, or by 8% of the pre-campaign mean. Although the data from the start of the available period (around the 2008 presidential election) suggests that language was highly negative during that time as well, negative language then decreased continuously during Obama’s tenure. However, this trend came to an end with the June 2015 discontinuity, and the level of negative language remained high in the subsequent period, indicating persistent negativity in political discourse not only during election campaigns, but also in day-to-day politics. Similar effects were observed for specific subtypes of negative language (anger, anxiety, and sadness), as well as for swearing terms. Qualitatively, the patterns are universal, emerging within each party and within strata of speaker prominence. Quantitatively, the language of Democrats, more prominent speakers, Congress members, and members of the opposition party is, ceteris paribus, overall more negative, but the shift at the discontinuity remains (and more strongly so for Republicans) when adjusting for these biographic attributes.hft y g y j g g These population-level effects are not the results of systematic shifts in the distribution of quoted politicians, but are mirrored at the individual level for a majority of the most-quoted politicians. Moreover, the effect size drops by 40% when omitting Donald Trump’s quotes from the analysis, and by 50% when weighing each speaker equally, rather than by the number of their quoted utterances (but the effect remains highly significant in both cases). Both findings imply that prominent speakers—and particularly Donald Trump—have disproportionately, though not exclusively, contributed to the rise in negative language.i g y g g g Taken together, these results objectively confirm the subjective impression held by most Americans1: recent years have indeed seen a profound and lasting change toward a more negative tone in US politicians’ language as reflected in online news, with the 2016 primary campaigns acting as a turning point. Moreover, contrary to some commentators’ assessment13,14, Donald Trump’s appearance in the political arena was linked to a directional change, rather than a continuation of previously existing trends in political tone. www.nature.com/scientificreports/ Whether these effects are fully driven by changes in politicians’ behavior or whether they are exacerbated by a shifting selection bias on behalf of the media remains an important open question (see “Discussion”). Either way, the results presented here have implications for how we see both the past and the future of US politics. Regarding the past, they emphasize the symptoms of growing toxicity in US politics from a new angle. Regarding the future, they highlight the danger of a positive feedback loop of negativity. Results To quantify negative language in quotes, we used Linguistic Inquiry and Word Count (LIWC)36, which provides a dictionary of words belonging to various linguistic, psychological, and topical categories, and whose validity has been established by numerous studies in different contexts and domains38–40. We computed a negative- emotion score for each of the 24 million quotes via the percentage of the quote’s constituent words that belong to LIWC’s negative emotion category. Analogously, we computed scores for LIWC’s three subcategories of the negative emotion category—anger, anxiety, and sadness—as well as for the swear words category. We collectively refer to these five categories as “negative language”. As a robustness check, we replicated the analysis using the dictionary provided by Empath41 instead of LIWC. In contrast to LIWC, the categories in Empath’s dictionary were not hand-crafted but generated by a deep-learning model based on a small set of seed words. The results, which are consistent with those from the LIWC-based analysis, are provided in Figs. S1, S2, S3, and S4, and in Supplementary Table S1. pp y In order to obtain a time series for each word category, we averaged individual quote scores by month. By giving each quote the same weight when averaging, we obtain quote-level aggregates; by giving each speaker the same weight, speaker-level aggregates. (Formal definitions in “Materials and methods”.) Quote-level aggregates give more weight to more frequently quoted politicians, and thus better capture the overall tone of political language as reflected in the news. Whenever, on the contrary, we reason about politicians, rather than about the overall media climate created by all politicians’ joint output, we use speaker-level aggregates as the more appropriate aggregation.i pp p gg g Table 1 summarizes the prevalence of the above five word categories via the means and standard deviations of the respective quote-level aggregates during the pre-campaign period (September 2008 through May 2015). Word category Mean µ Std. dev. σ 1/µ σ/µ Negative emotion 0.01853 0.00090 54 4.8% Anger 0.00647 0.00055 155 8.4% Anxiety 0.00295 0.00022 339 7.3% Sadness 0.00351 0.00014 285 4.1% Swear words 0.00043 0.00015 2329 33.9% Table 1. Overall statistics of LIWC negative-language word categories. Means µ and standard deviations σ were calculated over monthly quote-level aggregates from the pre-campaign period (September 2008–May 2015). The value n = 1/µ in the fourth column implies that, in an average quote, on average every n-th word belongs to the respective category. www.nature.com/scientificreports/ Obama (if Trump had T quotes and Obama had O quotes in a given month, the respective red bar covers a fraction T/(T + O) of the full y-range). Whereas the frequency of negative emotion words had decreased continuously during the first 6.5 years of Obama’s tenure, it suddenly and lastingly increased in June 2015, when Trump’s primary campaign started and his quote share began to surpass Obama’s. (b) Regression analysis: The black points again show the fraction of negative emotion words, but now as z-scores (i.e., after subtracting the pre-campaign mean and dividing by the pre-campaign standard deviation). In red, we plot regression lines for the periods before and after June 2015. The coefficients of the ordinary least squares regression model yt = α0 + β0 t + α it + β it t + εt (where t is the number of months since June 2015, and it indicates whether t ≥0 ; cf. Eq. (1)) quantify the slopes of both lines, as well as the sudden increase of α = 1.6 pre-campaign standard deviations coinciding with the discontinuity in June 2015 ( t = 0 ), as visualized. https://doi.org/10.1038/s41598-023-36839-1 Scientific Reports \| (2023) 13:10495 \| Table 1. Overall statistics of LIWC negative-language word categories. Means µ and standard deviations σ were calculated over monthly quote-level aggregates from the pre-campaign period (September 2008–May 2015). The value n = 1/µ in the fourth column implies that, in an average quote, on average every n-th word belongs to the respective category. The coefficients of variation, σ/µ , shown in the fifth column, allow to easily translate pre-campaign standard deviations (as shown on the y-axes of time series plots) into fractions of pre- campaign means. The most frequent words per category are listed in Supplementary Tables S2 and S3. Results (k–o) Quote- level aggregation by party performs the analysis of (a–e), but separately for quotes from Democrats vs. Republicans (coefficients omitted for clarity; cf. Supplementary Tables S8 and S9). Significance of regression coefficients: p < 0.001 , p < 0.01 , * p < 0.05 . We observe drastic shifts toward a more negative tone at the modeled June 2015 discontinuity (Trump’s campaign start). Figure 2. Temporal evolution of negative language. Columns correspond to negative-language word categories from LIWC; rows correspond to aggregation methods for computing monthly averages. Points show monthly averages, expressed as pre-campaign z-scores (i.e., subtracting pre-campaign mean from raw frequency values, and dividing by pre-campaign standard deviation). Lines (with 95% confidence intervals) were obtained via ordinary least squares regression, with coefficients shown in legends (cf. Eq. (1) and Fig. 1b for interpretation of coefficients; tabular summary in Supplementary Tables S4, S6, S8, S9). (a–e) Quote-level aggregation micro- averages over all quotes per month, i.e., speakers have weight proportional to their number of quotes in the respective month. Panel (a) shows the same data as Fig. 1. (f–j) Speaker-level aggregation macro-averages by speaker, i.e., all speakers with at least one quote in a given month have equal weight in that month. (k–o) Quote- level aggregation by party performs the analysis of (a–e), but separately for quotes from Democrats vs. Republicans (coefficients omitted for clarity; cf. Supplementary Tables S8 and S9). Significance of regression coefficients: *p < 0.001 , p < 0.01 , * p < 0.05 . We observe drastic shifts toward a more negative tone at the modeled June 2015 discontinuity (Trump’s campaign start). We observe that one in 54 words expresses a negative emotion; one in 155, anger; one in 339, anxiety; and one in 285, sadness. Swear words, at a rate of one in 2329, are least common. Given this wide range of frequencies, and in order to make effect sizes comparable across word categories, we standardize all monthly scores category-wise by subtracting the respective pre-campaign mean and dividing by the respective pre-campaign standard devia- tion. To facilitate the comparability and interpretability of results, standardization always uses the means and standard deviations computed on quote-level aggregates involving all speakers, even in analyses of speaker-level aggregates or of quote-level aggregates when omitting individual speakers. The resulting effect sizes, in units of pre-campaign standard deviations, become more palpable when expressed as multiples of the corresponding pre-campaign means. Results The coefficients of variation, σ/µ , shown in the fifth column, allow to easily translate pre-campaign standard deviations (as shown on the y-axes of time series plots) into fractions of pre- campaign means. The most frequent words per category are listed in Supplementary Tables S2 and S3. https://doi.org/10.1038/s41598-023-36839-1 Scientific Reports \| (2023) 13:10495 \| www.nature.com/scientificreports/ Figure 2. Temporal evolution of negative language. Columns correspond to negative-language word categories from LIWC; rows correspond to aggregation methods for computing monthly averages. Points show monthly averages, expressed as pre-campaign z-scores (i.e., subtracting pre-campaign mean from raw frequency values, and dividing by pre-campaign standard deviation). Lines (with 95% confidence intervals) were obtained via ordinary least squares regression, with coefficients shown in legends (cf. Eq. (1) and Fig. 1b for interpretation of coefficients; tabular summary in Supplementary Tables S4, S6, S8, S9). (a–e) Quote-level aggregation micro- averages over all quotes per month, i.e., speakers have weight proportional to their number of quotes in the respective month. Panel (a) shows the same data as Fig. 1. (f–j) Speaker-level aggregation macro-averages by speaker, i.e., all speakers with at least one quote in a given month have equal weight in that month. (k–o) Quote- level aggregation by party performs the analysis of (a–e), but separately for quotes from Democrats vs. Republicans (coefficients omitted for clarity; cf. Supplementary Tables S8 and S9). Significance of regression coefficients: *p < 0.001 , p < 0.01 , * p < 0.05 . We observe drastic shifts toward a more negative tone at the modeled June 2015 discontinuity (Trump’s campaign start). Figure 2. Temporal evolution of negative language. Columns correspond to negative-language word categories from LIWC; rows correspond to aggregation methods for computing monthly averages. Points show monthly averages, expressed as pre-campaign z-scores (i.e., subtracting pre-campaign mean from raw frequency values, and dividing by pre-campaign standard deviation). Lines (with 95% confidence intervals) were obtained via ordinary least squares regression, with coefficients shown in legends (cf. Eq. (1) and Fig. 1b for interpretation of coefficients; tabular summary in Supplementary Tables S4, S6, S8, S9). (a–e) Quote-level aggregation micro- averages over all quotes per month, i.e., speakers have weight proportional to their number of quotes in the respective month. Panel (a) shows the same data as Fig. 1. (f–j) Speaker-level aggregation macro-averages by speaker, i.e., all speakers with at least one quote in a given month have equal weight in that month. Results A data-driven analysis, conducted ex post, revealed that June 2015 is in fact the optimal discontinuity for modeling the data: out of 140 regression models analogous to Eq. (1), but each using another one of the 140 months of our analysis period as the discontinuity, the model with the June 2015 discontinuity yielded the best fit for negative emotion words and anger (Supplementary Fig. S5). (For anxiety and sadness, slightly better fits were obtained when using different discontinuities; for swear words, a 2009 discontinuity led to a better fit due to outliers around that time; see Supplementary Fig. S5). Role of speaker prominence. When repeating the analysis using speaker-level, rather than quote-level, aggregates, i.e., weighing all speakers equally when averaging, all of the above effects persisted qualitatively, but were reduced quantitatively (Fig. 2f–j): in each of the five word categories, the immediate increase α at the June 2015 discontinuity dropped by between one-third and one-half, remaining significant for negative emotions, anger, and swear words ( p = 9.1 × 10−5, 0.0025, and 0.030, respectively), but becoming non-significant for anxiety and sadness ( p = 0.11 and 0.43, respectively). The change of slope observed in the quote-level analysis also persisted in the speaker-level analysis.hff The fact that speaker-level effects are weaker than quote-level effects indicates that prominent, highly quoted speakers contribute disproportionally to the increase in negative language. To confirm this conclusion more directly, we divided the speakers into four quartiles with respect to the number of quotes attributed to them, and repeated the speaker-level analysis for each quartile individually. Figure 3 shows (1) that the abrupt increase in negative emotion words emerges in all strata of speaker prominence except the least prominent stratum, and Figure 3. Role of speaker prominence. The set of 18,627 US politicians was split into four evenly-sized quartiles with respect to their total number of quotes (i.e., prominence); each panel shows the time series for negative emotion words obtained by performing monthly speaker-level aggregation on the respective quartile separately. That is, the figure shows the data of Fig. 2f after stratifying speakers by prominence. Lines (with 95% confidence intervals) were obtained via ordinary least squares regression, with coefficients in legends (cf. Eq. (1) and Fig. 1b for interpretation of coefficients) (tabular summary of regression coefficients in Supplementary Tables S11, S12, S13, and S14). Results (For anxiety and sadness, slightly better fits were obtained when using different discontinuities; for swear words, a 2009 discontinuity led to a better fit due to outliers around that time; see Supplementary Fig. S5). g pi g gi We first focus on the time series of quote-level aggregates (Fig. 2a–e). The regression coefficients indicate a significant, sudden increase in the relative frequency of negative emotion words (Fig. 2a) in June 2015, by α = 1.6 ( p = 4.1 × 10−6 ) pre-campaign standard deviations (SD), translating to a relative increase of 8% over the pre- campaign mean (cf. Table 1). All three subcategories of negative emotion words, as well as swear words, also saw significant jumps in June 2015: anger, by 1.3 SD ( +11% of the pre-campaign mean, p = 0.0015 , Fig. 2b); anxiety, by 1.5 SD ( +11% , p = 5.6 × 10−4 , Fig. 2c); sadness, by 0.86 SD ( +4% , p = 0.021 , Fig. 2d); and swear words, by 0.92 SD ( +31% , p = 2.9 × 10−6 , Fig. 2e). Note that the pre-campaign regression line for swear words (Fig. 2e) underestimates the values just before the discontinuity, mostly due to outliers at the left boundary (2008/09). For swear words, the actual June 2015 jump should thus be considered to be smaller than the estimate α . The June 2015 discontinuity was not only associated with a sudden increase in negative language, but also with a change in slope: whereas the frequency of negative emotion words (Fig. 2a) had steadily and significantly decreased over the first 6.5 years of Obama’s tenure by β0 = −0.023 SD per month ( p = 9.5 × 10−7 ), i.e., by about a quarter SD per year, this trend came to a halt in June 2015, with a (non-significantly) positive slope of β0 + β = 0.0076 (compound p = 0.21 ) from June 2015 onward. These results hold when removing outlier months whose quote- level aggregate score lies more than three standard deviations from the mean (Supplementary Fig. S1, S5 and S7). gg g pp y g We emphasize that the June 2015 discontinuity was chosen ex ante based on incoming hypotheses grounded in the general public’s subjective impression that Donald Trump’s entering the political scene had changed the tone of US politics1. Results For this purpose, the rightmost column of Table 1 lists coefficients of variation, i.e., ratios of standard deviations and means. Temporal evolution of negative language. Figure 2 visualizes the evolution of negative language between September 2008 and April 2020, with one row per aggregation method, one column per word category, and one point per monthly aggregate score. In order to quantify the shape of the curves, we fitted ordinary least squares linear regression models with a discontinuity in June 2015, the starting month of Donald Trump’s pri- mary campaign. For a given word category, we model the aggregate score yt for month t as https://doi.org/10.1038/s41598-023-36839-1 Scientific Reports \| (2023) 13:10495 \| www.nature.com/scientificreports/ yt = α0 + β0 t + α it + β it t + εt, (1) where t ∈{−81, . . . , 58} , with t = 0 corresponding to June 2015; it indicates whether t is located before vs. after the campaign start (i.e., it = 0 for t < 0 , and it = 1 for t ≥0 ); and εt is the residual error. As illustrated in Fig. 1b, the coefficient α captures the immediate jump coinciding with the campaign start, and β , the change in slope, such that β0 and β0 + β describe the slopes of the regression lines before and after the campaign start, respectively. Figure 2 plots the fitted regression lines, alongside 95% confidence intervals.ihfi where t ∈{−81, . . . , 58} , with t = 0 corresponding to June 2015; it indicates whether t is located before vs. after the campaign start (i.e., it = 0 for t < 0 , and it = 1 for t ≥0 ); and εt is the residual error. As illustrated in Fig. 1b, the coefficient α captures the immediate jump coinciding with the campaign start, and β , the change in slope, such that β0 and β0 + β describe the slopes of the regression lines before and after the campaign start, respectively. Figure 2 plots the fitted regression lines, alongside 95% confidence intervals.ihfi g pi g , gi We first focus on the time series of quote-level aggregates (Fig. 2a–e). The regression coefficients indicate a significant, sudden increase in the relative frequency of negative emotion words (Fig. Results 2a) in June 2015, by α = 1.6 ( p = 4.1 × 10−6 ) pre-campaign standard deviations (SD), translating to a relative increase of 8% over the pre- campaign mean (cf. Table 1). All three subcategories of negative emotion words, as well as swear words, also saw significant jumps in June 2015: anger, by 1.3 SD ( +11% of the pre-campaign mean, p = 0.0015 , Fig. 2b); anxiety, by 1.5 SD ( +11% , p = 5.6 × 10−4 , Fig. 2c); sadness, by 0.86 SD ( +4% , p = 0.021 , Fig. 2d); and swear words, by 0.92 SD ( +31% , p = 2.9 × 10−6 , Fig. 2e). Note that the pre-campaign regression line for swear words (Fig. 2e) underestimates the values just before the discontinuity, mostly due to outliers at the left boundary (2008/09). For swear words, the actual June 2015 jump should thus be considered to be smaller than the estimate α . The June 2015 discontinuity was not only associated with a sudden increase in negative language, but also with a change in slope: whereas the frequency of negative emotion words (Fig. 2a) had steadily and significantly decreased over the first 6.5 years of Obama’s tenure by β0 = −0.023 SD per month ( p = 9.5 × 10−7 ), i.e., by about a quarter SD per year, this trend came to a halt in June 2015, with a (non-significantly) positive slope of β0 + β = 0.0076 (compound p = 0.21 ) from June 2015 onward. These results hold when removing outlier months whose quote- level aggregate score lies more than three standard deviations from the mean (Supplementary Fig. S1, S5 and S7). We emphasize that the June 2015 discontinuity was chosen ex ante based on incoming hypotheses grounded in the general public’s subjective impression that Donald Trump’s entering the political scene had changed the tone of US politics1. A data-driven analysis, conducted ex post, revealed that June 2015 is in fact the optimal discontinuity for modeling the data: out of 140 regression models analogous to Eq. (1), but each using another one of the 140 months of our analysis period as the discontinuity, the model with the June 2015 discontinuity yielded the best fit for negative emotion words and anger (Supplementary Fig. S5). www.nature.com/scientificreports/ (1): (2) ygt = α0 + β0 t + α it + β it t + γ Democratg + δ Governmentg + ζ Congressg + η Femaleg + εgt, (2) where Democratg = 1 (or 0) if group g contains Democrat (or Republican) speakers, and analogously for Governmentg , Congressg , and Femaleg.ifi where Democratg = 1 (or 0) if group g contains Democrat (or Republican) speakers, and analogously for Governmentg , Congressg , and Femaleg.ifi g Inspecting the fitted coefficients (shown in Fig. 4 for quote-level aggregation; for speaker-level aggregation, see Supplementary Fig. S6), we make two observations: First, the sudden June 2015 jump α in negative language (Fig. 4b) appears even after adjusting for the four biographic attributes. Second, we observe systematic cor- relations of negative language with biographic attributes, whereby quotes by members of the opposition party, Congress members, and Democrats contain, ceteris paribus, significantly more negative language. Moreover, quotes by females contain more anxiety and sadness, and quotes by males, more negative emotion, anger, and swear words. The fact that quotes by members of the opposition party contain more negative language may reflect their role as corrective agents in the democratic process. The fact that quotes by Congress members contain more negative language echoes previous work that has highlighted high-ranked politicians’ role not only as deputies Figure 4. Biographic correlates of negative language. Coefficients (with 95% confidence intervals) of ordinary least squares regression (Eq. (2)) for modeling time series of word categories (quote-level aggregation; speaker- level aggregation in Supplementary Fig. S6) while adjusting for party affiliation ( γ ), the party’s federal role ( δ ), Congress membership ( ζ ), and gender ( η ) (tabular summary in Supplementary Tables S15 and S16). Positive coefficients mark word categories that are, ceteris paribus, used more frequently by Democrats than by Republicans, by members of the governing than by members of the opposition party, by Congress members than by others, or by females than by males (and vice versa for negative coefficients). We observe that quotes by members of the opposition party, Congress members, and Democrats contain significantly more negative language. Importantly, the sudden June 2015 jump in negative language ( α ) remains significant after adjusting for biographic attributes. Figure 4. Biographic correlates of negative language. Coefficients (with 95% confidence intervals) of ordinary least squares regression (Eq. www.nature.com/scientificreports/ www.nature.com/scientificreports/ (2) that quotes by more prominent speakers generally contain more negative emotion words. (Results for other word categories in Supplementary Fig. S3.) (2) that quotes by more prominent speakers generally contain more negative emotion words. (Results for other word categories in Supplementary Fig. S3.) Biographic correlates of negative language. The patterns identified above—a sudden increase in neg- ative language in June 2015 followed by a change in slope—hold across party lines, but are more pronounced for Republicans, as seen in Fig. 2k–o, which tracks the (quote-level) evolution of negative language over time, analogously to Fig. 2a–e, but separately for quotes by Democrats vs. Republicans (all α coefficients of Fig. 2k–o are positive; for Republicans, all are significant ( p < 0.05 ); for Democrats, the coefficients for anger ( α = 0.57 ) and sadness ( α = 0.43 ) are non-significant ( p > 0.05)). For instance, the party-wise estimates of the June 2015 increase in negative emotion words is α = 0.89 pre-campaign SD for Democrats ( p = 0.014 ) and α = 2.3 SD for Republicans ( p = 5.6 × 10−9). p (p ) We further considered the possibility that the distribution of speaker characteristics may have changed over time; e.g., members of one party or gender may have become more frequently quoted over time. To account for potential confounding due to such factors, we repeated the regression analysis with added control terms for four biographic attributes: party affiliation (Republican, Democrat), the party’s federal role (Opposition, Gov- ernment), Congress membership (Non-Congress, Congress), and gender (Male, Female; due to small sample size, we discarded speakers of a non-binary gender according to Wikidata). For a given month, party affiliation fully determines the party’s federal role, so for each month, the set of speakers can be partitioned into 23 = 8 speaker groups, one per valid attribute combination. We computed monthly aggregate scores ygt separately for each speaker group g, obtaining eight aggregate data points per month, and modeled them jointly using the following extended version of Eq. Results Significance of regression coefficients: *p < 0.001 , p < 0.01 , * p < 0.05 . We observe that the abrupt increase in negative emotion words emerges in all strata of speaker prominence except the least prominent stratum, and that quotes by more prominent speakers overall contain more negative emotion words. The figure focuses on one category of negative-language words (negative emotion); for the other four categories, see Supplementary Fig. S3. Figure 3. Role of speaker prominence. The set of 18,627 US politicians was split into four evenly-sized quartiles with respect to their total number of quotes (i.e., prominence); each panel shows the time series for negative emotion words obtained by performing monthly speaker-level aggregation on the respective quartile separately. That is, the figure shows the data of Fig. 2f after stratifying speakers by prominence. Lines (with 95% confidence intervals) were obtained via ordinary least squares regression, with coefficients in legends (cf. Eq. (1) and Fig. 1b for interpretation of coefficients) (tabular summary of regression coefficients in Supplementary Tables S11, S12, S13, and S14). Significance of regression coefficients: *p < 0.001 , p < 0.01 , * p < 0.05 . We observe that the abrupt increase in negative emotion words emerges in all strata of speaker prominence except the least prominent stratum, and that quotes by more prominent speakers overall contain more negative emotion words. The figure focuses on one category of negative-language words (negative emotion); for the other four categories, see Supplementary Fig. S3. https://doi.org/10.1038/s41598-023-36839-1 Scientific Reports \| (2023) 13:10495 \| www.nature.com/scientificreports/ We further illustrate (Supplementary Fig. S10) using as examples the two presidents (Barack Obama, Donald Trump), vice presidents (Joe Biden, Mike Pence), and runners-up (Mitt Romney, Hillary Clinton) from the study period. Four of these six speakers were associ- ated with a significant ( p < 0.05 ) positive α for negative emotion words, and none with a significant negative α . Interestingly, although both Donald Trump ( α = 3.6 , p = 0.026 ) and Barack Obama ( α = 3.7 , p = 0.0094 ) fol- lowed the population-wide pattern by increasing their frequency of negative emotion words in June 2015, their pre- and post-discontinuity slopes were opposite to the population-wide pattern: their language first became more negative, then less negative. g g Next, we sought evidence whether individual politicians contributed particularly strongly to the overall increase in negative language. We proceeded in an ablation study of the 50 most quoted speakers and all runners- up for the 2016 Democratic and Republican primaries, totaling 61 speakers. For each of these speakers, we repeated the quote-level regression (Fig. 2a–e) on a dataset consisting of all quotes except those from the respec- tive speaker. If α is particularly low when removing a given speaker, that speaker contributed particularly strongly to the overall June 2015 increase in negative language. Figure 6a shows that no single speaker’s removal leads to an important change in α for negative emotion words, with one exception: Donald Trump. By removing Donald Trump’s quotes, the June 2015 increase in negative emotion words drops by 40%, from α = 1.6 to α = 0.98 pre- campaign SD (more precisely, (1.622 −0.974)/1.622 = 0.40 ). When considering quotes by Republicans only (cf. Figure 5. Role of individual politicians: single-speaker study. Results of ordinary least squares regressions (Eq. (1)) fitted separately to the time series of each of the 200 most quoted speakers. (a–e) Each speaker’s α coefficient (capturing the size of the June 2015 jump, with 95% confidence intervals) as a function of the speaker’s number of quotes. Significant coefficients ( p < 0.05 ) in color, others in gray. (f–j) Fraction of speakers with positive α among the speakers with at least n quotes, as a function of n. www.nature.com/scientificreports/ (2)) for modeling time series of word categories (quote-level aggregation; speaker- level aggregation in Supplementary Fig. S6) while adjusting for party affiliation ( γ ), the party’s federal role ( δ ), Congress membership ( ζ ), and gender ( η ) (tabular summary in Supplementary Tables S15 and S16). Positive coefficients mark word categories that are, ceteris paribus, used more frequently by Democrats than by Republicans, by members of the governing than by members of the opposition party, by Congress members than by others, or by females than by males (and vice versa for negative coefficients). We observe that quotes by members of the opposition party, Congress members, and Democrats contain significantly more negative language. Importantly, the sudden June 2015 jump in negative language ( α ) remains significant after adjusting for biographic attributes. https://doi.org/10.1038/s41598-023-36839-1 Scientific Reports \| (2023) 13:10495 \| www.nature.com/scientificreports/ of their own party, but also as antagonists of the opposing party, epitomized in the finding that “ideological moderates won’t run”42 for Congress. of their own party, but also as antagonists of the opposing party, epitomized in the finding that “ideological moderates won’t run”42 for Congress. Role of individual politicians. In order to determine to what extent the above population-level effects mirror individual-level effects, we fitted regression models (cf. Eq. 1) to individual speakers’ time series and analyzed the corresponding α coefficients (i.e., the size of the June 2015 jump; for completeness, β in Supple- mentary Fig. S9). In order to avoid data sparsity issues, this analysis focuses on the 200 most-quoted speakers, with α plotted in Fig. 5a–e. Additionally, Fig. 5f–j plots the fraction of speakers with positive α among the speakers with at least n quotes, as a function of n. We observe that, although many individual α coefficients are non-significant ( p > 0.05 , gray confidence intervals in Fig. 5a–e), the majority of coefficients are positive, par- ticularly among the top most-quoted speakers (as manifested in the increasing curves of Fig. 5f–j). For instance, for negative emotion words (Fig. 5f), all of the top four most-quoted politicians have positive α . Among the top 50, 74% have positive α ; among the top 100, 63%; and among the top 200, 59%. In other words, the June 2015 discontinuity emerges not only by aggregating at the population level, but mirrors a disruption that can also be perceived in the most-quoted politicians’ individual language. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 6. Role of individual politicians: ablation study. Results from ordinary least squares regression (Eq. (1); quote-level aggregation) on data sets obtained by removing all quotes by one target speaker and retaining all quotes from all 18,626 other speakers. The 50 most quoted speakers and 11 other runner-up candidates for both party primaries were used as target speakers. Each point shows the α coefficient (capturing the size of the June 2015 jump, with 95% confidence intervals) obtained after removing the target speaker’s quotes from the analysis, as a function of the target speaker’s number of quotes. Dashed horizontal lines mark the coefficients obtained on the full data set without removing any speaker (cf. Fig. 2a–e). We observe that no single speaker’s removal leads to a notable change in α , except Donald Trump: e.g., by removing Trump’s quotes, α drops by 40% for negative emotion words (a). Figure 6. Role of individual politicians: ablation study. Results from ordinary least squares regression (Eq. (1); quote-level aggregation) on data sets obtained by removing all quotes by one target speaker and retaining all quotes from all 18,626 other speakers. The 50 most quoted speakers and 11 other runner-up candidates for both party primaries were used as target speakers. Each point shows the α coefficient (capturing the size of the June 2015 jump, with 95% confidence intervals) obtained after removing the target speaker’s quotes from the analysis, as a function of the target speaker’s number of quotes. Dashed horizontal lines mark the coefficients obtained on the full data set without removing any speaker (cf. Fig. 2a–e). We observe that no single speaker’s removal leads to a notable change in α , except Donald Trump: e.g., by removing Trump’s quotes, α drops by 40% for negative emotion words (a). Fig. 2k–o), α drops by 43% when removing Trump’s quotes, from 2.3 to 1.3 pre-campaign SD (Supplementary Tables S9 and S10). Put differently, by adding Donald Trump’s quotes, the June 2015 increase in negative emotion words is boosted by 63%. Note that this is not merely an artifact of Trump’s being quoted especially frequently: Obama was quoted about twice as frequently as Trump over the course of the 12-year period, yet removing his quotes does not notably affect the June 2015 increase in negative emotion words. Qualitatively similar results hold for the other word categories (cf. Fig. 6b–e), in particular for swear words (Fig. www.nature.com/scientificreports/ 6e; β in Supplementary Fig. S11 for completeness). Although the size of the June 2015 jump in negative emotion words decreased drastically when removing Trump’s quotes, note that it remained highly significant ( p = 0.0032 ). That is, Trump was the main, but not the sole, driver of the effect. Positive emotion words. Complementary to the five word categories related to negative language ana- lyzed above, we also conducted an exploratory analysis of positive language, as captured by LIWC’s positive emotion category. As seen in Fig. 7a–b, the time series of positive emotion words does not simply mirror that of negative emotion words. In particular, no particular changes are observed in June 2015. Rather, positive emotion words appear stable well into Trump’s term, and then decline. (The best regression fit is achieved when using July 2018 as the discontinuity, with a sharp drop in quality of fit after February 2019; Supplementary Fig. S12). As in the case of negative emotion words, the same pattern emerges within each party (Fig. 7c), with a more pronounced drop in positive emotion words for Democrats than for Republicans. www.nature.com/scientificreports/ We observe that, although many individual α coefficients are non-significant (a–e), the majority of coefficients are positive, particularly among the most-quoted speakers (as manifested in the increasing curves of (f–j)). That is, the June 2015 jump in negative language emerges even at the individual level for a majority of the most-quoted politicians. Figure 5. Role of individual politicians: single-speaker study. Results of ordinary least squares regressions (Eq. (1)) fitted separately to the time series of each of the 200 most quoted speakers. (a–e) Each speaker’s α coefficient (capturing the size of the June 2015 jump, with 95% confidence intervals) as a function of the speaker’s number of quotes. Significant coefficients ( p < 0.05 ) in color, others in gray. (f–j) Fraction of speakers with positive α among the speakers with at least n quotes, as a function of n. We observe that, although many individual α coefficients are non-significant (a–e), the majority of coefficients are positive, particularly among the most-quoted speakers (as manifested in the increasing curves of (f–j)). That is, the June 2015 jump in negative language emerges even at the individual level for a majority of the most-quoted politicians. https://doi.org/10.1038/s41598-023-36839-1 Scientific Reports \| (2023) 13:10495 \| Discussionh t Rather, our results show that political debate during Donald Trump’s entire term was characterized by a nega- tive tone, and they specifically point to Trump as a key driver of this development: when removing Trump’s quotes from the corpus, the magnitude of the June 2015 jump in the frequency of negative emotion words drops by 40%. Interestingly, Trump’s own negative tone (Supplementary Fig. S10(z)) followed long-term trends opposite to the population-wide trends, with an initial increase and a subsequent decrease (and with a sudden June 2015 increase akin to that of the population). But as his language was overall far more negative than the average (Trump’s mean monthly average was 8.0 pre-campaign standard deviations larger than the overall mean monthly average), he strongly skewed the overall tone toward the negative end when he moved to the center of the media’s attention47. Despite Trump’s disproportionate impact, the increase in negative language was, however, not due to Trump alone. It remained significant in various complementary analyses: when removing Trump’s quotes from the analysis, when giving equal weight to all speakers, when analyzing each party separately, and when analyzing the most-quoted speakers individually. The negative tone of others might be partly provoked by Trump’s state- ments and actions, but as we do not have access to a counterfactual world without Trump, our analysis cannot speak to this possibility.t p p y Our analysis also cannot disambiguate to what extent the observed shift in negative language is caused by a real shift in what politicians say vs. a shift in what online media choose to report. It is well known that media outlets are biased in what they report48–50, typically towards negative news that tend to increase engagement on readers’ behalf51, and this bias may have drifted during the 12 years analyzed here. Future work may investigate this possibility by comparing quotes reported in the media to complete records of certain politicians’ utterances in certain contexts, e.g., via Congressional records or public speeches52. (But whether the shift was caused by politicians or by the media, the effect would be identical: a more negative tone as perceived by news-reading citizens.) Individuals also have access to news from a plethora of media types, of which only a subset is covered by this study. Discussionh Nevertheless, despite the continued influence of traditional outlets and TV stations53, digital sources have become the primary means by which most individuals consume news, and this trend has been consistently on the rise in recent years54. y Moreover, although word-counting is a powerful tool for detecting broad trends in large textual data39,55, and although our results are robust to the specific choice of dictionary (Empath41 yields qualitatively identical results to LIWC36; Supplementary Figs. S1, S2, S3, S4, and S11, and Supplementary Table S1), word-counting is crude and insensitive to nuances in context56. Our findings should thus be considered a starting point and hypothesis generator for more detailed analyses based on a closer inspection of the text and context of quotes, e.g., using a combination of advanced natural language processing tools and human annotation in order to shed light on the relation between negative language and polarization and partisanship: e.g., has politicians’ tone become more negative specifically when they talk about opponents?h g pi y y pp The present study is also limited with respect to its time frame. Although Quotebank34 is the largest existing corpus of speaker-attributed quotes in terms of size and temporal extent, the 12 years it spans are but a short sliver of the United States’ long political history. It is thus unclear when the decrease in negative language at the begin- ning of the corpus (during the initial 6.5 years of Obama’s tenure) started. Gentzkow et al.20 observed a concurrent decrease in partisan language in Congressional speeches during Obama’s tenure (2009–2016), which might be yet another manifestation of the processes that underlie the initial decrease in negative language observed here. Gentzkow et al.’s corpus of Congressional speeches, however, spans a much longer time (144 years, 1873–2016), during which overall partisanship increased much more—especially from the 1980s onward—than it eventually decreased during Obama’s tenure. We must therefore consider the possibility that, analogously, the 2009–2015 decrease in negative language may have been merely a short anomaly in a longer increasing trend in negative language. (Unfortunately, Gentzkow et al.’s corpus ends with Obama’s tenure, so we cannot compare trends in negative quote language to trends in partisan Congress language during Trump’s tenure.) g q g g p g g g g p We saw that the June 2015 rise in negative language was not accompanied by a simultaneous drop in positive language. Discussionh The goal of this work has been to determine the accuracy of the average American’s subjective impression (as of mid 2019) that “the tone and nature of political debate in the United States has become more negative in recent years”, and that Donald Trump “has changed the tone and nature of political debate [...] for the worse”1. Based on an analysis of 24 million quotes uttered by 18,627 US politicians between 2008 and 2020, we conclude that both of the above impressions are largely correct. Figure 7. Temporal evolution of positive emotion words. Points show monthly averages, expressed as pre- campaign z-scores. One panel per aggregation method for computing monthly averages. We observe that positive emotion words appear stable well into Trump’s term, and then decline. Figure 7. Temporal evolution of positive emotion words. Points show monthly averages, expressed as pre- campaign z-scores. One panel per aggregation method for computing monthly averages. We observe that positive emotion words appear stable well into Trump’s term, and then decline. Scientific Reports \| (2023) 13:10495 \| https://doi.org/10.1038/s41598-023-36839-1 www.nature.com/scientificreports/ The tone of US politicians (as seen through the lens of online media) indeed became suddenly and signifi- cantly more negative with the start of the 2016 primary campaigns in June 2015, and the frequency of negative language remained elevated between then and the end of our study period (April 2020). Intriguingly, the shift at this incision point coincides with a similar abrupt shift in political polarization on online platforms43,44. The sudden increase in negative language reported here was not only significant, but also strong; e.g., the frequency of negative emotion words jumped up by 1.6 pre-campaign standard deviations, or by 8% of the pre-campaign mean. The disruption becomes particularly stark when contrasted with the first 6.5 years of Obama’s tenure, during which negative language had decreased steadily—at odds with a commonly held belief that Trump merely continued an older trend13,14. The potential of negativity, incivility, and fear as tools to support political campaigns has been long known45,46 and might explain the increase of negative language during the campaigns. It cannot, however, explain the fact that the boost in negative language continued for years after the campaigns had ended. Materials and methods For a given quote, we maintained only the name with the highest probability and consider it to indicate the speaker of the quote (a heuristic that was shown to have an accuracy of around 87%34). A speaker name may be ambiguous. In such cases, Quotebank does not attempt to disambiguate the name, but rather provides a list of all speakers to whom the name may refer, where speakers are identified by their unique ID from the Wikidata knowledge base35. In our analysis, we attributed quotes that were linked to ambiguous speaker names (less than 6% of all quotes, see below) to each speaker to whom the respective name may refer. q p p y To further clean the data set, we discarded quotes that were clearly non-verbal (e.g., consisting of URLs, HTML tags, or dates only). Moreover, on some days, Spinn3r.com, which provided the news articles for Quote- bank, failed to deliver content due to technical problems34. We therefore identified missing days as those having less than 10% of the median number of unique quotes and dropped eight (out of 140) months with 20 or more missing days: May 2010, June 2010, January 2016, March 2016, June 2016, October 2016, November 2016, Janu- ary 2017. Quotes by US politicians. Keeping only quotes attributed to US politicians (see above), we obtained 24 million quotes attributed to 18,627 unique US politicians. Out of these, 4487 were female, 14,140 male; 9390 were Democrats, 9237 Republicans; and 1790 were labeled as members of Congress. Out of an original set of 18,954 US politicians appearing in Quotebank, Wikidata listed 327 as (former) members of both parties, usually because they switched membership during their careers. Out of these 327, we manually checked the 21 politi- cians with over 10,000 quotes, of whom 16 could be unambiguously assigned to one party for the study period. The remaining 311 politicians were dropped from the analysis. The most prolific speakers were Barack Obama (1.5m quotes), Donald Trump (763k quotes), Mitt Romney (281k quotes), Hillary Clinton (230k quotes), George W. Bush (200k quotes), John McCain (161k quotes), and Joe Biden (127k quotes). For a list of the 30 most fre- quently quoted politicians, see Supplementary Table S17. Materials and methods US politicians. We considered as politicians all people for whom the Wikidata knowledge base35 (version of 27 October 2021) lists “politician” (Wikidata item Q82955) or a subclass thereof as an occupation (P106). Given our focus on the United States, we included only those politicians whose party affiliation (P102) was listed as Democrat (Q29552) or Republican (Q29468). We considered as members of Congress those for whom Wikidata listed a US Congress Bio ID (P1157), making no distinction between former and active members of Congress. Due to the small number of speakers of a non-binary gender, we included only speakers whose gender (P21) was listed in Wikidata as male (Q6581097) or female (Q6581072). Quotebank. The analyzed quotes were obtained from Quotebank34, a publicly available65 corpus of 235 mil- lion unique speaker-attributed quotes extracted from 127 million English news articles published between Sep- tember 2008 and April 2020, provided by the large-scale online media aggregation service Spinn3r.com. While Spinn3r.com collects and supplies content from a comprehensive set of news domains66, it also includes much content beyond news alone, including “social media, weblogs, news, video, and live web content”67. Therefore, Quotebank was extracted from a filtered data set consisting only of content from a set of about 17,000 online news domains, defined as the set of domains appearing at least once in the large News on the Web corpus68, which has been collecting large numbers of news articles from Google News and Bing News since 2010 and may thus be considered to provide a comprehensive list of English-language media outlets. We emphasize that the News on the Web corpus was only used for defining the set of news domains. It was not used for obtaining the news articles themselves, which originated from Spinn3r.com only. g p y We use the quote-centric (as opposed to the article-centric) version of Quotebank, which contains one entry per unique quote and aggregates information from all news articles in which the quote occurs. In constructing Quotebank, a machine learning algorithm (based on the large pre-trained BERT language model69) was used to infer, for each quote, a probability distribution over all speaker names mentioned in the text surrounding the quote (and an additional “no speaker” option), specifying each speaker’s estimated probability of having uttered the quote. Discussionh Rather, maybe in line with a general positivity bias in human language57, positive language remained stable until it eventually dropped during Trump’s term. What happened at this point is an open question that lies beyond the scope of this work. The phenomenon highlights, however, that positive and negative emotion words are not necessarily complementary. From the start of the 2016 primary campaigns through the first half of Donald Trump’s term, political tone was both highly positive and highly negative—akin to Trump’s own style, characterized by typical features of affective polarization such as positive self-representation and negative other- presentation28,29,31: “Sorry losers and haters, but my I.Q. is one of the highest -and you all know it! Please don’t feel so stupid or insecure,it’s not your fault”58.h y This said, a key contribution of this work is the conclusion that, despite Donald Trump’s key role in setting the tone of political debate, the shift towards a more negative tone permeates all of US politics. The consequences are tangible, as shown by research that highlights the detrimental effects of affective polarization on altruism59, trust60, and opinion formation61,62, and by polls showing that politics has become a stressful experience for Scientific Reports \| (2023) 13:10495 \| https://doi.org/10.1038/s41598-023-36839-1 www.nature.com/scientificreports/ Americans1,63, exacting an ever increasing toll on their physical, emotional, and social well-being5,64. Exposure to negative political language can also exert a notable impact on an individual’s affective state, leading to nega- tive emotion biases and influencing reasoning and decision-making2. Finding ways to break out of this cycle of negativity is one of the big challenges faced by the United States today. Materials and methods While it seems unreasonable that any single person could utter over 300 different quote-worthy statements a day, the large numbers can be explained by news outlets attending to different parts of a politician’s spoken output (for instance, a long sentence can be quoted in various ways). Although, as mentioned, ambiguous names led to some quotes being attributed to multiple speakers, this happened rarely: the vast majority (94.3%) of quotes were attributed to a single politician, 4.3% to two politi- cians, 1.2% to three politicians, and 0.13% to four politicians. Supplementary Fig. S14 shows the number of quotes and the number of unique speakers per month. Aggregation methods. Consider a fixed LIWC word category c and a fixed month t. Let S be the set of speakers with at least one quote during month t. Let Qs be the set of quotes attributed to speaker s ∈S during month t, and let Q = s∈S Qs be the set of all quotes from month t (the few quotes attributed to multiple speak- ers are included in Q once per speaker). Let ψ(q) be quote q’s score for word category c.hi p p ) ψ(q) q q g y Then, the quote-level aggregate score for word category c in month t is defined as Scientific Reports \| (2023) 13:10495 \| https://doi.org/10.1038/s41598-023-36839-1 www.nature.com/scientificreports/ and the speaker-level aggregate score, as That is, in quote-level aggregation, every speaker contributes with weight proportional to their number of yquote t = 1 \|Q\| q∈Q ψ(q) = s∈S \|Qs\| \|Q\| depends on s  1 \|Qs\| q∈Qs ψ(q)   average of speaker s , yspeaker t = 1 \|S\| s∈S  1 \|Qs\| q∈Qs ψ(q)  = s∈S 1 \|S\| constant  1 \|Qs\| q∈Qs ψ(q)   average of speaker s . yquote t = 1 \|Q\| q∈Q ψ(q) = s∈S \|Qs\| \|Q\| depends on s  1 \|Qs\| q∈Qs ψ(q)   average of speaker s , and the speaker-level aggregate score, as and the speaker-level aggregate score, as vel aggregate score, as yspeaker t = 1 \|S\| s∈S  1 \|Qs\| q∈Qs ψ(q)  = s∈S 1 \|S\| constant  1 \|Qs\| q∈Qs ψ(q)   average of speaker s . 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J.K. compiled the supplementary information with support from A.A., A.S., and R.W. R.W. provided compute resources. S.G. and R.W. were in charge of project administration. All authors reviewed the manuscript. All authors contributed to the revised version. Competing interests h p g The authors declare no competing interests. Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-023-36839-1. Correspondence and requests for materials should be addressed to R.W. Correspondence and requests for materials should be addressed to R.W. 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. 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https://openalex.org/W2559872258	https://biblio.ugent.be/publication/8507096/file/8507102.pdf	English	null	Atomic layer deposition of vanadium oxides for thin-film lithium-ion battery applications	RSC advances	2,016	cc-by	7,017	Department of Solid State Sciences, Ghent University, Krijgslaan 281 S1, 9000 Gent, Belgium. E-mail: felix.mattelaer@ugent.be † Electronic supplementary information (ESI) available: In situ XRD during the formation of crystalline lms for electrochemical testing, and cyclic voltammetry of the Pt substrate. See DOI: 10.1039/c6ra25742a Open Access Article. Published on 08 December 2016. Downloaded on 26/01/2017 16:10:59. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Felix Mattelaer,* Kobe Geryl, Geert Rampelberg, Thomas Dobbelaere, Jolien Dendooven and Christophe Detavernier Amorphous VO2 thin ﬁlms are deposited by atomic layer deposition (ALD) using tetrakis[ethylmethylamino] vanadium (TEMAV) as vanadium precursor and water or ozone as the oxygen source. The crystallisation and oxidation behaviour is investigated for diﬀerent oxygen partial pressures between ambient air and 3.7 Pa, resulting in phase formation diagrams on SiO2, TiN and Pt substrates, demonstrating a series of stable vanadium oxide phases in the VO2–V2O5 series. Most of the obtained phases exhibit lithium intercalation behaviour in the 1.5–4.5 V vs. Li+/Li potential range, and demonstrate high volumetric capacities in the order of V2O5 < VO2 (B) < V6O13 < V3O7 < V4O9, with the latter at more than twice the capacity of the best commercial cathode materials. Received 24th October 2016 Accepted 30th November 2016 DOI: 10.1039/c6ra25742a www.rsc.org/advances and interface intercalation dominates over the bulk diﬀusion.22 However, the thin-lm nature of these batteries inherently leads to a low energy density. This problem can be overcome by depositing these thin-lms on complex battery architectures leading to signicant enhancements in eﬀective surface area and thus in energy density per footprint area.23–27 Many depo- sition techniques have been used to deposit a variety of vana- dium oxides such as PVD,28,29 sol–gel29 and CVD,30 however, only few techniques have the high step-coverage of atomic layer deposition (ALD) required to coat the high-aspect-ratio struc- tures required in 3D thin-lm batteries.31 Several ALD processes exist for vanadium oxides.7,32–38 1 Introduction 1 The development of an ‘internet of things’ requires both wire- less sensor networks and autonomous microsystems.1,2 While the electronics, sensors and wireless communication systems abide Moore’s law, the energy storage devices lag behind.2,3 To advance from traditional secondary batteries to energy storage systems tuned for these micro-devices, all-solid state thin-lm batteries can provide the answer.4 While the removal of a liquid component in an all-solid state battery provides a huge advancement in battery safety and bio-compatibility,5 the thin- lm approach provides integrability to on-chip devices as well as improved charge–discharge kinetics. In this work, we will investigate the formation of vanadium oxide thin lms in the VO2–V2O5 series, based on the atomic layer deposition of amorphous VO2. We will show that we can obtain all vanadium oxide phases in the series by changing the post-deposition annealing conditions, and demonstrate elec- trochemical activity of diﬀerent phases. Due to the transition-metal nature of vanadium oxides and the high number of vanadium–oxygen ratio compositions, vanadium oxides are heavily investigated materials for appli- cations such as catalysis,6 optical switching7 and thin-lm batteries.8–17 Although V2O5 and VO2 (B) are the most investi- gated cathode materials in lithium ion battery research,8–17 all vanadium oxides in the Wadsley series (VnO2n+1) are related to one another18 and show promising electrochemical proper- ties.19,20 Even though vanadium oxides have a high theoretical energy density, they suﬀer from poor charge–discharge kinetics due to their moderate electronic conductivity and intrinsically low ionic diﬀusion.21 RSC Advances RSC Advances This journal is © The Royal Society of Chemistry 2016 2 Experimental The vanadium oxides were grown in an experimental high- vacuum ALD setup with a base pressure of 107 mbar with walls heated at 95 C.31,38,39 The ALD process used here was characterised earlier by Rampelberg et al.7,34–36,40 and is based on tetrakis[ethylmethylamino]vanadium (V(NC2H5CH3)4, or TEMAV) in combination with H2O and ozone. The precursor was held in a stainless steel container at 70 C with argon as a carrier gas. TEMAV and argon were pulsed at a pressure of 6 103 mbar. As reactant gases H2O and O3 were applied, pulsed at 7.5 103 mbar and 2 101 mbar, respectively. The ozone was produced with an OzoneLab™generator from a pure O2 gas ow, resulting in a minimal concentration of 150 mg ml1. In Upon going from a typical thin-lm battery (1 to 10 mm thick electrodes) to truly thin-lm electrodes (10 to 100 nm), the resistance for electronic current drops by orders of magnitude, This journal is © The Royal Society of Chemistry 2016 114658 \| RSC Adv., 2016, 6, 114658–114665 RSC Advances View Article Online RSC Advances View Article Online View Article Online Paper between pulses, reaction products and remaining precursor or reactant gas were evacuated using a turbomolecular pump to a pressure of <2.0 106 mbar. The lms were grown on silicon substrates covered with 100 nm thermal SiO2, 60 nm PVD TiN and 80 nm PVD Pt. Two thermal ALD processes were used here: the TEMAV–H2O process and the TEMAV–O3 process. To ensure similar conditions, both processes were performed at a substrate temperature of 150 C with saturated process parameters, as determined by earlier experiments.7,36 Pulse times for the TEMAV precursor were 5 and 4 s in the water and ozone processes. Water and ozone pulse times were 5 s on the SiO2 substrate. The Pt substrate inuenced the growth, as was also reported by Premkumar et al.,34 so higher ozone pulse times (8 s) were required to ensure uniform lms. The growth rate for the water process was 0.67 A per cycle. The growth rate for the ozone process was 1.05 A per cycle, a higher value than reported by Rampelberg et al.,7 but still lower than the reported value by Premkumar et al.34 was investigated using SEM, and images are shown in Fig. 1 for both lms on the Pt substrate. A very similar granular morphology is observed for both processes. Open Access Article. Published on 08 December 2016. Downloaded on 26/01/2017 16:10:59. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. X-Ray techniques were used to determine lm thickness (X- ray reectivity or XRR), crystallinity (X-ray diﬀraction or XRD) and lm composition and oxidation state (X-ray photoelectron spectroscopy or XPS). XRR and XRD were performed in a Bruker D8 Discover using a copper X-ray source (Cu-Ka radiation at 0.154 nm) and a point detector (for XRR) or a linear detector (for XRD). To determine the lm crystallinity during annealing in a controlled ambient, in situ XRD was performed using a linear detector and a controlled ambient/temperature system. XPS was performed using Al-Ka radiation (0.834 nm) under a take-oﬀ angle of 45 in a high-vacuum chamber (2 109 mbar). A resolution of 0.108 eV was obtained. Even though the morphology and oxidation state of the as- deposited lms are shown to be quite similar, a diﬀerence in lm density was found by evaluating the V XRF counts, nor- malised to the Si-substrate XRF counts to account for surface and distance factors. When these values are compared to the lm thicknesses measured by XRR, it is clear from Fig. 3 that there is more vanadium per unit thickness in the H2O-deposited lms than in the O3-deposited lm, a direct proof that the lms grown by utilizing the water process are denser when compared to those grown from the ozone process, as is consistent with XRR and XRF measurements performed in earlier work. The lm morphology was probed using a Bruker Dimension Edge atomic force microscope (AFM) and lm rms roughnesses were determined from 1 mm 1 mm AFM images. Electrochemical measurements were performed in an Ar- lled glove box (O2 < 1 ppm, H2O < 1 ppm) with a Metrohm Autolab PGSTAT302 connected to a three-electrode setup. Electrical contact to the TiN or Pt current collector was made by contacting the side of the sample with silver paste to a Cu foil. Lithium ribbon (99.9%, Sigma Aldrich) was used as counter and reference electrodes, and 1 M LiClO4 in propylene carbonate (99%, io-li-tec) was used as Li+ electrolyte solution. Cyclic vol- tammetry (CV) measurements were performed at a 10 mV s1 sweep rate between 1.5 and 4.5 V vs. Li+/Li, or relevant potential boundaries. Galvanostatic charge–discharge measurements were performed to examine the capacity and thin lm kinetics at varying current densities. 3.2 VOx crystallisation and oxidation state control for 2 # x # 2.5 The as-deposited lms using the TEMAV ALD processes are found to be amorphous by XRD with vanadium in a 4+ oxidation state. By post-ALD annealing in ambients with carefully controlled oxygen partial pressures, vanadium oxides in the VnO2n+1 series could be reached. The crystallisation was Fig. 1 Morphology of the as-deposited VOx ﬁlms on the Pt substrate as measured by SEM, showing the H2O-based (left) and O3-based (right) ALD ﬁlms. This journal is © The Royal Society of Chemistry 2016 2 Experimental These results imply that the choice of process barely inuences the as-deposited lm morphology and topology. Next, the lm composition was investigated using XPS. Since vanadium oxides are reported to be very sensitive to the pref- erential sputtering of oxygen during depth proling with XPS, only the surface spectra are evaluated to determine the composition and the oxidation state.42 The measured spectra were calibrated to the O 1s signals at 530.0 eV, since calibrating using the C 1s peak is not ideal for vanadium oxides.41 As the V 2p core level binding energy depends strongly on its oxidation state,7,42 tting can be used to determine the oxidation state of the as-deposited lms. Furthermore, the separation of the V 2p3/2 peak from the O 1s VOx-component at 530.0 eV was also used to compare the two as-deposited ALD lms.42 Fig. 2 shows the results of this analysis, alongside a V2O5 reference, using the reported binding energies for the V2p components: 515.8 and 523.2 eV for the V2p3/2 and V2p1/2, and 517.2 and 524.5 eV for the V2p3/2 and V2p1/2 for the V4+ and V5+ components, respec- tively. Both as-deposited lms clearly have vanadium in the 4+ oxidation state. 3.1 VOx thin lm deposition by thermal ALD The binding energies are referenced to O 1s calibrated at 530.0 eV.41 Fig. 2 XPS spectra for the VOx ﬁlm surface: (a) a crystalline V2O5 reference ﬁlm, (b) as-deposited ﬁlm using the water-based ALD process and (c) as-deposited ﬁlm using the ozone-based ALD process. The binding energies are referenced to O 1s calibrated at 530.0 eV.41 The binding energies are referenced to O 1s calibrated at 530.0 eV. Fig. 3 Relation between the XRF vanadium counts (normalised to the Si signal) and the measured ﬁlm thickness (XRR) for diﬀerent ﬁlm thicknesses for both processes. The higher values for the H2O-based ALD ﬁlms indicate a higher VOx density for these ﬁlms. The dashed lines are a guide to the eye to illustrate this. This article is licensed under a Crea Fig. 4 Crystallisation of the as-deposited VOx thin ﬁlms on the SiO2 substrate, as studied by in situ XRD in varying oxygen partial pressure at a heating rate of 0.25 C s1. Symbols indicate VOx crystal states (2 # x # 2.5), as shown in the legend (top left). Fig. 3 Relation between the XRF vanadium counts (normalised to the Si signal) and the measured ﬁlm thickness (XRR) for diﬀerent ﬁlm thicknesses for both processes. The higher values for the H2O-based ALD ﬁlms indicate a higher VOx density for these ﬁlms. The dashed lines are a guide to the eye to illustrate this. a 3.7 Pa oxygen partial pressure is discussed here (top section of Fig. 4(b)). One can observe that the lm is initially amorphous by the lack of diﬀraction peaks. When heating in a controlled ambient, the lm crystallises into VO2 (B) just above 400 C, and nally oxidizes between 500 and 600 C to V6O13. monitored using in situ XRD and the oxidation states were in accordance to the crystal state, as was conrmed by XPS on selected quenches (not shown here). Ex situ XRD on selected quenches conrmed the crystal states at intermediate temper- atures. The crystallisation of the thin lms was investigated on three substrates: SiO2, Pt and TiN. The results of the in situ XRD measurements are summarized in ‘phase formation diagrams’, as shown in Fig. 5 for the crys- tallisation and oxidation on a SiO2 substrate. This journal is © The Royal Society of Chemistry 2016 3.1 VOx thin lm deposition by thermal ALD First, the properties of the as-deposited amorphous lms were investigated. The rms roughness of the as-deposited lms on a Pt substrate, as measured by AFM, was 1.95 and 1.77 nm for the H2O- and O3-based lms, respectively. The Pt substrate was also measured, and showed a roughness of 1.75 nm, so the deposition of a 10 nm lm on the substrate barely increases the roughness, indicating smooth lms. The surface morphology Fig. 1 Morphology of the as-deposited VOx ﬁlms on the Pt substrate as measured by SEM, showing the H2O-based (left) and O3-based (right) ALD ﬁlms. This journal is © The Royal Society of Chemistry 2016 RSC Adv., 2016, 6, 114658–114665 \| 114659 View Article Online RSC Advances Paper Fig. 2 XPS spectra for the VOx ﬁlm surface: (a) a crystalline V2O5 reference ﬁlm, (b) as-deposited ﬁlm using the water-based ALD process and (c) as-deposited ﬁlm using the ozone-based ALD process. The binding energies are referenced to O 1s calibrated at 530.0 eV.41 Fig. 3 Relation between the XRF vanadium counts (normalised to the Si signal) and the measured ﬁlm thickness (XRR) for diﬀerent ﬁlm thicknesses for both processes. The higher values for the H2O-based ALD ﬁlms indicate a higher VOx density for these ﬁlms. The dashed lines are a guide to the eye to illustrate this. Open Access Article. Published on 08 December 2016. Downloaded on 26/01/2017 16:10:59. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 2 XPS spectra for the VOx ﬁlm surface: (a) a crystalline V2O5 reference ﬁlm, (b) as-deposited ﬁlm using the water-based ALD process and (c) as-deposited ﬁlm using the ozone-based ALD process. The binding energies are referenced to O 1s calibrated at 530.0 eV.41 Fig. 4 Crystallisation of the as-deposited VOx thin ﬁlms on the SiO2 substrate, as studied by in situ XRD in varying oxygen partial pressure at a heating rate of 0.25 C s1. Symbols indicate VOx crystal states (2 # x # 2.5), as shown in the legend (top left). g. 2 ferenc ocess he bin Open Access Article. Published on 08 December 2016. Downloaded on 26/01/2017 16:10:59. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 2 XPS spectra for the VOx ﬁlm surface: (a) a crystalline V2O5 reference ﬁlm, (b) as-deposited ﬁlm using the water-based ALD process and (c) as-deposited ﬁlm using the ozone-based ALD process. 114660 \| RSC Adv., 2016, 6, 114658–114665 3.1 VOx thin lm deposition by thermal ALD These should not be interpreted as thermodynamic phase diagrams, since no constant-temperature steps were made to allow lm stabilisa- tion to the equilibrium state, but as phase formation diagrams displaying the kinetic path the lms go through while being heated at 0.25 C s1 in the ambients under study (He with oxygen partial pressure of 3.7, 7.4, 14.8, 29.6 and 48.1 Pa, and ambient air). First, we will discuss the results on the SiO2 substrate. The systematic study of the crystallisation behaviour of the ALD VOx on the SiO2 substrate is presented in Fig. 4(a) and (b) for the H2O- and O3-ALD deposited lms, respectively. As an example, the phase evolution of the ozone-grown lm during annealing in This journal is © The Royal Society of Chemistry 2016 114660 \| RSC Adv., 2016, 6, 114658–114665 RSC Advances View Article Online RSC Advances View Article Online RSC Advances View Article Online Paper Paper Open Access Article. Published on 08 December 2016. Downloaded on 26/01/2017 16:10:59. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 5 Phase formation diagrams for the formation of crystalline VOx phases (2 # x # 2.5), based on the in situ XRD measurements (Fig. 4) at 0.25 C s1 for He ambients with oxygen partial pressures of 3.7, 7.4, 14.8, 29.6 and 48.1 Pa, and in ambient air. The same set of symbols is used as in Fig. 4 except for the extrema, namely VO2 and V2O5 states, since these dominate the phase formation diagrams to a large extent. le. Published on 08 December 2016. Downloaded on 26/01/2017 16:10:59. article is licensed under a Creative Commons Attribution 3.0 Unported Licence. octahedral sites. The diﬀerence lies in the mutual orientation of the fourfold axis of the oxygen octahedra, being aligned or perpendicular for the VO2 (B) and VO2 (R), respectively.43 This translates into a diﬀerence in structure as well as in density: VO2 (R) has a higher density (4.67 g cm3) than VO2 (B) (4.03 g cm3).44 Earlier, we showed that the water-based process leads to growth of a higher density amorphous VOx layer than the ozone process. The high density amorphous layer deposited using the H2O-based ALD process crystallizes into the high density VO2 (R) phase, while the lower density amorphous layer deposited using the ozone process crystallizes initially into the lower density VO2 (B) phase. 3.1 VOx thin lm deposition by thermal ALD So, by choice of ALD process, we can choose to crystallise the lm in either the VO2 (B) or VO2 (M1) phase for electrochemical testing. Open Access Article. Published on 08 December 2016. Downloaded on 26/01/2017 16:10:59. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. We hypothesize that the use of ozone in the ALD process also has an inuence on the oxidation behaviour. Even though XPS shows that these lms have the same oxidation state for the vanadium (4+), we observe a lower temperature for the intro- duction of higher oxidation state vanadium oxides for the ozone grown lms. For the lowest oxygen partial pressure we obtain phase-pure VO2 for both lms. For higher oxygen partial pres- sures, we see the presence of V4O9 upon crystallisation of the ozone grown lms, having a higher oxidation state. For the water-grown lms this was not the case, and we obtained phase- pure VO2 for all oxygen partial pressures examined (except for air), before further oxidation occurred. Even though the oxida- tion state is the same, a stronger oxidant is used during the ozone-based ALD process, which could incorporate more oxygen into the lms. Another hypothesis is based on the fact that the lms are less dense when using the ozone process. This could allow faster oxygen diﬀusion into these lms, enhancing the oxidation kinetics during post-ALD annealing. Fig. 5 Phase formation diagrams for the formation of crystalline VOx phases (2 # x # 2.5), based on the in situ XRD measurements (Fig. 4) at 0.25 C s1 for He ambients with oxygen partial pressures of 3.7, 7.4, 14.8, 29.6 and 48.1 Pa, and in ambient air. The same set of symbols is used as in Fig. 4 except for the extrema, namely VO2 and V2O5 states, since these dominate the phase formation diagrams to a large extent. The in situ XRD data provides a wealth of information on the oen quite complex phase formation sequence. On SiO2 substrates, phase-pure regions were only observed for the VO2, V6O13 and V2O5 phases. A clear trend emerges when examining the inuence of the oxygen partial pressure. When going through both formation diagrams in Fig. 5 from low to high oxygen partial pressure, we see that phases emerge and disap- pear in the order of increasing oxidation state: VO2–V6O13– V4O9–V3O7–V2O5 with respective average oxidation states for the V of 4–4.33–4.5–4.67–5. 3.1 VOx thin lm deposition by thermal ALD The inuence of the temperature is more complex since the phases have no time to settle to an equilibrium state at each temperature, so thin-lm kinetics will complicate this behaviour. However, we can see that in general with increasing temperature the average oxidation state rises to higher values, showing high-temperature stability for the higher oxides. The crystallisation of the lms on the Pt and TiN substrates was examined next, since a current collector is required to test the vanadium oxides as LIB electrode materials. The results are shown in Fig. 6. Similar trends were observed, with again formation of the VO2 (B) or VO2 (M1) as rst phases for the ozone and water-based ALD, respectively, and the V2O5 phase occurring at high-temperature and/or high oxygen ambients. However, one can note two main diﬀerences when comparing these phase formation diagrams to those on SiO2. Firstly, the SiO2 formation diagrams are much richer in phase zones than their counterparts on Pt and TiN. Furthermore, we observed that on SiO2 the transformation to higher oxidation state vanadium oxides occurs at lower temperatures than on the other investigated substrates. In the case of a TiN substrate, the vanadium oxide phases with the highest oxidation states appear at the highest temperatures, or not at all, when compared to SiO2 and Pt. The Pt case lies in between both other cases. Here, we see the inuence of the TiN in the substrate. We hypothesise that the TiN-substrate acts as an oxygen drain, extracting oxygen from the lm, and thus preventing further oxidation of the vanadium oxides, as was also observed by the presence of TiO2 rutile diﬀraction peaks in the XRD scans taken aer the oxidation experiments. The SiO2 is completely oxidized already, so it does not act as an oxygen drain, lowering the oxygen pressure and temperatures required to oxidise the vanadium Furthermore, we can see the remarkable inuence of the as- deposited lm on the initial crystallisation of the amorphous layers. Since all parameters are equal except for the ALD process gas during the ALD process (ozone or water), this translates to an inuence of the latter on the initial crystallisation. The ozone-based ALD crystallizes into the VO2 (B)-phase, while that via the water-based process crystallizes into the high- temperature stable (>68 C) VO2 (R)-state (which settles to the VO2 (M1) state when quenched in that phase). This journal is © The Royal Society of Chemistry 2016 3.1 VOx thin lm deposition by thermal ALD Both these phases are based on a bcc lattice with vanadium on the This journal is © The Royal Society of Chemistry 2016 RSC Adv., 2016, 6, 114658–114665 \| 114661 Paper View Article Online View Article Online RSC Advances Fig. 6 Phase formation diagrams for the formation of the VOx crystal states (2 # x # 2.5) on TiN substrates (left) and on Pt substrates (right), at 0.25 C s1 for He ambients with oxygen partial pressure of 3.7, 7.4, 14.8, 29.6 and 48.1 Pa, and in ambient air. The absence of the melt-line on the left ﬁgure is related to the presence of rutile TiO2 from the substrate, complicating the analysis of the VO2 (R) phase on the TiN substrate at high temperatures. d on 08 December 2016. Downloaded on 26/01/2017 16:10:59. nsed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 08 December 2016. Downloaded on 26/01/2017 16:10:59. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 6 Phase formation diagrams for the formation of the VOx crystal states (2 # x # 2.5) on TiN substrates (left) and on Pt substrates (right), at 0.25 C s1 for He ambients with oxygen partial pressure of 3.7, 7.4, 14.8, 29.6 and 48.1 Pa, and in ambient air. The absence of the melt-line on the left ﬁgure is related to the presence of rutile TiO2 from the substrate, complicating the analysis of the VO2 (R) phase on the TiN substrate at high temperatures. Fig. 7 Cyclic voltammetry on the selected vanadium oxide thin ﬁlms, performed at 10 mV s1 in a three-electrode setup with lithium as counter and reference electrodes, and 1 M LiClO4 in PC as electrolyte. Potential ranges were chosen according to activity. Table 1 Conversion paths from the as-deposited ﬁlms to their crys- tallised and oxidized forms on the Pt-substrate Table 1 Conversion paths from the as-deposited ﬁlms to their crys- tallised and oxidized forms on the Pt-substrate Phase ALD reactant Ambient Temperature (C) VO2 (B) O3 He + 3.7 Pa O2 420 VO2 (M1) H2O He + 18 Pa O2 450 V6O13 O3 He + 3.7 Pa O2 550 V4O9 H2O Ambient air 356 V3O7 O3 He + 48 Pa O2 560 V2O5 H2O or O3 Ambient air 500 oxides. The Pt substrate has a TiN layer underneath. 3.1 VOx thin lm deposition by thermal ALD Since oxygen can diﬀuse through the Pt layer, the underlying TiN can still get oxidized, as was conrmed by XRD. However, this oxidation is delayed by the presence of the Pt layer. This causes both observed diﬀerences: the phase-richness in the formation diagrams likely also occurs for TiN or Pt substrates, but at higher oxygen partial pressures than examined here, when the supply of oxygen is higher than the harvesting eﬀect of the TiN. 3.3 Electrochemical characterisation of the possible VOx phases (2 # x # 2.5) Micron-sized LMO is shown as a model bulk cathode, (red circles)46 and more general bulk cathodes (shaded left corner)45 from literature are shown for comparison. Paper Creative Commons Attribution 3.0 Unported Licence. oxidations † and results are summarised in Table 1 Only V3O Fig. 8 Measured delithiation capacity of the examined ﬁlms on a logarithmic-linear scale (solid symbols). The data was extrapolated (dashed lines) resulting in a good match to the data (R2 > 0.98 Micron-sized LMO is shown as a model bulk cathode, (red circles)4 and more general bulk cathodes (shaded left corner)45 from literature are shown for comparison. Table 2 Delithiation capacities for the examined thin ﬁlms, calculated by charging and discharging the ﬁlms at varying current density ove several orders of magnitude and extrapolating the measured capacit to 1C (Fig. 8), which corresponds to the current necessary to reach the theoretical capacity in 1 hour. Volumetric capacities of commercia cathode materials are included for comparison45 Potential (V vs. Li+/Li) Volum. capacit (mA h cm3) This work VO2 (M1) 1.5–4.0 No activity VO2 (B) 1.5–4.0 805 (1C) V6O13 1.5–4.0 894 (1C) V4O9 1.5–4.0 1380 (1C) V3O7 1.5–4.0 1255 (1C) V2O5 (/LiV2O5) 2.9–3.5 488 (1C) V2O5 (/Li3V2O5) 1.5–4.0 810 (1C) Commercial cathode materials LiCoO2 3.8 550 LiMn2O4 4.1 596 LiFePO4 3.4 589 LiNi0.33Mn0.33Co0.33O2 3.7 600 LiNi0.8Co0.15Al0.05O2 3.8 700 Paper Open Access Article. Published on 08 December 2016. Downloaded on 26/01/2017 16:10:59. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Paper one used for the formation diagrams. Cyclic voltammetry at 10 mV s1 was performed down to 1.5 V vs. Li+/Li to examine the electrochemical intercalation behaviour of the diﬀerent vana- dium oxides. As can be seen in Fig. 7, all phases show a clear lithiation and delithiation behaviour. However, even though the VO2 (M1) appeared to show activity, initial galvanostatic charge– discharge tests showed only very low electrochemical storage capacity. Furthermore, some peaks of the cyclic voltammogram appear closely related to the electrochemical activity of the Pt substrate, as shown in ESI.† Due to the absence of storage of lithium into these lms, VO2 (M1) was not examined in-depth further. All other lms showed a clear lithium insertion/ extraction behaviour, indicating lithium can be stored in these lms reversibly. 3.3 Electrochemical characterisation of the possible VOx phases (2 # x # 2.5) Micron-sized LMO is shown as a model bulk cathode, (red circles)46 and more general bulk cathodes (shaded left corner)45 from literature are shown for comparison. Fig. 8 Measured delithiation capacity of the examined ﬁlms on a logarithmic-linear scale (solid symbols). The data was extrapolated (dashed lines) resulting in a good match to the data (R2 > 0.98). Micron-sized LMO is shown as a model bulk cathode, (red circles)46 and more general bulk cathodes (shaded left corner)45 from literature are shown for comparison. Table 2 Delithiation capacities for the examined thin ﬁlms, calculated by charging and discharging the ﬁlms at varying current density over several orders of magnitude and extrapolating the measured capacity to 1C (Fig. 8), which corresponds to the current necessary to reach the theoretical capacity in 1 hour. Volumetric capacities of commercial cathode materials are included for comparison45 oxidations,† and results are summarised in Table 1. Only V3O7 was not obtainable in a phase-pure form on the Pt substrate, but the oxidation path indicated in Table 1 shows only a small fraction of the V3O7 lm crystallised to V2O5, as was conrmed by XRD and XPS. This makes the V3O7 almost phase-pure, and will be denoted further simply as V3O7. The path to V6O13 does not match the phase formation diagram, since phase-pure V6O13 was obtained by performing a ramp slower than the oxidations,† and results are summarised in Table 1. Only V3O7 was not obtainable in a phase-pure form on the Pt substrate, but the oxidation path indicated in Table 1 shows only a small fraction of the V3O7 lm crystallised to V2O5, as was conrmed by XRD and XPS. This makes the V3O7 almost phase-pure, and will be denoted further simply as V3O7. The path to V6O13 does not match the phase formation diagram, since phase-pure V6O13 was obtained by performing a ramp slower than the 3.3 Electrochemical characterisation of the possible VOx phases (2 # x # 2.5) Fig. 7 Cyclic voltammetry on the selected vanadium oxide thin ﬁlms, performed at 10 mV s1 in a three-electrode setup with lithium as counter and reference electrodes, and 1 M LiClO4 in PC as electrolyte. Potential ranges were chosen according to activity. All of the obtained crystalline vanadium oxides (VO2 (B), VO2 (M1), V6O13, V4O9, V3O7 and V2O5) were tested as LIB electrodes on Pt substrates. Film crystallisation was monitored by in situ XRD to ensure corresponding phase formations and This journal is © The Royal Society of Chemistry 2016 114662 \| RSC Adv., 2016, 6, 114658–114665 RSC Advances View Article Online RSC Advances View Article Online oxidations,† and results are summarised in Table 1. Only V3O7 was not obtainable in a phase-pure form on the Pt substrate, but the oxidation path indicated in Table 1 shows only a small fraction of the V O lm crystallised to V O as was conrmed Fig. 8 Measured delithiation capacity of the examined ﬁlms on a logarithmic-linear scale (solid symbols). The data was extrapolated (dashed lines) resulting in a good match to the data (R2 > 0.98). Micron-sized LMO is shown as a model bulk cathode, (red circles)46 and more general bulk cathodes (shaded left corner)45 from literature are shown for comparison. Table 2 Delithiation capacities for the examined thin ﬁlms, calculated by charging and discharging the ﬁlms at varying current density over several orders of magnitude and extrapolating the measured capacity to 1C (Fig. 8), which corresponds to the current necessary to reach the theoretical capacity in 1 hour. Volumetric capacities of commercial cathode materials are included for comparison45 Potential (V vs. Li+/Li) Volum. capacity (mA h cm3) This work VO2 (M1) 1.5–4.0 No activity VO2 (B) 1.5–4.0 805 (1C) V6O13 1.5–4.0 894 (1C) V4O9 1.5–4.0 1380 (1C) V3O7 1.5–4.0 1255 (1C) V2O5 (/LiV2O5) 2.9–3.5 488 (1C) V2O5 (/Li3V2O5) 1.5–4.0 810 (1C) Commercial cathode materials LiCoO2 3.8 550 LiMn2O4 4.1 596 LiFePO4 3.4 589 LiNi0.33Mn0.33Co0.33O2 3.7 600 LiNi0.8Co0.15Al0.05O2 3.8 700 Paper Open Access Article. Published on 08 December 2016. Downloaded on 26/01/2017 16:10:59. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 8 Measured delithiation capacity of the examined ﬁlms on a logarithmic-linear scale (solid symbols). The data was extrapolated (dashed lines) resulting in a good match to the data (R2 > 0.98). This journal is © The Royal Society of Chemistry 2016 3.3 Electrochemical characterisation of the possible VOx phases (2 # x # 2.5) y Galvanostatic charging and discharging was performed on the lms to determine the available storage capacity and compare it to V2O5, which is the most investigated form of vanadium oxide and is generally considered to be the most promising phase. The lms were cycled at varying current densities between 0.1C and 100C, and the capacity was extrap- olated to the 1C capacity to allow direct comparison between the diﬀerent VOx phases, as shown in Fig. 8. Extrapolation was performed by tting a linear relation to the logarithm of the capacity plotted to the logarithm of the C-rate, which provided ts with R2 > 0.98, demonstrating the validity of this approach. Table 2 summarizes the extrapolated capacities at 1C. Both Table 2 and Fig. 8 clearly demonstrate that, although V2O5 is the most investigated vanadium oxide for LIB cathodes, the capacity of the stable region (which corresponds to insertion of one lithium into V2O5), has a limited volumetric capacity. This is related to the low density of this V2O5 (3.36 g cm3) compared to that of commercial lithium-ion cathodes such as LiCoO2 (4.9 g cm3).45 All of the other investigated vanadium oxide phases have capacities well above the commercial cathodes, which is spearheaded by LiNi0.33Mn0.33Co0.33O2 (NMC) with a capacity of 600 mA h cm3. The higher capacity for other vanadium oxides has an origin in increased material density and insertion range. First, the densities of the lower oxidation state vanadium oxides are higher than that of V2O5, leading to a higher volumetric capacity density; V3O7, V4O9, V6O13 and the VO2 (B) lms have densities of 3.61, 3.78, 3.91 and 4.0 g cm3, respectively. Second, while V2O5 can only be used in the narrow range that leads to LiV2O5, i.e. reduction of vanadium from V5+ to an average oxidation state of V4.5+, the vanadium in the series of vanadium oxides tested here changes its oxidation state much more, ranging from a change in average oxidation state of 0.62 for VO2 (B) to almost 1.2 for V4O9. So, while V2O5 and VO2 (B) are generally considered the most promising cathode materials, vanadium oxides in between them exhibit higher volumetric capacities. Fig. 8 Measured delithiation capacity of the examined ﬁlms on a logarithmic-linear scale (solid symbols). The data was extrapolated (dashed lines) resulting in a good match to the data (R2 > 0.98). en Access Article. Published on 08 December 2016. Downloaded on 26/01/2017 16:10:59. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence 17 N. Ganganagappa and A. Siddaramanna, Mater. Charact., 2012, 68, 58–62. 18 H. A. Wriedt, Bull. Alloy Phase Diagrams, 1989, 10, 271–277. Acknowledgements 19 M. S. Whittingham, Chem. Rev., 2004, 104, 4271–4302. 20 D. W. Murphy, P. A. Christian, F. J. DiSalvo and J. V. Waszczak, Inorg. Chem., 1979, 18, 2800–2803. The authors acknowledge the IWT-Vlaanderen, BOF-UGent (GOA 01G01513), M-ERA LaminaLion, SBO SoS-Lion and the Hercules foundation for nancial support. The authors also thank N. Deroo and Dr Ir. K. Devloo-Casier for the XPS measurements, and O. Janssens for assistance with the SEM and EDX measurements. J. D. and T. D. acknowledge the Flemish FWO for a postdoctoral and aspirant fellowship, respectively. Finally, the authors would like to thank the Air Liquide Advanced Materials team to provide us with the TEMAV precursor. 21 X. Huang, X. Rui, H. H. Hng and Q. Yan, Part. Part. Syst. Charact., 2015, 32, 276–294. 22 J. Jiang, Y. Li, J. Liu, X. Huang, C. Yuan and X. W. D. Lou, Adv. Mater., 2012, 24, 5166–5180. 23 J. F. M. Oudenhoven, L. Baggetto and P. H. L. Notten, Adv. Energy Mater., 2011, 1, 10–33. 24 Y. Wang and G. Cao, Adv. Mater., 2008, 20, 2251–2269. 25 L. Baggetto, R. A. H. Niessen, F. Roozeboom and P. H. L. Notten, Adv. Funct. Mater., 2008, 18, 1057–1066. 26 J. W. Long, B. Dunn, D. R. Rolison and H. S. White, Chem. Rev., 2004, 104, 4463–4492. Paper diﬀer in density. By annealing these lms in controlled ambi- ents with varying oxygen partial pressures, we demonstrated that all crystalline phases between VO2 and V2O5 could be ob- tained. The substrate was found to inuence the oxidation and crystallisation behaviour, with TiN acting as an oxygen drain delaying the oxidation. All obtained crystalline phases were characterised electrochemically, and showed activity as lithium- ion electrodes. We demonstrated that, while V2O5 is the most widely investigated vanadium-based cathode material, also VO2 (B), V6O13, V4O9 and V3O7 can be used as cathodes, with capacities up to 1380 mA h cm3 (V4O9), more than twice the volumetric capacity for commercial cathode materials such as NMC. 12 Y. Liu, M. Clark, Q. Zhang, D. Yu, D. Liu, J. Liu and G. Cao, Adv. Energy Mater., 2011, 1, 194–202. 13 C. Leger, S. Bach, P. Soudan and J.-P. Pereira-Ramos, J. Electrochem. Soc., 2005, 152, 236–241. 14 Y. S. Cohen and D. Aurbach, Electrochem. Commun., 2004, 6, 536–542. 15 A. Pan, H. B. Wu, L. Yu, T. Zhu and X. W. D. Lou, ACS Appl. Mater. Interfaces, 2012, 4, 3874–3879. 16 S.-L. Chou, J.-Z. Wang, J.-Z. Sun, D. Wexler, M. Forsyth, H.-K. Liu, D. R. MacFarlane and S.-X. Dou, Chem. Mater., 2008, 20, 7044–7051. 4 Conclusions We demonstrated the deposition of two diﬀerent forms of amorphous VO2 using two diﬀerent ALD chemistries based on the same TEMAV precursor. 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https://openalex.org/W4232911073	https://www.atmos-chem-phys.net/16/2359/2016/acp-16-2359-2016.pdf	English	null	Air quality and radiative impacts of Arctic shipping emissions in the summertime in northern Norway: from the local to the regional scale	null	2,015	cc-by	20,228	orrespondence to: Louis Marelle (louis.marelle@latmos.ipsl.fr) and Jennie L. Thomas (jennie.thom ouis Marelle (louis.marelle@latmos.ipsl.fr) and Jennie L. Thomas (jennie.thomas@latmos.ipsl.fr) Received: 15 April 2015 – Published in Atmos. Chem. Phys. Discuss.: 7 July 2015 Revised: 12 January 2016 – Accepted: 1 February 2016 – Published: 29 February 2016 Abstract. In this study, we quantify the impacts of ship- ping pollution on air quality and shortwave radiative ef- fect in northern Norway, using WRF-Chem (Weather Re- search and Forecasting with chemistry) simulations com- bined with high-resolution, real-time STEAM2 (Ship Traf- ﬁc Emissions Assessment Model version 2) shipping emis- sions. STEAM2 emissions are evaluated using airborne mea- surements from the ACCESS (Arctic Climate Change, Econ- omy and Society) aircraft campaign, which was conducted in the summer 2012, in two ways. First, emissions of ni- trogen oxides (NOx) and sulfur dioxide (SO2) are derived for speciﬁc ships by combining in situ measurements in ship plumes and FLEXPART-WRF plume dispersion modeling, and these values are compared to STEAM2 emissions for the same ships. Second, regional WRF-Chem runs with and without STEAM2 ship emissions are performed at two differ- ent resolutions, 3 km × 3 km and 15 km × 15 km, and evalu- ated against measurements along ﬂight tracks and average campaign proﬁles in the marine boundary layer and lower troposphere. These comparisons show that differences be- tween STEAM2 emissions and calculated emissions can be quite large (−57 to +148 %) for individual ships, but that WRF-Chem simulations using STEAM2 emissions repro- duce well the average NOx, SO2 and O3 measured during ACCESS ﬂights. The same WRF-Chem simulations show that the magnitude of NOx and ozone (O3) production from ship emissions at the surface is not very sensitive (< 5 %) to the horizontal grid resolution (15 or 3 km), while sur- face PM10 particulate matter enhancements due to ships are moderately sensitive (15 %) to resolution. The 15 km reso- lution WRF-Chem simulations are used to estimate the re- gional impacts of shipping pollution in northern Norway. Our results indicate that ship emissions are an important source of pollution along the Norwegian coast, enhancing 15-day-averaged surface concentrations of NOx (∼+80 %), SO2 (∼+80 %), O3 (∼+5 %), black carbon (∼+40 %), and PM2.5 (∼+10 %). The residence time of black carbon origi- nating from shipping emissions is 1.4 days. Over the same 15-day period, ship emissions in northern Norway have a global shortwave (direct + semi-direct + indirect) radiative effect of −9.3 mWm−2. 1 Introduction Shipping is an important source of air pollutants and their precursors, including carbon monoxide (CO), nitrogen ox- ides (NOx), sulfur dioxide (SO2), volatile organic com- pounds (VOCs) as well as organic carbon (OC) and black carbon (BC) aerosols (Corbett and Fischbeck, 1997; Corbett and Köhler, 2003). It is well known that shipping emissions have an important inﬂuence on air quality in coastal regions, Air quality and radiative impacts of Arctic shipping emissions in the summertime in northern Norway: from the local to the regional scale Louis Marelle1,2, Jennie L. Thomas1, Jean-Christophe Raut1, Kathy S. Law1, Jukka-Pekka Jalkanen3, Lasse Johansson3, Anke Roiger4, Hans Schlager4, Jin Kim4, Anja Reiter4, and Bernadett Weinzierl4,5 1LATMOS/IPSL, UPMC Univ. Paris 06 Sorbonne Universités, UVSQ, CNRS, Paris, France 2TOTAL S.A, Direction Scientiﬁque, Tour Michelet, 92069 Paris La Defense, France 3Finnish Meteorological Institute, Helsinki, Finland Louis Marelle1,2, Jennie L. Thomas1, Jean-Christophe Raut1, Kathy S. Law1, Jukka-Pekka Jalk Lasse Johansson3, Anke Roiger4, Hans Schlager4, Jin Kim4, Anja Reiter4, and Bernadett Wein 1LATMOS/IPSL, UPMC Univ. Paris 06 Sorbonne Universités, UVSQ, CNRS, Paris, France 2TOTAL S.A, Direction Scientiﬁque, Tour Michelet, 92069 Paris La Defense, France 3Finnish Meteorological Institute, Helsinki, Finland 4Institut für Physik der Atmosphäre, Deutsches Zentrum für Luft- und Raumfahrt (DLR), Oberpfaffenhofen, Germany 5Ludwig Maximilians Universität (LMU), Meteorologisches Institut, 80333, München, Germany espondence to: Louis Marelle (louis.marelle@latmos.ipsl.fr) and Jennie L. Thomas (jennie.thomas@ L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions Ships produced or heavily modiﬁed recently must also comply to lower NOx emissions factors limits, reducing emission fac- tors (in g kWh−1) by approximately −10 % (after 2000) and another −15 % (after 2011) compared to ships built before year 2000 (IMO, 2010). Jonson et al. (2015) showed that the creation of the North Sea and Baltic Sea SECAs was effective in reducing current pollution levels in Europe, and that fur- ther NOx and sulfur emission controls in these regions could help to achieve strong health beneﬁts by 2030 by reducing PM levels. on air quality in a now relatively pristine region (e.g., Granier et al., 2006), and will inﬂuence both Arctic and global cli- mate (Dalsøren et al., 2013; Lund et al., 2012). In addition, it has recently been shown that routing international mar- itime trafﬁc through the Arctic, as opposed to traditional routes through the Suez and Panama canals, will result in warming in the coming century and cooling on the long term (150 years). This is due to the opposite sign of impacts due to reduced SO2 linked to IMO regulations and reduced CO2 and O3 associated with fuel savings from using these shorter Arctic routes (Fuglestvedt et al., 2014). In addition, sulfate is predicted to cause a weaker cooling effect for the northern routes (Fuglestvedt et al., 2014). g Although maritime trafﬁc is relatively minor at present in the Arctic compared to global shipping, even a small number of ships can signiﬁcantly degrade air quality in regions where other anthropogenic emissions are low (Aliabadi et al., 2015; Eckhardt et al., 2013). Dalsøren et al. (2007) and Ødemark et al. (2012) have shown that shipping emissions also inﬂu- ence air quality and climate along the Norwegian and Rus- sian coasts, where current Arctic ship trafﬁc is the largest. Both studies (for years 2000 and 2004) were based on emis- sion data sets constructed using ship activity data from the AMVER (Automated Mutual-Assistance VEssel Rescue sys- tem) and COADS (Comprehensive Ocean–Atmosphere Data Set) data sets. However, the AMVER data set is biased to- wards larger vessels (> 20 000 t) and cargo ships (Endresen et al., 2003), and both data sets have limited coverage in Eu- rope (Miola and Ciuffo, 2011). L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions Future Arctic shipping is expected to have important impacts In addition to its impacts on air quality, maritime traf- ﬁc already contributes to climate change, by increasing the concentrations of greenhouse gases (CO2, O3) and aerosols (SO4, OC, BC) (Capaldo et al., 1999; Endresen et al., 2003). The current radiative forcing of shipping emissions is neg- ative and is dominated by the cooling inﬂuence of sulfate aerosols formed from SO2 emissions (Eyring et al., 2010). However, due to the long lifetime of CO2 compared to sul- fate, shipping emissions warm the climate in the long term (after 350 years; Fuglestvedt et al., 2009). In the future, global shipping emissions of SO2 are expected to decrease due to IMO regulations, while global CO2 emissions from shipping will continue to grow due to increased trafﬁc. This combination is expected to cause warming relative to the present day (Fuglestvedt et al., 2009; Dalsøren et al., 2013). In addition to their global impacts, shipping emissions are of particular concern in the Arctic, where they are projected to increase in the future as sea ice declines (for details of fu- ture sea ice, e.g., Stroeve et al., 2011). Decreased summer sea ice, associated with warmer temperatures, is progressively opening the Arctic region to transit shipping, and projec- tions indicate that new trans-Arctic shipping routes should be available by mid-century (Smith and Stephenson, 2013). Other shipping activities are also predicted to increase, in- cluding shipping associated with oil and gas extraction (Pe- ters et al., 2011). Sightseeing cruises have increased signif- icantly during the last decades (Eckhardt et al., 2013), al- though it is uncertain whether or not this trend will continue. Future Arctic shipping is expected to have important impacts In this study, we aim to quantify the impacts of shipping along the Norwegian coast in July 2012, using airborne mea- surements from the ACCESS (Arctic Climate Change, Econ- omy and Society) aircraft campaign (Roiger et al., 2015). This campaign (Sect. 2) took place in summer 2012 in north- ern Norway, and was primarily dedicated to the study of lo- cal pollution sources in the Arctic, including pollution origi- nating from shipping. ACCESS measurements are combined with two modeling approaches, described in Sect. 3. First, we use the Weather Research and Forecasting (WRF) model to drive the Lagrangian particle dispersion model FLEXPART- WRF run in forward mode to predict the dispersion of ship www.atmos-chem-phys.net/16/2359/2016/ Atmos. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions 2360 often enhancing ozone (O3) and increasing aerosol concen- trations (e.g., Endresen et al., 2003). Corbett et al. (2007) and Winebrake et al. (2009) showed that aerosol pollution from ships might be linked to cardiopulmonary and lung dis- eases globally. Because of their negative impacts, shipping emissions are increasingly subjected to environmental regu- lations. The International Maritime Organization (IMO) has designated several regions as Sulfur Emission Control Areas (SECAs; including the North Sea and Baltic Sea in Europe), where low sulfur fuels must be utilized to minimize the air quality impacts of shipping on particulate matter (PM) lev- els. The sulfur content in ship fuels in SECAs was limited to 1 % by mass in 2010, decreasing to 0.1 % in 2015, while the global average is 2.4 % (IMO, 2010). Less strict sulfur emis- sion controls (0.5 %) will also be implemented worldwide, at the latest in 2025, depending on current negotiations. Ships produced or heavily modiﬁed recently must also comply to lower NOx emissions factors limits, reducing emission fac- tors (in g kWh−1) by approximately −10 % (after 2000) and another −15 % (after 2011) compared to ships built before year 2000 (IMO, 2010). Jonson et al. (2015) showed that the creation of the North Sea and Baltic Sea SECAs was effective in reducing current pollution levels in Europe, and that fur- ther NOx and sulfur emission controls in these regions could help to achieve strong health beneﬁts by 2030 by reducing PM levels. often enhancing ozone (O3) and increasing aerosol concen- trations (e.g., Endresen et al., 2003). Corbett et al. (2007) and Winebrake et al. (2009) showed that aerosol pollution from ships might be linked to cardiopulmonary and lung dis- eases globally. Because of their negative impacts, shipping emissions are increasingly subjected to environmental regu- lations. The International Maritime Organization (IMO) has designated several regions as Sulfur Emission Control Areas (SECAs; including the North Sea and Baltic Sea in Europe), where low sulfur fuels must be utilized to minimize the air quality impacts of shipping on particulate matter (PM) lev- els. The sulfur content in ship fuels in SECAs was limited to 1 % by mass in 2010, decreasing to 0.1 % in 2015, while the global average is 2.4 % (IMO, 2010). Less strict sulfur emis- sion controls (0.5 %) will also be implemented worldwide, at the latest in 2025, depending on current negotiations. L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions More recently, ship emissions using new approaches have been developed that use ship ac- tivity data more representative of European maritime trafﬁc, based on the AIS (Automatic Identiﬁcation System) ship po- sitioning system. These include the STEAM2 (Ship Trafﬁc Emissions Assessment Model version 2) shipping emissions, described in Jalkanen et al. (2012) and an Arctic-wide emis- sion inventory described in Winther et al. (2014). To date, quantifying the impacts of Arctic shipping on air quality and climate has also been largely based on global model studies, which are limited in horizontal resolution. In addition, there have not been speciﬁc ﬁeld measurements focused on Arc- tic shipping that could be used to study the local inﬂuence of shipping emissions in the European Arctic and to validate model predicted air quality impacts. In addition to its impacts on air quality, maritime traf- ﬁc already contributes to climate change, by increasing the concentrations of greenhouse gases (CO2, O3) and aerosols (SO4, OC, BC) (Capaldo et al., 1999; Endresen et al., 2003). The current radiative forcing of shipping emissions is neg- ative and is dominated by the cooling inﬂuence of sulfate aerosols formed from SO2 emissions (Eyring et al., 2010). However, due to the long lifetime of CO2 compared to sul- fate, shipping emissions warm the climate in the long term (after 350 years; Fuglestvedt et al., 2009). In the future, global shipping emissions of SO2 are expected to decrease due to IMO regulations, while global CO2 emissions from shipping will continue to grow due to increased trafﬁc. This combination is expected to cause warming relative to the present day (Fuglestvedt et al., 2009; Dalsøren et al., 2013). In addition to their global impacts, shipping emissions are of particular concern in the Arctic, where they are projected to increase in the future as sea ice declines (for details of fu- ture sea ice, e.g., Stroeve et al., 2011). Decreased summer sea ice, associated with warmer temperatures, is progressively opening the Arctic region to transit shipping, and projec- tions indicate that new trans-Arctic shipping routes should be available by mid-century (Smith and Stephenson, 2013). Other shipping activities are also predicted to increase, in- cluding shipping associated with oil and gas extraction (Pe- ters et al., 2011). Sightseeing cruises have increased signif- icantly during the last decades (Eckhardt et al., 2013), al- though it is uncertain whether or not this trend will continue. 2 The ACCESS aircraft campaign Fresh ship emissions were sampled less than 4 h after emis- sion. In addition to the single-plume ﬂights, the 19 July 2012 ACCESS ﬂight targeted aged ship emissions in the marine boundary layer near Trondheim. Data collected during the 11 and 12 July 2012 ﬂights are used to derive emissions from operating ships (Sect. 4), and data from the four ﬂights (11, 12, 19, and 25 July 2012) are used to evaluate regional chemical transport simulations investigating the impacts of shipping in northern Norway (Sect. 5). Other ﬂights from the ACCESS campaign were not used in this study because their ﬂight objectives biased the measurements towards other emissions sources (e.g., oil platforms in the Norwegian Sea) or because they included limited sampling in the boundary layer (ﬂights north to Svalbard and into the Arctic free tropo- sphere; Roiger et al., 2015). The ACCESS aircraft campaign took place in July 2012 from Andenes, Norway (69.3◦N, 16.1◦W); it included character- ization of pollution originating from shipping (four ﬂights) as well as other local Arctic pollution sources (details are available in the ACCESS campaign overview paper; Roiger et al., 2015). The aircraft (DLR Falcon 20) payload in- cluded a wide range of instruments measuring meteorolog- ical variables and trace gases, described in detail by Roiger et al. (2015). Brieﬂy, O3 was measured by UV (ultravio- let) absorption (5 % precision, 0.2 Hz), nitrogen oxide (NO), and nitrogen dioxide (NO2) by chemiluminescence and pho- tolytic conversion (10 % precision for NO, 15 % for NO2; 1 Hz), and SO2 by chemical ionization ion trap mass spec- trometry (20 % precision; 0.3 to 0.5 Hz). Aerosol size dis- tributions between 60 nm and 1 µm were measured using a Ultra-High Sensitivity Aerosol Spectrometer Airborne. The four ﬂights focused on shipping pollution took place on 11, 12, 19, and 25 July 2012 and are shown in Fig. 1a (details on the 11 and 12 July 2012 ﬂights shown in Fig. 1b). The three ﬂights on 11, 12, and 25 July 2012 sampled pollu- tion from speciﬁc ships (referred to as single-plume ﬂights). During these ﬂights, the research aircraft repeatedly sam- pled relatively fresh emissions from one or more ships dur- ing ﬂight legs at constant altitudes, at several distances from the emission source, and in some cases at different altitudes. L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions 2361 Table 1. Description of the ships sampled during the ACCESS ﬂights on 11 and 12 July 2012. emissions. FLEXPART-WRF results are used in combina- tion with ACCESS aircraft measurements in Sect. 4 to derive emissions of NOx and SO2 for speciﬁc ships sampled dur- ing ACCESS. The derived emissions are compared to emis- sions from the STEAM2 model for the same ships. Then, we perform simulations with the WRF-Chem model, including STEAM2 ship emissions, in order to examine in Sect. 5 the local (i.e., at the plume scale) and regional impacts of ship- ping pollution on air quality and shortwave radiative effects along the coast of northern Norway. Ship name Vessel type Gross Fuel type tonnage (tons) Wilson Leer Cargo ship 2446 Marine gas oil Costa Deliziosa Passenger ship 92 720 Heavy fuel oil Wilson Nanjing Cargo ship 6118 Heavy fuel oil Alaed∗ Cargo ship 7579 Heavy fuel oil ∗Ship present in STEAM2, not targeted during the campaign. ∗Ship present in STEAM2, not targeted during the campaign. 2 The ACCESS aircraft campaign In this study, measurements from these single-plume ﬂights are used in combination with ship plume dispersion simu- lations (described in Sects. 3.1 and 4.1) to estimate emis- sions from individual ships. This method relies on knowing the precise locations of the ships during sampling. Because those locations are not known for the ship emissions sam- pled on 25 July 2012 ﬂight, emissions are only calculated for the three ships targeted during the 11 and 12 July ﬂights (the Costa Deliziosa, Wilson Leer, and Wilson Nanjing), and for an additional ship (the Alaed) sampled during the 12 July ﬂight, whose location could be retrieved from the STEAM2 shipping emission inventory (presented in Sect. 3.3). Table 1 gives more information about these four ships, one large cruise ship and three cargo ships. On 11 and 12 July 2012, the research aircraft sampled fresh ship emissions within the boundary layer, during ﬂight legs at low altitudes (< 200 m). L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions Chem. Phys., 16, 2359–2379, 2016 L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions Atmospheric process WRF-Chem option Planetary boundary layer MYNN (Nakanishi and Niino, 2006) Surface layer MM5 Similarity scheme, Carlson–Boland viscous sublayer (Zhang and Anthes, 1982; Carlson and Boland, 1978) Land surface Uniﬁed Noah land-surface model (Chen and Dudhia, 2001) Microphysics Morrison (Morrison et al., 2009) Shortwave radiation Goddard (Chou and Suarez, 1999) Longwave radiation RRTM (Mlawer et al., 1997) Cumulus parameterization Grell-3D (Grell and Devenyi, 2002) Photolysis Fast-J (Wild et al., 2000) Gas phase chemistry CBM-Z (Zaveri and Peters, 1999) Aerosol model MOSAIC 8 bins (Zaveri et al., 2008) density from the CTRL3 simulation (Sects. 3.2 and 5.1). The NOx lifetime was estimated to be 12 h on 11 July and 5 h on 12 July. The SO2 lifetime was not taken into account, consis- tent with the ﬁndings of Lee et al. (2011), who reported a life- time of ∼20 h over the mid-Atlantic during summer, which is signiﬁcantly longer than the ages of plumes measured dur- ing ACCESS. The FLEXPART-WRF output consists of par- ticle positions, each associated with a pollutant mass; these particles are mapped onto a 3-D output grid (600 m × 600 m, with 18 vertical levels between 0 and 1500 ma.s.l.) to derive ﬁelds of volume mixing ratios every minute. Since emissions are assumed to be constant with time, and since our simu- lations only take into account transport processes depending linearly on concentrations, the intensity of these mixing ratio ﬁelds also depend linearly on the emission strength chosen for the simulation. Therefore, the model results can be scaled a posteriori to represent any constant emission ﬂux value. 6 h. This WRF meteorological simulation is referred to as the MET simulation. Ship emissions are represented in the FLEXPART-WRF plume dispersion simulations as moving 2 m × 2 m × 2 m box sources, whose locations are updated every 10 s along the ship trajectory (routes shown in Fig. 1b). In all, 1000 particles are released every 10 s into these volume sources, representing a constant emission ﬂux with time of an inert tracer. During the ACCESS ﬂights, targeted ships were mov- ing at relatively constant speeds during the ∼3 h of the ﬂight, meaning that fuel consumption and emission ﬂuxes are likely to be constant during the ﬂights if environmental conditions (wind speed, waves, and currents) were not varying strongly. FLEXPART-WRF takes into account a simple exponential decay using a prescribed lifetime. L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions 2362 Figure 1. WRF and WRF-Chem domain (a) outer domains used for the MET, CTRL, and NOSHIP runs. ACCESS ﬂight tracks during 11, 12, 19a (a – denotes that this was the ﬁrst ﬂight that occurred on this day, ﬂight 19b – the second ﬂight was dedicated to hydrocarbon extraction facilities) and 25 July 2012 ﬂights are shown in color. (b) Inner domain used for the CTRL3 and NOSHIPS3 simulations, with the tracks of the four ships sampled during the 11 and 12 July 2012 ﬂights (routes extracted from the STEAM2 inventory). Figure 1. WRF and WRF-Chem domain (a) outer domains used for the MET, CTRL, and NOSHIP runs. ACCESS ﬂight tracks during 11, 12, 19a (a – denotes that this was the ﬁrst ﬂight that occurred on this day, ﬂight 19b – the second ﬂight was dedicated to hydrocarbon extraction facilities) and 25 July 2012 ﬂights are shown in color. (b) Inner domain used for the CTRL3 and NOSHIPS3 simulations, with the tracks of the four ships sampled during the 11 and 12 July 2012 ﬂights (routes extracted from the STEAM2 inventory). Table 2. Parameterizations and options used for the WRF and WRF-Chem simulations. Atmospheric process WRF-Chem option Planetary boundary layer MYNN (Nakanishi and Niino, 2006) Surface layer MM5 Similarity scheme, Carlson–Boland viscous sublayer (Zhang and Anthes, 1982; Carlson and Boland, 1978) Land surface Uniﬁed Noah land-surface model (Chen and Dudhia, 2001) Microphysics Morrison (Morrison et al., 2009) Shortwave radiation Goddard (Chou and Suarez, 1999) Longwave radiation RRTM (Mlawer et al., 1997) Cumulus parameterization Grell-3D (Grell and Devenyi, 2002) Photolysis Fast-J (Wild et al., 2000) Gas phase chemistry CBM-Z (Zaveri and Peters, 1999) Aerosol model MOSAIC 8 bins (Zaveri et al., 2008) Table 2. Parameterizations and options used for the WRF and WRF-Chem simulations Table 2. Parameterizations and options used for the WRF and WRF-Chem simulations. Table 2. Parameterizations and options used for the WRF and WRF-Chem simulations. 3.1 FLEXPART-WRF and WRF Plume dispersion simulations are performed with FLEXPART-WRF for the four ships presented in Ta- ble 1, in order to estimate their emissions of NOx and SO2. FLEXPART-WRF (Brioude et al., 2013) is a version of the Lagrangian particle dispersion model FLEXPART (Stohl et al., 2005), driven by meteorological ﬁelds from the mesoscale weather forecasting model WRF (Skamarock et al., 2008). In order to drive FLEXPART-WRF, a meteoro- logical simulation was performed with WRF version 3.5.1, from 4 to 25 July 2012, over the domain presented in Fig. 1a. The domain (15 km × 15 km horizontal resolution with 65 vertical eta levels between the surface and 50 hPa) covers most of northern Norway (∼62 to 75◦N) and includes the region of all ACCESS ﬂights focused on ship emissions. The ﬁrst week of the simulation (4 to 10 July included) is used for model spin-up. WRF options and parameterizations used in these simulations are shown in Table 2. Meteorological initial and boundary conditions are obtained from the FNL (abbreviation for “ﬁnal”) analysis from NCEP (National Centers for Environmental Prediction). The simulation is also nudged to FNL winds, temperature, and humidity every www.atmos-chem-phys.net/16/2359/2016/ Atmos. Chem. Phys., 16, 2359–2379, 2016 L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions Ship emissions can continue to rise after leaving the ex- haust, due to their vertical momentum and buoyancy. This was taken into account in the FLEXPART-WRF simula- tions by calculating effective injection heights for each tar- geted ship, using a simple plume rise model (Briggs, 1965). This model takes into account ambient temperature and wind speed, as well as the volume ﬂow rate and temperature at the ship exhaust, to calculate a plume injection height above the ship stack. Ambient temperature and wind speed val- ues at each ship’s position are obtained from the WRF sim- ulation. We use an average of measurements by Lyyranen et al. (1999) and Cooper (2001) for the exhaust tempera- ture of the four targeted ships (350 ◦C). The volume ﬂows at the exhaust are derived for each ship using CO2 emis- sions from the STEAM2 ship emission model (STEAM2 emissions described in Sect. 3.3). Speciﬁcally, CO2 emis- sions from STEAM2 for the four targeted ships are con- verted to an exhaust gas ﬂow based on the average com- position of ship exhaust gases measured by Cooper (2001) and Petzold et al. (2008). Average injection heights, includ- ing stack heights and plume rise, are found to be approxi- mately 230 m for the Costa Deliziosa, 50 m for the Wilson Nanjing, 30 m for the Wilson Leer, and 65 m for the Alaed. In order to estimate the sensitivity of plume dispersion to these calculated injection heights, two other simulations are per- formed for each ship, where injection heights are decreased and increased by 50 %. Details of the FLEXPART-WRF runs and how they are used to estimate emissions are presented in Sect. 4. vated aerosols, and wet scavenging within and below clouds. Aerosol activation changes the cloud droplet number concen- trations and cloud droplet radii in the Morrison microphysics scheme, thus inﬂuencing cloud optical properties (ﬁrst indi- rect aerosol effect). Aerosol activation in MOSAIC also in- ﬂuences cloud lifetime by changing precipitation rates (sec- ond indirect aerosol effect). Chemical initial and boundary conditions are taken from the global chemical-transport model MOZART-4 (model for ozone and related chemical tracers version 4; Emmons et al., 2010). In our simulations, the dry deposition routine for trace gases (Wesely, 1989) was modiﬁed to improve dry deposi- tion on snow, following the recommendations of Ahmadov et al. (2015). L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions The seasonal variation of dry deposition was also updated to include a more detailed dependence of dry deposition parameters on land use, latitude, and date, which was already in use in WRF-Chem for the MOZART-4 gas- phase mechanism. Anthropogenic emissions (except ships) are taken from the HTAPv2 (Hemispheric transport of air pollution version 2) inventory (0.1◦× 0.1◦resolution). Bulk VOCs are speciated for both shipping and anthropogenic emissions, based on Murrells et al. (2010). Ship VOC emis- sions are speciated using the “other transport” sector (trans- port emissions, excluding road transport) and anthropogenic VOC emissions are speciated using the average speciation for the remaining sectors. DMS emissions are calculated follow- ing the methodology of Nightingale et al. (2000) and Saltz- man et al. (1993). The oceanic concentration of DMS in the Norwegian Sea in July, taken from Lana et al. (2011), is 5.8×10−6 molm−3. Other biogenic emissions are calculated online by the MEGAN (Model of Emissions of Gases and Aerosols from Nature; Guenther et al., 2006) model within WRF-Chem. Sea salt emissions are also calculated online within WRF-Chem. L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions In our case, the lifetime of NOx relative to reaction with OH was estimated using results from WRF-Chem simulations presented in Sect. 3.2. Speciﬁcally, we use OH concentrations, temperature, and air www.atmos-chem-phys.net/16/2359/2016/ Atmos. Chem. Phys., 16, 2359–2379, 2016 2363 L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions 3.2 WRF-Chem In order to estimate the impacts of shipping on air quality and radiative effects in northern Norway, simulations are per- formed using the 3-D chemical transport model WRF-Chem (Weather Research and Forecasting model, including chem- istry, Grell et al., 2005; Fast et al., 2006). WRF-Chem has been used previously by Molders et al. (2010) to quantify the inﬂuence of ship emissions on air quality in southern Alaska. Table 2 summarizes all the WRF-Chem options and param- eterizations used in the present study, detailed brieﬂy below. The gas phase mechanism is the carbon bond mechanism, version Z (CBM-Z; Zaveri and Peters, 1999). The version of the mechanism used in this study includes dimethylsulﬁde (DMS) chemistry. Aerosols are represented by the 8 bin sec- tional MOSAIC (Model for Simulating Aerosol Interactions and Chemistry; Zaveri et al., 2008) mechanism. Aerosol op- tical properties are calculated by a Mie code within WRF- Chem, based on the simulated aerosol composition, con- centrations, and size distributions. These optical properties are linked with the radiation modules (aerosol direct effect), and this interaction also modiﬁes the modeled dynamics and can affect cloud formation (semi-direct effect). The sim- ulations also include cloud–aerosol interactions, represent- ing aerosol activation in clouds, aqueous chemistry for acti- The WRF-Chem simulations performed in this study are summarized in Table 3. The CTRL simulation uses the set- tings and emissions presented above, as well as ship emis- sions produced by the model STEAM2 (Sect. 3.3). The NO- SHIPS simulation is similar to CTRL, but does not include ship emissions. The NOSHIPS and CTRL simulations are carried out from 4 to 25 July 2012, over the 15 km × 15 km simulation domain presented in Fig. 1a. The CTRL3 and NO- SHIPS3 simulations are similar to CTRL and NOSHIPS, but are run on a smaller 3 km × 3 km resolution domain, shown in Fig. 1b, from 10 to 13 July 2012. The CTRL3 and NO- SHIPS3 simulations are not nudged to FNL and do not in- clude a subgrid parameterization for cumulus due to their high resolution. Boundary conditions for CTRL3 and NO- SHIPS3 are taken from the CTRL and NOSHIPS simulations (using one-way nesting within WRF-Chem) and are updated every hour. The CTRL and CTRL3 simulations are not nudged to the reanalysis ﬁelds in the boundary layer, in order to ob- tain a more realistic boundary layer structure. However, comparison with ACCESS meteorological measurements www.atmos-chem-phys.net/16/2359/2016/ www.atmos-chem-phys.net/16/2359/2016/ Atmos. Chem. Phys., 16, 2359–2379, 2016 2364 L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions Table 3. Description of WRF and WRF-Chem simulations. Name Description Period Remarks MET WRF meteorological simulation, 15 km × 15 km res- olution (d01) 4–25 July 2012 Nudged to FNL CTRL WRF-Chem simulation, HTAPv2 anthropogenic emissions, STEAM2 ship emissions, online MEGAN biogenic emissions, online DMS and sea salt emis- sions, 15 km × 15 km horizontal resolution (d01) 4–25 July 2012 Nudged to FNL in the free troposphere only NOSHIPS CTRL without STEAM2 emissions, 15 km × 15 km horizontal resolution (d01) 4–25 July 2012 Nudged to FNL in the free troposphere only CTRL3 CTRL setup and emissions, 3 km × 3 km horizontal resolution (d02) 10–12 July 2012 Boundary conditions from CTRL No nudging No cumulus parameterization NOSHIPS3 NOSHIPS setup and emissions, 3 km × 3 km hori- zontal resolution (d02) 10–12 July 2012 Boundary conditions from NOSHIPS No nudging No cumulus parameterization L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emission 2364 Table 3. Description of WRF and WRF-Chem simulations. shows that on 11 July 2012 this leads to an overestimation of marine boundary layer wind speeds (normalized mean bias = +38 %). Since wind speed is one of the most critical parameters in the FLEXPART-WRF simulations, we decided to drive FLEXPART-WRF with the MET simulation instead of using CTRL or CTRL3. In the MET simulation, results are also nudged to FNL in the boundary layer in order to reproduce wind speeds (normalized mean bias of +14 % on 11 July 2012). All CTRL, NOSHIPS, CTRL3, NOSHIPS3 and MET simulations agree well with meteorological mea- surements during the other ACCESS ship ﬂights. grid every 15 min for the CTRL3 simulation, and were re- gridded on the WRF-Chem simulation grids. Shipping emis- sions of NOx, SO2, black carbon, and organic carbon are presented in Fig. 2 for the 15 km × 15 km simulation do- main (emissions totals during the simulation period are indi- cated within the ﬁgure panels). For comparison, the HTAPv2 emissions (without shipping emissions) are also shown. Ship emissions are, on average, located in main shipping lanes along the Norwegian coastline. However, they also include less traveled routes, which are apparent closer to shore. www.atmos-chem-phys.net/16/2359/2016/ Other anthropogenic emissions are mainly located along the Nor- wegian coast (mostly in southern Norway) or farther inland and to the south in Sweden and Finland. Over the whole domain, NOx and OC emissions from shipping are approxi- mately one-third of total anthropogenic NOx and OC emis- sions, but represent a lower proportion of anthropogenic SO2 and BC emissions (5 and 10 %, respectively). However, other anthropogenic emissions are not co-located with shipping emissions, which represent an important source further north along the coast, as many ships are in transit between Euro- pean ports and Murmansk in Russia. Very strong SO2 emis- sions in Russia are included in the model domain (in the area highlighted in Fig. 2d), associated with smelting activities that occur on the Russian Kola Peninsula (Virkkula et al., 1997; Prank et al., 2010). The Kola Peninsula emissions rep- resent 79 % of the total HTAPv2 SO2 emissions in the do- main. 4 Ship emission evaluation In this section, emissions of NOx and SO2 are determined for the four ships sampled during ACCESS ﬂights (shown in Ta- ble 1). We compare airborne measurements in ship plumes and concentrations predicted by FLEXPART-WRF plume dispersion simulations. In order to derive emission ﬂuxes, good agreement between measured and modeled plume lo- cations is required (discussed in Sect. 4.1). The methods, derived emissions values for the four ships, and comparison with STEAM2 emissions are presented in Sect. 4.2. Figure 2. (a, c, e, g) STEAM2 ship emissions and (b, d, f, h) HTAPv2 anthropogenic emissions (without ships) of (a, b) NOx, (c, d) SO2, (e, f) BC, and (g, h) OC in kg km−2 over the CTRL and NOSHIPS WRF-Chem domain, during the simulation period (00:00 UTC 4 July 2012 to 00:00 UTC 26 July 2012). On panel (d), the location of the intense Kola Peninsula SO2 emissions is high- lighted by a gray box. The emissions totals for the simulation period are noted in each panel. L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions Figure 2. (a, c, e, g) STEAM2 ship emissions and (b, d, f, h) HTAPv2 anthropogenic emissions (without ships) of (a, b) NOx, (c, d) SO2, (e, f) BC, and (g, h) OC in kg km−2 over the CTRL and NOSHIPS WRF-Chem domain, during the simulation period (00:00 UTC 4 July 2012 to 00:00 UTC 26 July 2012). On panel (d), the location of the intense Kola Peninsula SO2 emissions is high- lighted by a gray box. The emissions totals for the simulation period are noted in each panel. treated in the same way together with all unidentiﬁed ships, using the exhaust parameters of the Wilson Leer and local meteorological conditions to estimate injection heights. This means that, for the Costa Deliziosa, Alaed and Wilson Nan- jing, the plume rise model is used in CTRL with exhaust parameters from a smaller ship (the Wilson Leer) than in CTRL3. Because of this, emission injection heights for these ships are lower in CTRL (0 to 30 m) than in CTRL3 (230 m for the Costa Deliziosa, 50 m for the Wilson Nanjing, 30 m for the Wilson Leer, and 65 m for the Alaed). Primary aerosol emissions from STEAM2 (BC, OC, SO4, and ash) are distributed into the eight MOSAIC aerosol bins in WRF-Chem, according to the mass size distribution mea- sured in the exhaust of ships equipped with medium-speed diesel engines by Lyyranen et al. (1999). The submicron mode of this measured distribution is used to distribute pri- mary BC, OC, and SO= 4, while the coarse mode is used to distribute exhaust ash particles (represented as “other inor- ganics” in MOSAIC). 3.3 High-resolution ship emissions from STEAM2 STEAM2 is a high-resolution, real-time bottom-up shipping emissions model based on AIS positioning data (Jalkanen et al., 2012). STEAM2 calculates fuel consumption for each ship based on its speed, engine type, fuel type, vessel length, and propeller type. The model can also take into account the effect of waves, and distinguishes ships at berth, maneuver- ing ships, and cruising ships. Contributions from weather ef- fects were not included in this study, however. The presence of AIS transmitters is mandatory for large ships (gross ton- nage > 300 t) and voluntary for smaller ships. Emissions from STEAM2 are compared with emissions derived from measurements for individual ships in Sect. 4. STEAM2 emissions of CO, NOx, OC, BC (technically ele- mental carbon in STEAM2), sulfur oxides (SOx), SO4, and exhaust ashes are also used in the WRF-Chem CTRL and CTRL3 simulations. SOx are emitted as SO2 in WRF-Chem, and NOx are emitted as 94 % NO, and 6 % NO2 (EPA, 2000). VOC emissions are estimated from STEAM2 CO emissions using a bulk VOC / CO mass ratio of 53.15 %, the ratio used in the Arctic ship inventory from Corbett et al. (2010). STEAM2 emissions were generated on a 5 km × 5 km grid every 30 min for the CTRL simulation, and on a 1 km × 1 km STEAM2 emissions are based on AIS signals that are transmitted to base stations on shore that have a limited range of 50–90 km, which explains why the emissions pre- sented in Fig. 2 only represent near-shore trafﬁc. In addi- tion, our study is focused on shipping emissions in north- ern Norway, therefore STEAM2 emissions were only gener- ated along the Norwegian coast. As a result, ship emissions in the northern Baltic and along the northwestern Russian coast are not included in this study. However, these missing www.atmos-chem-phys.net/16/2359/2016/ Atmos. Chem. Phys., 16, 2359–2379, 2016 2365 4.1 Ship plume representation in FLEXPART-WRF and comparison with airborne measurements FLEXPART-WRF plume dispersion simulations driven by the MET simulation are performed for the four ships sam- pled during ACCESS (Sect. 3.1). The MET simulation agrees well with airborne meteorological measurements on both days (shown in the Supplement, Fig. S1) in terms of wind direction (mean bias of −16◦on 11 July, +6◦on 12 July) and wind speed (normalized mean bias of +14 % on 11 July, −17 % on 12 July). Figure 3 shows the comparison between maps of the measured NOx and plume locations predicted by FLEXPART-WRF. This ﬁgure also shows the typical mean- dering pattern of the plane during ACCESS, measuring the same ship plumes several times as they age, while moving further away from the ship (Roiger et al., 2015). Wilson Leer and Costa Deliziosa plumes were sampled during two dif- ferent runs at two altitudes on 11 July 2012, and presented in Fig. 3a and b (z = 49 m) and Fig. 3c and d (z = 165 m). During the second altitude level on 11 July (Fig. 3c and d) the Wilson Leer was farther south and the Costa Deliziosa had moved further north. Therefore, the plumes are farther shipping emissions are much lower than other anthropogenic sources inside the model domain. In the CTRL and CTRL3 simulations, ship emissions are injected in altitude using the plume rise model presented in Sect. 3.1. Stack height and exhaust ﬂuxes are unknown for most of the ships present in the STEAM2 emissions, which were not speciﬁcally targeted during ACCESS. For these ships, exhaust parameters for the Wilson Leer (∼6000 gross tonnage) are used as a compro- mise between the smaller ﬁshing ships (∼40 % of Arctic shipping emissions; Winther et al., 2014), and larger ships like the ones targeted during ACCESS. In the CTRL3 sim- ulation, the four ships targeted during ACCESS are usually alone in a 3 km × 3 km grid cell, which enabled us to treat these ships separately and to inject their emissions in alti- tude using individual exhaust parameters (Sect. 3.1). In the CTRL simulation, there are usually several ships in the same 15 km × 15 km grid cell, and the four targeted ships were 4.2 Ship emission derivation and comparison with STEAM2 This last ship was identiﬁed during the post-campaign analysis, and we were able to ex- tract its location and emissions from the STEAM2 inventory in order to perform the plume dispersion simulations shown here. The NOx and FLEXPART-WRF predicted plume loca- tions are in good agreement for both ships. apart than during the ﬁrst pass at 49 m. Modeled and mea- sured plume locations agree well for the ﬁrst run (z = 49 m). For the second run (z = 165 m), the modeled plume for the Costa Deliziosa is, on average, located 4.7 km to the west of the measured plume. This displacement is small considering that, at the end of this ﬂight leg, the plume was being sampled ∼80 km away from its source. This displacement is caused by biases in the simulation (MET) used to drive the plume dispersion model (−16◦for wind direction, +14 % for wind speed). On 12 July 2012, the aircraft targeted emissions from the Wilson Nanjing ship (Fig. 3e and f), but also sampled the plume of another ship, the Alaed. This last ship was identiﬁed during the post-campaign analysis, and we were able to ex- tract its location and emissions from the STEAM2 inventory in order to perform the plume dispersion simulations shown here. The NOx and FLEXPART-WRF predicted plume loca- tions are in good agreement for both ships. apart than during the ﬁrst pass at 49 m. Modeled and mea- sured plume locations agree well for the ﬁrst run (z = 49 m). For the second run (z = 165 m), the modeled plume for the Costa Deliziosa is, on average, located 4.7 km to the west of the measured plume. This displacement is small considering that, at the end of this ﬂight leg, the plume was being sampled Ei = E × R tend i tbegin i (SO2(t) −SO2background)dt R tend i tbegin i Tracer(t)dt × MSO2 Mair (1) Ei = E × R tend i tbegin i (SO2(t) −SO2background)dt R tend i tbegin i Tracer(t)dt × MSO2 Mair (1) In Eq. L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions 2366 Figure 3. Left panels: ACCESS airborne NOx measurements be- tween (a) 16:00 and 16:35 UTC, 11 July 2012 (ﬂight leg at Z ∼ 49 m), (c) 16:52 and 18:08 UTC, 11 July 2012 (Z ∼165 m), and (e) 10:53 and 11:51 UTC, 12 July 2012 (Z ∼46 m). Right panels: corresponding FLEXPART-WRF plumes (relative air tracer mix- ing ratios): (b, d) Wilson Leer and Costa Deliziosa plumes and (f) Wilson Nanjing and Alaed plumes. FLEXPART-WRF plumes are shown for the closest model time step and vertical level. Modeled air tracer mixing ratios are interpolated in space and time to the aircraft location, and compared with airborne NOx and SO2 measurements (Fig. 4). Each peak in Fig. 4 corresponds to the aircraft crossing the ship plume once dur- ing the meandering pattern before turning around for an ad- ditional plume crossing. Figure 4a and b only show measure- ments for the ﬁrst altitude level at z = 49 m on 11 July 2012 (results for the second altitude level are shown in the Supple- ment in Fig. S2). As expected from the comparison shown in Fig. 3, modeled peaks are co-located with measured peaks in Fig. 4. The model is also able to reproduce the gradual de- crease of concentrations measured in the plume of the Wilson Nanjing on Fig. 4c–e, as the plane ﬂies further away from the ship and the plume gets more dispersed. These peak concen- trations vary less for the measured and modeled plume of the Costa Deliziosa (Fig. 4a and b). Measured plumes are less concentrated for the Wilson Leer since it is a smaller vessel, and for the Alaed because its emissions were sampled further away from their source. www.atmos-chem-phys.net/16/2359/2016/ www.atmos-chem-phys.net/16/2359/2016/ Atmos. Chem. Phys., 16, 2359–2379, 2016 4.2 Ship emission derivation and comparison with STEAM2 In this section, we describe the method for deriving ship emissions of NOx and SO2 using FLEXPART-WRF and measurements. This method relies on the fact that in the FLEXPART-WRF simulations presented in Sect. 3.1, there is a linear relationship between the constant emission ﬂux of the tracer chosen for the simulation and the tracer concentra- tions in the modeled plume. The only source of non-linearity that cannot be taken into account is changes in the emission source strength, which is assumed to be constant in time for the plumes sampled. Given that the ship and meteorologi- cal conditions were consistent during sampling (shown in the Supplement, Fig. S1), we expect that these effects would be very small. In our simulations, this constant emission ﬂux is picked at E = 0.1 kgs−1 and is identical for all ships. This initial value E is scaled for each ship by the ratio of the mea- sured and modeled areas of the peaks in concentration corre- sponding to plume crossings, as shown in Fig. 4. Equation (1) shows how SO2 emissions are derived by this method. Figure 3. Left panels: ACCESS airborne NOx measurements be- tween (a) 16:00 and 16:35 UTC, 11 July 2012 (ﬂight leg at Z ∼ 49 m), (c) 16:52 and 18:08 UTC, 11 July 2012 (Z ∼165 m), and (e) 10:53 and 11:51 UTC, 12 July 2012 (Z ∼46 m). Right panels: corresponding FLEXPART-WRF plumes (relative air tracer mix- ing ratios): (b, d) Wilson Leer and Costa Deliziosa plumes and (f) Wilson Nanjing and Alaed plumes. FLEXPART-WRF plumes are shown for the closest model time step and vertical level. apart than during the ﬁrst pass at 49 m. Modeled and mea- sured plume locations agree well for the ﬁrst run (z = 49 m). For the second run (z = 165 m), the modeled plume for the Costa Deliziosa is, on average, located 4.7 km to the west of the measured plume. This displacement is small considering that, at the end of this ﬂight leg, the plume was being sampled ∼80 km away from its source. This displacement is caused by biases in the simulation (MET) used to drive the plume dispersion model (−16◦for wind direction, +14 % for wind speed). On 12 July 2012, the aircraft targeted emissions from the Wilson Nanjing ship (Fig. 3e and f), but also sampled the plume of another ship, the Alaed. 4.2 Ship emission derivation and comparison with STEAM2 The number of peaks used to derive emis- sions for each ship is N = 13 for the Costa Deliziosa, N = 4 for the Wilson Leer, N = 8 for the Wilson Nanjing (N = 4 for SO2) and N = 5 for the Alaed. The derived emissions of NOx (equivalent NO2 mass ﬂux in kg day−1) and SO2 are given in Table 4. The emissions extracted from the STEAM2 inventory for the same ships during the same time period are also shown. STEAM2 SO2 emissions are higher than the value derived for the Costa Deliziosa, and lower than the value derived for the Wilson Nanjing. NOx emissions from STEAM2 are higher than our calculations for all ships. In STEAM2, the NOx emission fac- tor is assigned according to IMO MARPOL (marine pollu- tion) Annex VI requirements (IMO, 2008) and engine revolu- tions per minute (RPM), but all engines subject to these lim- its must emit less NOx than this required value. For the Wil- son Leer, two calculated values are reported: one calculated by averaging the estimates from the four measured peaks, and one value where an outlier value was removed before calculating the average. During the 11 July ﬂight, the Wilson Leer was traveling south at an average speed of 4.5 ms−1, with relatively slow tailwinds of 5.5 ms−1. Because of this, the dispersion of this ship’s plume on this day could be sensi- tive to small changes in modeled wind speeds, and calculated emissions are more uncertain. 4. (a, c, d) NOx and (b, e) SO2 aircraft measureme Figure 4. (a, c, d) NOx and (b, e) SO2 aircraft measurements Figure 4. (a, c, d) NOx and (b, e) SO2 aircraft measurements (black) compared to FLEXPART-WRF air tracer mixing ratios in- terpolated along ﬂight tracks, for the plumes of the (a, b) Costa Deliziosa and Wilson Leer on 11 July 2012 (ﬁrst constant altitude level (Z ∼49 m), also shown in Fig. 3a) and (c, d, e) Wilson Nan- jing and Alaed on 12 July 2012. Panel (d) shows the same results as panel (c) in detail. Since model results depend linearly on the emission ﬂux chosen a priori for each ship, model results have been scaled so that peak heights are comparable to the measurements. The most important difference between the inventory NOx and our estimates is ∼150 % for the Costa Deliziosa. 4.2 Ship emission derivation and comparison with STEAM2 (1), SO2(t) is the measured SO2 mixing ratio (pptv), SO2background is the background SO2 mixing ratio for each peak, Tracer(t) is the modeled tracer mixing ratio interpo- lated along the ACCESS ﬂight track (pptv), tbegin i and tend i are the beginning and end time of peak i (modeled or measured, in s) and MSO2 and Mair are the molar masses of SO2 and air (gmol−1). This method produces a different SO2 emis- sion ﬂux value Ei (kgs−1) for each of the i = 1 to N peaks corresponding to all the crossings of a single ship plume www.atmos-chem-phys.net/16/2359/2016/ Atmos. Chem. Phys., 16, 2359–2379, 2016 L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions 2367 Figure 4. (a, c, d) NOx and (b, e) SO2 aircraft measurements (black) compared to FLEXPART-WRF air tracer mixing ratios in- terpolated along ﬂight tracks, for the plumes of the (a, b) Costa Deliziosa and Wilson Leer on 11 July 2012 (ﬁrst constant altitude level (Z ∼49 m), also shown in Fig. 3a) and (c, d, e) Wilson Nan- jing and Alaed on 12 July 2012. Panel (d) shows the same results as panel (c) in detail. Since model results depend linearly on the emission ﬂux chosen a priori for each ship, model results have been scaled so that peak heights are comparable to the measurements. heights if the peak area in tracer concentration changes by more than 50 % in the injection height sensitivity runs. Us- ing a lower threshold of 25 % alters the ﬁnal emission es- timates by less than 6 %. Peaks sensitive to the calculated injection height typically correspond to samplings close to the ship, where the plumes are narrow. An intense SO2 peak most likely associated with the Costa Deliziosa and sampled around 17:25 UTC on 11 July 2012 is also excluded from the calculations, because this large increase in SO2 in an older, diluted part of the ship plume suggests contamination from another source. SO2 emissions are not determined for the Wilson Leer and the Alaed, since SO2 measurements in their plumes are too low to be distinguished from the background variability. For the same reason, only the higher SO2 peaks (four peaks > 1 ppbv) were used to derive emissions for the Wilson Nanjing. 4.2 Ship emission derivation and comparison with STEAM2 Rea- sons for large discrepancy in predicted and measured NOx emissions of Costa Deliziosa were investigated in more de- tail. A complete technical description of Costa Deliziosa was not available, but her sister vessel Costa Luminosa was described at length recently (RINA, 2010). The details of Costa Luminosa and Costa Deliziosa are practically identi- cal and allow for in-depth analysis of emission modeling. With complete technical data, the STEAM2 SOx and NOx emissions of Costa Deliziosa were estimated to be 2684 and 5243 kg day−1, respectively, whereas our derived estimates indicate 2399 and 2728 kg day−1 (difference of +12 % for SOx and +92 % for NOx). The good agreement for SOx in- dicates that the power prediction at vessel speed reported in by the aircraft. These N different estimates are averaged to- gether to reduce the uncertainty in the estimated SO2 emis- sions. A similar approach is used to estimate NOx emissions. The background mixing ratios were determined by applying a 30 s running average to the SO2 and NOx measurements. Background values were then determined manually from the ﬁltered time series. For each NOx peak, an individual back- ground value was identiﬁed and used to determine the NOx enhancement for the same plume. For SO2, a single back- ground value was used for each ﬂight leg (constant altitude). In order to reduce sensitivity to the calculated emission injection heights, FLEXPART-WRF peaks that are sensitive to a ±50 % change in injection height are excluded from the analysis. Results are considered sensitive to injection www.atmos-chem-phys.net/16/2359/2016/ 4.3 Comparison of STEAM2 to other shipping emission inventories for northern Norway We compare in Table 5 the July emission totals for NOx, SO2, BC, OC and SO= 4 in northern Norway (latitudes 60.6 to 73◦N, longitudes 0 to 31◦W) for STEAM2 and four other shipping emission inventories used in previous studies inves- tigating shipping impacts in the Arctic. We include emissions from the Winther et al. (2014), Dalsøren et al. (2009, 2007), and Corbett et al. (2010) inventories. The highest shipping emissions in the region of northern Norway are found in the STEAM2 and Winther et al. (2014) inventories, which are both based on 2012 AIS ship activity data (Sect. 3.3 for a de- scription of the methodology used for STEAM2). We note that, except for OC, the emissions are higher in the Winther et al. (2014) inventory because of the larger geographical coverage: Winther et al. (2014) used both ground-based and satellite retrieved AIS signals, whereas the current study is restricted to data received by ground based AIS stations (cap- turing ships within 50 to 90 km of the Norwegian coastline). Despite lower coverage, the horizontal and temporal resolu- tions are better described in land-based AIS networks than satellite AIS data. The terrestrial AIS data used in this study is thus more comparable to the spatial extent and temporal resolution of the measurements collected close to the Nor- wegian coast. STEAM2 is the only inventory including sul- fate emissions, which account for SO2 to SO= 4 conversion in the ship exhaust. Ship emissions from Dalsøren et al. (2009) and Corbett et al. (2010) are based on ship activity data from 2004, when marine trafﬁc was lower than in 2012. Further- more, the gridded inventory from Corbett et al. (2010) does not include emissions from ﬁshing ships, which represent close to 40 % of Arctic shipping emissions (Winther et al., 2014). These emissions could not be precisely distributed geospatially using earlier methodologies, since ﬁshing ships do not typically follow a simple course (Corbett et al., 2010). Dalsøren et al. (2007) emissions for coastal shipping in Nor- p g p g The case of Costa Deliziosa underlines the need for accu- rate and up-to-date technical data for ships when bottom-up emission inventories are constructed. It also necessitates the inclusion of the effect of emission abatement technologies in ship emission inventories. Furthermore, model predictions for individual vessels are complicated by external contribu- tions, like weather and sea currents, affecting vessel perfor- mance. L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions Table 4. NOx and SO2 emissions estimated from FLEXPART-WRF and ACCESS measurements, compared with STEAM2 emissions. Values in parentheses indicate the relative difference between STEAM2 and calculated values. SO2 emissions were not calculated for the Wilson Leer and Alaed since the measured SO2 concentrations in the plumes were too low above background. Ship name NOx calculated NOx from STEAM2 SO2 calculated SOx from STEAM2 from measurements (kg day−1) from measurements (kg day−1) (kg day−1) (kg day−1) Costa Deliziosa 2728 6767/5243a (+148/+92 %a) 2399 3285/2684a (+37/+12 %a) Wilson Leer 167/82b 287 (+72/+250 %b) NA 88 (NA) Wilson Nanjing 561 602 (+7 %) 504 219 (−57 %) Alaed 1362 1809 (+33 %) NA 1130 (NA) a The second value corresponds to STEAM2 calculations using complete technical data from the Costa Deliziosa sister ship Costa Luminosa. b Value with outliers removed. on average in the Norwegian Sea during ACCESS than for individual ships. AIS and associated fuel ﬂow is well predicted by STEAM2, but emissions of NOx are twice as high as the value derived from measurements. In case of Costa Deliziosa, the NOx emission factor of 10.5 gkWh−1 for a tier II compliant ves- sel with 500 RPM engine is assumed by STEAM2. Based on the measurement-derived value, a NOx emission factor of 5.5 gkWh−1 would be necessary, which is well below the tier II requirements. It was reported recently (IPCO, 2015) that NOx emission reduction technology was installed on Costa Deliziosa, but it is unclear whether this technology was in place during the airborne measurement campaign in 2012. www.atmos-chem-phys.net/16/2359/2016/ Atmos. Chem. Phys., 16, 2359–2379, 2016 2368 5 Modeling the impacts of ship emissions along the Norwegian coast In this section, WRF-Chem, using STEAM2 ship emissions, is employed to study the inﬂuence of ship pollution on at- mospheric composition along the Norwegian coast, at both the local (i.e., at the plume scale) and regional scale. As shown in Fig. 4, shipping pollution measured during AC- CESS is inhomogeneous, with sharp NOx and SO2 peaks in thin ship plumes, emitted into relatively clean background concentrations. The measured concentrations are on spa- tial scales that can only be reproduced using very high- resolution WRF-Chem simulations (a few kilometers of hor- izontal resolution), but such simulations can only be per- formed for short periods and over small domains. Therefore, high-resolution simulations cannot be used to estimate the regional impacts of shipping emissions. In order to bridge the scale between measurements and model runs that can be used to make conclusions about the regional impacts of ship- ping pollution, we compare in Sect. 5.1 WRF-Chem simu- lations using STEAM2 ship emissions, at 3 km × 3 km res- olution (CTRL3) and at 15 km × 15 km resolution (CTRL). Speciﬁcally, we show in Sect. 5.1 that both the CTRL3 and CTRL simulations reproduce the average regional inﬂuence of ships on NOx, O3, and SO2, compared to ACCESS mea- surements. In Sect. 5.2 we use the CTRL simulation to quan- tify the regional contribution of ships to surface pollution and shortwave radiative ﬂuxes in northern Norway. WRF-Chem results from CTRL and CTRL3 for surface (∼0 to 30 m) NOx and O3 are shown in Fig. 5. On 11 and 12 July, the aircraft speciﬁcally targeted plumes from the Wil- son Leer, Costa Deliziosa, Wilson Nanjing and, in addition, sampled emissions from the Alaed, identiﬁed later during the post-campaign analysis (Fig. 3). All these ships are individ- ually present in the STEAM2 emissions inventory (Sect. 4 and Table 4). Emissions from these ships, as well as from other vessels traveling in that area, are clearly resolved in the CTRL3 model results for NOx (Fig. 5a and e). Ship NOx emissions are smoothed out in the CTRL run, seen in Fig. 5b and f, and the individual ship plumes cannot be clearly dis- tinguished in the NOx surface concentrations. The predicted surface O3 concentrations are shown in Fig. 5c, d, g, and h. On the 11 and 12 July 2012, titration of O3 by NOx from fresh ship emissions can be identiﬁed in Fig. L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions 2369 Table 5. July emission totals in northern Norway (60.6–73◦N, 0 to 31◦W) of NOx, SO2, BC, OC, and SO= 4 in different ship emission inventories. Table 5. July emission totals in northern Norway (60.6–73◦N, 0 to 31◦W) of NOx, SO2, BC, OC, and SO= 4 in different ship emission inventories ls in northern Norway (60.6–73◦N, 0 to 31◦W) of NOx, SO2, BC, OC, and SO= 4 in different ship emissio Table 5. July emission totals in northern Norway (60.6–73◦N, 0 to 31◦W) of NOx, SO2, BC, OC, and SO= 4 in different ship emission inventories. Inventory Year NOx (kt) SO2 (kt) BC (t) OC (t) SO= 4 (t) STEAM2 2012 7.1 2.4 48.1 123.4 197.3 Winther et al. (2014) 2012 9.3 3.4 47.7 82.9 – Dalsøren et al. (2009) 2004 3.1 1.9 7.3 24.5 – Corbett et al. (2010) 2004 2.4 1.6 10.6 32.5 – Dalsøren et al. (2007) 2000 5.5 1.1 24. 479.3 – 5.1 Model evaluation from the plume scale to the regional scale wegian waters are estimated based on Norwegian shipping statistics for the year 2000, and contain higher NOx, BC, and OC emissions, but less SO2, than the 2004 inventories. This comparison indicates that earlier ship emission inven- tories usually contain lower emissions in this region, which can be explained by the current growth in shipping trafﬁc in northern Norway. This means that up-to-date emissions are required in order to assess the current impacts of shipping in this region. It is well known that ship plumes contain ﬁne-scale features that cannot be captured by most regional or global chem- ical transport models. This ﬁne plume structure inﬂuences the processing of ship emissions, including O3 and aerosol formation, which are non-linear processes that largely de- pend on the concentration of species inside the plume. Some models take into account the inﬂuence of the instantaneous mixing of ship emissions in the model grid box by includ- ing corrections to the O3 production and destruction rates (Huszar et al., 2010) or take into account plume ageing be- fore dilution by using corrections based on plume chemistry models (Vinken et al., 2011). Here, we take an alternative approach by running the model at a sufﬁcient resolution to distinguish individual ships in the Norwegian Sea (CTRL3 run at 3 km × 3 km resolution), and at a lower resolution (CTRL run at 15 km × 15 km resolution). It is clear that a 3 km × 3 km horizontal resolution is not sufﬁciently small to capture all small-scale plume processes. However, by com- paring the CTRL3 simulation to ACCESS measurements, we show in this section that this resolution is sufﬁcient to resolve individual ship plumes and to reproduce some of the plume macroscopic properties. The CTRL and CTRL3 simulations (presented in Table 3) are then compared to evaluate if non- linear effects are important for this study period and region. 4.3 Comparison of STEAM2 to other shipping emission inventories for northern Norway However, the STEAM2 emission model is based on AIS real-time positioning data, which has a much better cov- erage than activity data sets used to generate older shipping emission inventories (e.g., COADS and AMVER). These earlier data sets also have known biases for ships of speciﬁc sizes or types. In addition, components of the STEAM2 in- ventory, such as fuel consumption, engine loads, and emis- sion factors have already been studied in detail in the Baltic Sea by Jalkanen et al. (2009, 2012) and Beecken et al. (2015). Beecken et al. (2015) compared STEAM2 emission factors to measurements for ∼300 ships in the Baltic Sea. Their re- sults showed that, while important biases were possible for individual ships, STEAM2 performed much better on aver- age for a large ﬂeet. In the Baltic Sea, STEAM2 NOx emis- sion factors were found to be biased by +4 % for passen- ger ships, based on 29 ships, and −11 % for cargo ships, based on 118 ships. For SOx, the biases were respectively +1 and +14 % for the same ships. Therefore, we expect that the large discrepancy in NOx for one individual ship (the Costa Deliziosa) has only a small impact on the total regional emis- sions generated by STEAM2. The results presented later in Sect. 5.1 also indicate that STEAM2 likely performs better www.atmos-chem-phys.net/16/2359/2016/ Atmos. Chem. Phys., 16, 2359–2379, 2016 5 Modeling the impacts of ship emissions along the Norwegian coast 5c and g for the 3 km run (areas indicated by black arrows on Fig. 5c www.atmos-chem-phys.net/16/2359/2016/ Atmos. Chem. Phys., 16, 2359–2379, 2016 L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions 2370 Figure 5. Snapshots of model predicted surface NOx and O3 from the CTRL3 (3 km) simulation (a, c, e, g) and the CTRL (15 km) simula- tion (b, d, f, h) during the ﬂights on 11 and 12 July 2012. Model results for the CTRL3 simulation are shown over the full model domain. CTRL run results are shown over the same region for comparison. The aircraft ﬂight tracks are indicated in blue. On panels (c) and (g), black arrows indicate several areas of O3 titration due to high NOx from ships. (i, j) NOx and (k, l) O3 2-day average surface enhancements (00:00 UTC 11 July 2012 to 00:00 UTC 13 July 2012) due to shipping emissions, (i, k) CTRL3 simulation, (j, l) CTRL simulation. The 2-day average enhancements of NOx and O3 over the whole area are given below each respective panel. Figure 5. Snapshots of model predicted surface NOx and O3 from the CTRL3 (3 km) simulation (a, c, e, g) and the CTRL (15 km) simula- tion (b, d, f, h) during the ﬂights on 11 and 12 July 2012. Model results for the CTRL3 simulation are shown over the full model domain. CTRL run results are shown over the same region for comparison. The aircraft ﬂight tracks are indicated in blue. On panels (c) and (g), black arrows indicate several areas of O3 titration due to high NOx from ships. (i, j) NOx and (k, l) O3 2-day average surface enhancements (00:00 UTC 11 July 2012 to 00:00 UTC 13 July 2012) due to shipping emissions, (i, k) CTRL3 simulation, (j, l) CTRL simulation. The 2-day average enhancements of NOx and O3 over the whole area are given below each respective panel. To further investigate the ability of these different model runs to represent single ship plumes, we compare measured NOx, SO2, and O3 along the ﬂight track on 11 July 2012 with WRF-Chem predictions (Fig. 6). Corresponding results for 12 July 2012 are shown in the Supplement (Fig. S3). Large enhancements of NOx and SO2 are seen during plume crossings in measurements, as already noted in Sect. 4. 5 Modeling the impacts of ship emissions along the Norwegian coast For comparison with WRF-Chem, we have averaged the mea- sured data using a 56 s running average, equivalent to the aircraft crossing 6 km (two model grid cells) at its average speed during this ﬂight (107 ms−1). Using a running aver- age takes into account plume dilution in grid cells, as well as additional smoothing introduced when modeled results are spatially interpolated onto the ﬂight track. The CTRL3 sim- ulation captures both the width and magnitude of NOx and SO2 peaks, suggesting that the individual plumes are cor- rectly represented in space and time. During the second part of the ﬂight (17:20 UTC), the model does not reproduce two intense measured SO2 peaks. We already noted in Sect. 4.2 that measurements in this part of the ﬂight might be contami- nated by another source. In contrast, the CTRL run has wider NOx and SO2 peaks and lower peak concentrations, because of dilution in larger grids. Another difference between the simulations is the treatment of plume rise (Sect. 3.3), such and g). However, evidence for O3 titration quickly disap- pears away from the fresh emissions sources. In contrast, O3 titration is not apparent in the CTRL run. However, NOx and O3 patterns and average surface concentrations are very similar. This is illustrated in the lower panels, showing 2- day-averaged NOx and O3 enhancements due to ships in the CTRL3 (CTRL3 – NOSHIPS3) and CTRL (CTRL – NO- SHIPS) simulations. The results show that changing the hor- izontal resolution from 3 km × 3 km (1 km × 1 km emissions, 15 min emissions injection) to 15 km × 15 km (5 km × 5 km emissions, 1 h emissions injection) does not have a large in- ﬂuence on the domain-wide average NOx (−3.2 %) or O3 (+0.08 ppbv, +4.2 %) enhancements due to ships. This is in agreement with earlier results by Cohan et al. (2006), who showed that regional model simulations at similar resolu- tions (12 km) were sufﬁcient to reproduce the average O3 response. Results by Vinken et al. (2011) suggest that sim- ulations at a lower resolution more typical of global models (2◦× 2.5◦) would lead to an overestimation of O3 produc- tion from ships in this region by 1 to 2 ppbv. L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions After the ﬁrst run at 49 m, a vertical proﬁle was performed (16:35 to 16:45 UTC) providing information about the vertical structure of the boundary layer. Figure 7. Observed background-corrected PM1 enhancements in the plume of the Costa Deliziosa on 11 July 2012 (black squares), compared to modeled PM1 enhancements in ship plumes (in red), extracted along the ﬂight track (CTRL3 – NOSHIPS3 PM1). A 56 s averaging window is applied to the measured data to simulate dilu- tion in the model grid. Flight altitude is given as dashed black line. Figure 7. Observed background-corrected PM1 enhancements in the plume of the Costa Deliziosa on 11 July 2012 (black squares), compared to modeled PM1 enhancements in ship plumes (in red), extracted along the ﬂight track (CTRL3 – NOSHIPS3 PM1). A 56 s averaging window is applied to the measured data to simulate dilu- tion in the model grid. Flight altitude is given as dashed black line. (shown in the Supplement, Fig. S3), with lower model biases for O3 but a stronger overestimation of NOx. Figure 6. Time series of measured O3 and NOx on 11 July 2012 compared to model results extracted along the ﬂight track for the CTRL and CTRL3 runs. Observations are in black, the CTRL run is in red, and the CTRL3 run is in green. A 56 s averaging window is applied to the measured data for model comparison (approximately the time for the aircraft to travel 2 × 3 km). Flight altitude is given as a dashed gray line. After the ﬁrst run at 49 m, a vertical proﬁle was performed (16:35 to 16:45 UTC) providing information about the vertical structure of the boundary layer. In order to evaluate modeled aerosols in ship plumes, mod- eled aerosols are evaluated using size distributions measured during the 11 July 2012 ﬂight. Size distributions are inte- grated to estimate submicron aerosol mass (PM1), assuming a density of 1700 kgm−3 and spherical particles. This indi- cates that observed PM1 enhancements in plumes (∼0.1 to 0.5 µgm−3) are relatively low compared to background PM1 (∼0.7 to 1.1 µgm−3), because of the presence of high sea salt concentrations in the marine boundary layer (54 % of the modeled background PM1 during ship plume sampling is sea salt in NOSHIPS3). L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions 2371 L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions 2371 Figure 6. Time series of measured O3 and NOx on 11 July 2012 compared to model results extracted along the ﬂight track for the CTRL and CTRL3 runs. Observations are in black, the CTRL run is in red, and the CTRL3 run is in green. A 56 s averaging window is applied to the measured data for model comparison (approximately the time for the aircraft to travel 2 × 3 km). Flight altitude is given as a dashed gray line. After the ﬁrst run at 49 m, a vertical proﬁle was performed (16:35 to 16:45 UTC) providing information about the vertical structure of the boundary layer. Figure 7. Observed background-corrected PM1 enhancements in the plume of the Costa Deliziosa on 11 July 2012 (black squares), compared to modeled PM1 enhancements in ship plumes (in red), extracted along the ﬂight track (CTRL3 – NOSHIPS3 PM1). A 56 s averaging window is applied to the measured data to simulate dilu- tion in the model grid. Flight altitude is given as dashed black line. (shown in the Supplement, Fig. S3), with lower model biases for O3 but a stronger overestimation of NOx. In order to evaluate modeled aerosols in ship plumes, mod- eled aerosols are evaluated using size distributions measured during the 11 July 2012 ﬂight. Size distributions are inte- grated to estimate submicron aerosol mass (PM1), assuming a density of 1700 kgm−3 and spherical particles. This indi- cates that observed PM1 enhancements in plumes (∼0.1 to 0.5 µgm−3) are relatively low compared to background PM1 (∼0.7 to 1.1 µgm−3), because of the presence of high sea salt concentrations in the marine bo ndar la er (54 % of the L. Marelle et al.: Air quality and radiative impacts of Arct Figure 6. Time series of measured O3 and NOx on 11 July 2012 compared to model results extracted along the ﬂight track for the CTRL and CTRL3 runs. Observations are in black, the CTRL run is in red, and the CTRL3 run is in green. A 56 s averaging window is applied to the measured data for model comparison (approximately the time for the aircraft to travel 2 × 3 km). Flight altitude is given as a dashed gray line. L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions Because of this, comparing modeled and observed in-plume PM1 directly would be mostly represen- tative of background aerosols, especially sea salt, which is not the focus of this paper. Figure 7 shows the comparison between modeled and measured enhancements in PM1 in the plume of the Costa Deliziosa (11 July 2012), removing from the model and measurements the contribution from sea salt and other aerosols not associated with shipping. Similarly to Fig. 6, a 56 s moving average was applied to the measure- ment (representing plume dilution in the model grid). This comparison indicates a generally good agreement between modeled and measured PM1 enhancements in ship plumes. There is a discrepancy between the model and the measure- ments for the ﬁrst two PM1 plumes measured close to the ships (around 16:05 UTC), which could be an artifact of the limited resolution of this simulation (3 km). If these peaks are excluded, the model slightly overestimates peak PM1 en- hancements in ship plumes (+26 %). Since this enhancement is modeled as 80 % SO= 4, this overestimation can be linked to the +37 % overestimation of SO2 emissions for the Costa Deliziosa in STEAM2 (Table 4). that the Costa Deliziosa plume is located at lower altitudes in CTRL than in CTRL3. The CTRL3 simulation tends to overestimate NOx in ship plumes, which is in agreement with the results shown in Table 4, indicating that STEAM2 NOx emissions are overestimated for the ships targeted dur- ing ACCESS. This overestimation is unlikely to be caused by chemistry issues, since an overestimated NOx lifetime would lead to comparatively larger biases at the end of the constant altitude runs, when older parts of the plume were sampled. Figure 6b shows O3 during the same ﬂight. The CTRL3 simulation reproduces the ozone variability better than the CTRL run, but both runs perform relatively well on average (mean bias = −3 ppbv during the constant altitude legs). This negative bias is due to a small underestimation in the background ozone, which could be caused by a num- ber of reasons, including the boundary chemical conditions from the MOZART4 model, photolysis rates, cloud proper- ties and locations, ozone deposition, and/or emissions. Both measurements and CTRL3 results show evidence of O3 titra- tion in the most concentrated NOx plumes, where ozone is 1.5 to 3 ppbv lower than out of the plumes. L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions However, precise quantiﬁcation of this titration is difﬁcult because these val- ues are the same order of magnitude as the spatial variability of O3 outside of the plumes. O3 titration is not apparent in the CTRL run. Results are similar for the 12 July 2012 ﬂight Analysis of O3 maps, average surface enhancements due to ships (Fig. 5) and analysis of model results along ﬂight tracks (Fig. 6) show that both runs capture the NOx and O3 concentrations in this region reasonably well. Further- www.atmos-chem-phys.net/16/2359/2016/ 5 Modeling the impacts of ship emissions along the Norwegian coast The inﬂuence of model resolution on surface aerosol concentrations is also moderate, and PM10 due to ships are 15 % lower on average in CTRL than in CTRL3 (not shown here). and g). However, evidence for O3 titration quickly disap- pears away from the fresh emissions sources. In contrast, O3 titration is not apparent in the CTRL run. However, NOx and O3 patterns and average surface concentrations are very similar. This is illustrated in the lower panels, showing 2- day-averaged NOx and O3 enhancements due to ships in the CTRL3 (CTRL3 – NOSHIPS3) and CTRL (CTRL – NO- SHIPS) simulations. The results show that changing the hor- izontal resolution from 3 km × 3 km (1 km × 1 km emissions, 15 min emissions injection) to 15 km × 15 km (5 km × 5 km emissions, 1 h emissions injection) does not have a large in- ﬂuence on the domain-wide average NOx (−3.2 %) or O3 (+0.08 ppbv, +4.2 %) enhancements due to ships. This is in agreement with earlier results by Cohan et al. (2006), who showed that regional model simulations at similar resolu- tions (12 km) were sufﬁcient to reproduce the average O3 response. Results by Vinken et al. (2011) suggest that sim- ulations at a lower resolution more typical of global models (2◦× 2.5◦) would lead to an overestimation of O3 produc- tion from ships in this region by 1 to 2 ppbv. The inﬂuence of model resolution on surface aerosol concentrations is also moderate, and PM10 due to ships are 15 % lower on average in CTRL than in CTRL3 (not shown here). www.atmos-chem-phys.net/16/2359/2016/ Atmos. Chem. Phys., 16, 2359–2379, 2016 L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions Atmos. Chem. Phys., 16, 2359–2379, 2016 www.atmos-chem-phys.net/16/2359/2016/ Atmos. Chem. Phys., 16, 2359–2379, 2016 2372 L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions Figure 8. Average vertical proﬁles of (a) NOx, (b) SO2, (c) O3 and (d) PM2.5 observed during the four ACCESS ship ﬂights (in black, with error bars showing standard deviations), and interpolated along the ACCESS ﬂight tracks in the CTRL simulation (red line) and in the NOSHIPS simulation (blue line). For PM2.5 only simulation results are shown. L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions 2372 Figure 8. Average vertical proﬁles of (a) NOx, (b) SO2, (c) O3 and (d) PM2.5 observed during the four ACCESS ship ﬂights (in black, with error bars showing standard deviations), and interpolated along the ACCESS ﬂight tracks in the CTRL simulation (red line) and in the NOSHIPS simulation (blue line). For PM2.5 only simulation results are shown. typically activated as cloud condensation nuclei (shown in Fig. S4 in the Supplement). more, Fig. 7 shows that PM1 enhancements in ship plumes are well reproduced in the CTRL3 simulation, and we found that PM10 production from ships over the simulation domain was not very sensitive to resolution. This suggests that the CTRL simulation is sufﬁcient to assess the impacts of ship emissions at a larger scale during July 2012. This is inves- tigated further by comparing modeled NOx, SO2, and O3 in the CTRL and NOSHIPS simulations with the average ver- tical proﬁles (200–1500 m) measured during four ACCESS ﬂights from 11 to 25 July 2012 (ﬂights shown in Fig. 1a); this comparison is shown in Fig. 8. Modeled vertical proﬁles of PM2.5 are also shown in Fig. 8. This comparison allows us to estimate how well CTRL represents the average impact of shipping over a larger area and a longer period. NOx concentrations are overestimated in the parts of the proﬁle strongly inﬂuenced by shipping emissions. This is in agreement with the ﬁndings of Sect. 4.2, showing that STEAM2 NOx emissions were overestimated for the ships sampled during ACCESS. However, the CTRL simulation performs well on average, suggesting that the STEAM2 in- ventory is able to represent the average NOx emissions from ships along the northern Norwegian coast during the study period. The bias for SO2 is very low compared to results from Eyring et al. www.atmos-chem-phys.net/16/2359/2016/ (2007), which showed that global models signif- icantly underestimated SO2 in the polluted marine boundary layer in July. Since aerosols from ships contain mostly sec- ondary sulfate formed from SO2 oxidation, the validation of modeled SO2 presented in Fig. 8 also gives some conﬁdence in our aerosol results compared to earlier studies investigat- ing the air quality and radiative impacts of shipping aerosols. We therefore use the 15 km × 15 km CTRL run for further analysis of the regional inﬂuence of ships on pollution and the shortwave radiative effect in this region in Sect. 5.2. Figure 8 shows that the NOSHIPS simulation signiﬁcantly underestimates NOx and SO2, and moderately underesti- mates O3 along the ACCESS ﬂights, indicating that ship emissions are needed to improve the agreement between the model and observations. In the CTRL simulation, NOx, SO2, and O3 vertical structure and concentrations are generally well reproduced, with normalized mean biases of +14.2, −6.8, and −7.0 %, respectively. Correlations between mod- eled (CTRL) and measured proﬁles are signiﬁcant for NOx and O3 (r2 = 0.82 and 0.90). However, the correlation is very low between measured and modeled SO2 (r2 = 0.02), and it is not improved compared to the NOSHIPS simulation. Ships have the largest inﬂuence on NOx and SO2 proﬁles, a moder- ate inﬂuence on O3 and do not strongly inﬂuence PM2.5 pro- ﬁles along the ACCESS ﬂights. However, this small increase in PM2.5 corresponds to a larger relative increase in sulfate concentrations and in particle numbers in the size ranges 5.2.1 Surface air pollution from ship emissions in northern Norway The regional-scale impacts of ships on surface atmospheric composition in northern Norway are estimated by calculat- ing the 15-day (00:00 UTC, 11 July 2012 to 00:00 UTC, 26 July 2012) average difference between the CTRL and NO- SHIPS simulations. Figure 9 shows maps of these anoma- Atmos. Chem. Phys., 16, 2359–2379, 2016 www.atmos-chem-phys.net/16/2359/2016/ L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions 2373 L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions 2373 Figure 9. 15-day average (00:00 UTC 11 July 2012 to 00:00 UTC 26 July 2012) of (top) absolute and (bottom) relative surface enhancements (CTRL – NOSHIPS) in (a, d) SO2, (b, e) NOx, and (c, e) O3 due to ship emissions in northern Norway from STEAM2. Figure 9. 15-day average (00:00 UTC 11 July 2012 to 00:00 UTC 26 July 2012) of (top) absolute and (bottom) relative surface enhancements (CTRL – NOSHIPS) in (a, d) SO2, (b, e) NOx, and (c, e) O3 due to ship emissions in northern Norway from STEAM2. lies at the surface, for SO2, NOx, and O3. Ship emissions have the largest inﬂuence on surface NOx and SO2 concen- trations, with 75 to 100 % increases along the coast. Average O3 increases from shipping are ∼6 % (∼1.5 ppbv) in the coastal regions, with slightly lower enhancements (∼1 ppbv, ∼4 %,) further inland over Sweden. lies at the surface, for SO2, NOx, and O3. Ship emissions have the largest inﬂuence on surface NOx and SO2 concen- trations, with 75 to 100 % increases along the coast. Average O3 increases from shipping are ∼6 % (∼1.5 ppbv) in the coastal regions, with slightly lower enhancements (∼1 ppbv, ∼4 %,) further inland over Sweden. lutions. For example Vinken et al. (2011) estimated that in- stant dilution of shipping NOx emissions in 2◦× 2.5◦model grids leads to a 1 to 2 ppbv overestimation in ozone in the Norwegian and Barents seas during July 2005. This effect could explain a large part of the difference in O3 enhance- ments from shipping between the simulations of Ødemark et al. (2012) (2.8◦× 2.8◦resolution) and the simulations pre- sented in this paper (15 km × 15 km resolution). Dalsøren et al. 5.2.1 Surface air pollution from ship emissions in northern Norway (2007) studied the impact of maritime traf- ﬁc in northern Norway in the summer using ship emission estimates for the year 2000. They found, for July 2000, a 1 to 1.5 % increase in surface O3 from coastal shipping in Nor- wegian waters. However, unlike the present study, the esti- mate of Dalsøren et al. (2007) did not include the impact of international transit shipping along the Norwegian coast. Our estimated impact on O3 in this region (6 % and 1.5 ppbv increase) is about half of the one determined by Ødemark et al. (2012) (12 % and 3 ppbv), for the total Arctic ﬂeet in the summer (June–Aug–Sept) 2004, using ship emissions for the year 2004 from Dalsøren et al. (2009). It is important to note that we expect lower impacts of shipping in studies based on earlier years, because of the continued growth of shipping emissions along the Norwegian coast (as discussed in Sect. 4.3 and illustrated in Table 5). However, stronger or lower emissions do not seem to completely explain the differ- ent modeled impacts. Ødemark et al. (2012) found that Arctic ships had a strong inﬂuence on surface O3 in northern Nor- way for relatively low 2004 shipping emissions. This could be explained by the different processes included in both mod- els, or by different meteorological situations in the two stud- ies based on two different meteorological years (2004 and 2012). However, it is also likely that the higher O3 in the Ødemark et al. (2012) study could be caused, in part, by non- linear effects associated with global models run at low reso- The impact of ships in northern Norway on surface PM2.5, BC, and SO= 4 during the same period is shown in Fig. 10. The impact on PM2.5 is relatively modest, less than 0.5 µgm−3. However, these values correspond to an important relative increase of ∼10 % over inland Norway and Sweden because of the low background PM2.5 in this region. Over the sea sur- face, the relative effect of ship emissions is quite low because of higher sea salt aerosol background. Aliabadi et al. (2014) have observed similar increases in PM2.5 (0.5 to 1.9 µgm−3) in air masses inﬂuenced by shipping pollution in the remote Canadian Arctic. In spite of the higher trafﬁc in northern Norway, we ﬁnd lower values than Aliabadi et al. (2014) be- cause results in Fig. 5.2.1 Surface air pollution from ship emissions in northern Norway 10 are smoothed by the 15-day average. Impacts on surface sulfate and BC concentrations are quite large, reaching up to 20 and 50 %, respectively. We note that Eckhardt et al. (2013) found enhancements in summertime equivalent BC of 11 % in Svalbard from cruise ships alone. As expected, absolute SO= 4 and BC enhancements in our sim- ulations are higher in the southern part of the domain, where ship emissions are the strongest. We estimated the lifetime (residence time) of BC originating from ship emissions us- ing the method presented in Samset et al. (2014). This resi- dence time is deﬁned as the ratio of the average BC burden from ships divided by the average BC emissions in STEAM2 www.atmos-chem-phys.net/16/2359/2016/ Atmos. Chem. Phys., 16, 2359–2379, 2016 www.atmos-chem-phys.net/16/2359/2016/ Atmos. Chem. Phys., 16, 2359–2379, 2016 L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions 2374 2374 L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions Figure 10. 15-day average (00:00 UTC 11 July 2012 to 00:00 UTC 26 July 2012) of (top) absolute and (bottom) relative surface enhancements (CTRL – NOSHIPS) in (a, d) PM2.5, (b, e) BC and (c, f) SO= 4 due to ship emissions in northern Norway from STEAM2. Figure 10. 15-day average (00:00 UTC 11 July 2012 to 00:00 UTC 26 July 2012) of (top) absolute and (bottom) relative surface enhancements (CTRL – NOSHIPS) in (a, d) PM2.5, (b, e) BC and (c, f) SO= 4 due to ship emissions in northern Norway from STEAM2. lations do not include the effect of BC on snow, since this effect is not currently included in WRF-Chem. during the simulation. Using this method, we ﬁnd a BC life- time of 1.4 days. This short lifetime can be explained by the negative sea level pressure anomalies over northern Norway during the ACCESS campaign (Roiger et al., 2015), which indicates more rain and clouds than normal during summer. Given this short lifetime, BC is not efﬁciently transported away from the source region. The shortwave radiative effect at TOA of in-domain ship emissions is −1.77 Wm−2 (15-day average). We multiply this value by the area of our simulation domain to obtain a forcing value in watts (W), and divide it by the surface area of the Earth in order to obtain an equivalent global radia- tive effect in mWm−2 that can be compared to results from global studies. This equivalent global radiative effect at TOA is −9.3 mWm−2. This value is strongly negative, indicating that ship emissions cause a net cooling effect in this region (likely due to sulfate) despite the strong relative increase in BC concentrations from shipping emissions (up to +50 %, Fig. 10). This can be explained by the fact that these strong relative enhancements in BC correspond to low absolute val- ues (at most 20 ngm−3) above very low background concen- trations. L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions These results also indicate that shipping emissions comprise a signiﬁcant source of NOx and SO2 at low altitudes during the ACCESS ﬂights, even though speciﬁc ship plume sampling near the surface was excluded from these proﬁles. Pollution sampled during these ﬂights thus represents shipping pollution that had time to mix vertically in the marine boundary layer and is more rep- resentative of the regional pollution from shipping in northern Norway. These results are in agreement with the recent evaluation of STEAM2 in the Baltic Sea by Beecken et al. (2015), which showed that STEAM2 per- formed well for an average ﬂeet (∼200 ships), despite biases for individual ships. 2. Regional model representation of ship plumes and their local-scale inﬂuence – WRF-Chem runs includ- ing shipping emissions from STEAM2 are performed at 15 km × 15 km and 3 km × 3 km horizontal resolu- tions, and compared with airborne measurements of NOx and ozone. The high-resolution simulation is bet- ter at reproducing measured NOx peaks and suggests some ozone titration in ship plumes, but the NOx and ozone enhancements due to ships in both simulations are within less than 5 % of each other when aver- aged over the whole domain and simulation period. The 3 km × 3 km simulation also reproduces observed PM1 enhancements in ship plumes. Surface PM10 enhance- ments due to ships are 15 % higher in the 3 km × 3 km resolution simulation. L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions 200 µgm−2 along the Norwegian coast, about half of the value (250 to 300 µgm−2) found by Ødemark et al. (2012). This means that the stronger effect found here is not due to increased sulfate concentrations from larger emissions, but is likely due to the way aerosol–cloud interactions are treated in both models: the indirect aerosol effect was calculated by Ødemark et al. (2012) based on parameterizations of the relationship between clouds droplet numbers and aerosol mass, whereas the MOSAIC aerosol module used in this study explicitly treats aerosol activation within clouds and their impacts on cloud properties (Yang et al., 2011). It is also important to note here that the indirect radiative effect of shipping emissions is uncertain and that the difference between the estimate of Ødemark et al. (2012) and the one in this work can also be explained by these uncertainties. Based on the work of Eyring et al. (2007), Lauer et al. (2007), and Fuglestvedt et al. (2008), Eyring et al. (2010) estimated that the radiative forcing of global shipping emissions was −0.408 Wm−2, but found an uncertainty range of ±0.425 Wm−2. Ødemark et al. (2012) considered that the uncertainty in the indirect effect in their simulations was the same as the uncertainty in the global indirect forcing of aerosols as estimated by the IPCC (Forster et al., 2007, Table 2.12). Using this method, Ødemark et al. (2012) estimated a range of [−3.9 mWm−2, −1.3 mWm−2] for the global and annual indirect effect of Arctic shipping emissions. It is important to better understand and constrain this effect, which would require more aerosol measurements in shipping lanes (including number concentrations and aerosol compositions in ship plumes) and more model case studies. same ships by the STEAM2 emission model. Results show that STEAM2 overestimates NOx emissions for the four ships sampled during ACCESS. SO2 emissions are also determined for two ships. Large biases are pos- sible for individual ships in STEAM2, especially for ships for which there is incomplete technical data or where emission reduction techniques have been em- ployed. Nevertheless, combining WRF-Chem simula- tions and STEAM2 emissions leads to reasonable pre- dictions of NOx, SO2, and O3 compared to ACCESS proﬁles in the lower troposphere (normalized mean bi- ases of +14.2, −6.8, and −7.0 %, respectively). 5.2.2 Shortwave radiative effect of ship emissions in northern Norway The present-day climate effect of ship emissions is mostly due to aerosols, especially sulfate, which cool the climate through their direct and indirect effects (Capaldo et al., 1999). However, large uncertainties still exist concerning the magnitude of the aerosol indirect effects (Boucher et al., 2013). In this section, we determine the total shortwave ra- diative effect of ships by calculating the difference between the top-of-atmosphere (TOA) upwards shortwave (0.125 to 10 µm wavelengths) radiative ﬂux in the CTRL and the NO- SHIPS simulations. Since the CTRL and NOSHIPS simu- lations take into account aerosol–radiation interactions and their feedbacks (the so-called direct and semi-direct effects) as well as cloud–aerosol interactions (indirect effects), this quantity represents the sum of modeled direct, semi-direct and indirect effects from aerosols associated with ship emis- sions. Yang et al. (2011) and Saide et al. (2012) showed that including cloud aerosol couplings in WRF-Chem improved signiﬁcantly the representation of simulated clouds, indicat- ing that the indirect effect was relatively well simulated using CBM-Z/MOSAIC chemistry within WRF-Chem. Our calcu- The radiative effect calculated in this study, −9.3 mWm−2, is similar to the estimate by Ødemark et al. (2012), who found a direct and indirect shortwave effect of aerosols from Arctic-wide shipping in July 2004 of −10.4 mWm−2. However, since the present study only represents the effect of shipping along the Norwegian coast, this implies that current ship emissions in northern Norway have a stronger effect in this study than in Ødemark et al. (2012), which was based on ship emissions from Dalsøren et al. (2009) corresponding to 24 % less SO2 emis- sions than STEAM2. Higher emissions in our simulations could explain the stronger local shortwave effect of Arctic ships, since this effect is mostly associated with the direct and indirect effect of sulfate aerosols. However, the total sulfate column due to ship emissions in our study is 100 to www.atmos-chem-phys.net/16/2359/2016/ Atmos. Chem. Phys., 16, 2359–2379, 2016 2375 6 Conclusions The focus of this work, linking modeling and measure- ments, is to better quantify regional atmospheric impacts of ships in northern Norway in July 2012. The study relies on measurements from the ACCESS aircraft campaign, emis- sions evaluation, and regional modeling in order to evaluate both individual ship plumes and their regional-scale effects. STEAM2 emissions, which represent individual ships based on high-resolution AIS ship positioning data, are compared with emissions for speciﬁc ships derived from measurements and plume dispersion modeling using FLEXPART-WRF. Re- gional WRF-Chem simulations run with and without ship emissions are performed at two different resolutions to quan- tify the surface air quality changes and radiative effects from ship emissions in northern Norway in July 2012. The most important conclusions from our study are 3. Average inﬂuence of ship pollution in July 2012 – the difference between runs with and without ship emis- sions are compared with campaign average proﬁles (ex- cluding ﬂights focused on oil platforms, smelters, and biomass burning emissions from outside the simula- tion domain). Including STEAM2 emissions reduces the mean bias between measured and modeled trace gases NOx, SO2, and O3. At the surface, ship emissions enhance 15-day-averaged concentrations along the Nor- wegian coast by approximately 80 % for NOx, 80 % for SO2, 5 % for O3, 40 % for BC, and 10 % for PM2.5, suggesting that these emissions are already having an impact on atmospheric composition in this region. Re- gional model results presented in this study predict 3. Average inﬂuence of ship pollution in July 2012 – the difference between runs with and without ship emis- sions are compared with campaign average proﬁles (ex- cluding ﬂights focused on oil platforms, smelters, and biomass burning emissions from outside the simula- tion domain). Including STEAM2 emissions reduces the mean bias between measured and modeled trace gases NOx, SO2, and O3. At the surface, ship emissions enhance 15-day-averaged concentrations along the Nor- wegian coast by approximately 80 % for NOx, 80 % for SO2, 5 % for O3, 40 % for BC, and 10 % for PM2.5, suggesting that these emissions are already having an impact on atmospheric composition in this region. Re- gional model results presented in this study predict 1. Validation of the STEAM2 emissions – emissions of NOx and SO2 are determined for individual ships, by comparing airborne measurements with plume disper- sion modeling results. L. Marelle et al.: Air quality and radiative impacts of Arctic shipping emissions 2376 lower ozone production from ships compared to cer- tain earlier studies using global models. However, it is known that global models run at low resolution tend to overestimate ozone production (underestimate ozone titration) from fresh ship emissions because of non- linearities introduced when diluting concentrated emis- sions from ships into coarse model grid cells. (Helmholtz-Hochschul-Nachwuchsforschergruppe AerCARE). We thank the pilots, engineers, and scientists from the DLR ﬂight department for their excellent support during the ﬁeld campaign. We also thank the Wilsonship management for reporting the positions of their vessels during the campaign period. We thank Jerome Brioude (CIRES) for the development of FLEXPART-WRF and advice on how to derive ship emissions, and the WRF-Chem community for making the model available. Computing resources were provided by IDRIS HPC resources under the allocation 2014– 017141 under GENCI, and by the IPSL CICLAD/CLIMSERV mesocenter. We thank Stig Dalsøren, Jesper Heile Christensen and James Corbett for providing their ship emission data sets. We thank the EDGAR team for compiling the HTAPv2 emissions (http://edgar.jrc.ec.europa.eu/htap_v2/index.php?SECURE=123). 4. Inﬂuence on the radiative budget – northern Norwegian ship emissions contribute −9.3 mWm−2 to the global shortwave radiative budget of ship emissions, includ- ing semi-direct and indirect effects. These results are more signiﬁcant than found previously in a study using a global model that did not explicitly resolve aerosol ac- tivation in clouds. This suggests that global models may be underestimating the radiative impacts of shipping in this region. 4. Inﬂuence on the radiative budget – northern Norwegian ship emissions contribute −9.3 mWm−2 to the global shortwave radiative budget of ship emissions, includ- ing semi-direct and indirect effects. These results are more signiﬁcant than found previously in a study using a global model that did not explicitly resolve aerosol ac- tivation in clouds. This suggests that global models may be underestimating the radiative impacts of shipping in this region. Edited by: J. G. 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https://openalex.org/W4362558116	https://www.mdpi.com/2076-393X/11/4/788/pdf?version=1680526580	English	null	Lessons Learned and Future Perspectives for Rotavirus Vaccines Switch in the World Health Organization, Regional Office for Africa	Vaccines	2,023	cc-by	4,300	Brief Report Lessons Learned and Future Perspectives for Rotavirus Vaccines Switch in the World Health Organization, Regional Ofﬁce for Africa Inacio Mandomando 1,2,3 , Augusto Messa, Jr. 1 , Joseph Nsiari-Muzeyi Biey 4, Gilson Paluku 5, Mutale Mumba 6 and Jason M. Mwenda 7,* 1 Centro de Investigação em Saúde de Manhiça (CISM), Maputo P.O. Box 1929, Mozambique 2 Instituto Nacional de Saúde (INS), Maputo P.O. Box 3943, Mozambique 3 ISGlobal, Hospital Clínic, Universitat de Barcelona, 08036 Barcelona, Spain 4 Inter Country Support Team (IST) for West Africa, Regional Ofﬁce for Africa, World Health Organization (WHO), Ouagadougou 03 BP 7019, Burkina Faso 5 Inter Country Support Team (IST) for Central Africa, World Health Organization, Libreville P.O. Box 820, Gabon 6 Inter Country Support Team (IST) for East and Southern Africa, Regional Ofﬁce for Africa, World Health Organization, Harare P.O. Box 5160, Zimbabwe 7 Regional Ofﬁce for Africa, World Health Organization (WHO), Brazzaville P.O. Box 06, Congo * Correspondence: mwendaj@who.int 1 Centro de Investigação em Saúde de Manhiça (CISM), Maputo P.O. Box 1929, Mozambique 2 g ( ), g g , 5 Inter Country Support Team (IST) for Central Africa, World Health Organization, Libreville P.O. Box 820, Gabon Abstract: Background: Following the World Health Organization (WHO) recommendation, 38/47 coun- tries have introduced rotavirus vaccines into the program of immunization in the WHO Regional Ofﬁce for Africa (WHO/AFRO). Initially, two vaccines (Rotarix and Rotateq) were recommended and recently two additional vaccines (Rotavac and Rotasiil) have become available. However, the global supply challenges have increasingly forced some countries in Africa to switch vaccine products. Therefore, the recent WHO pre-qualiﬁed vaccines (Rotavac, Rotasiil) manufactured in India, offer alternatives and reduce global supply challenges related to rotavirus vaccines; Methods: Using a questionnaire, we administered to the Program Managers, Expanded Program for Immunization, we collected data on vaccine introduction and vaccine switch and the key drivers of the decisions for switching vaccines products, in the WHO/AFRO. Data was also collected fromliterature review and the global new vaccine introduction status data base maintained by WHO and other agencies. Results: Of the 38 countries that introduced the vaccine, 35 (92%) initially adopted Rotateq or Rotarix; and 23% (8/35) switched between products after rotavirus vaccine introduction to either Rotavac (n = 3), Rotasiil (n = 2) or Rotarix (n = 3). Three countries (Benin, Democratic Republic of Congo and Nigeria) introduced the rotavirus vaccines manufactured in India. The decision to either introduce or switch to the Indian vaccines was predominately driven by global supply challenges or supply shortage. Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Citation: Mandomando, I.; Augusto Messa, Jr.; Biey, J.N.-M.; Paluku, G.; Mumba, M.; Mwenda, J.M. Lessons Learned and Future Perspectives for Rotavirus Vaccines Switch in the World Health Organization, Regional Ofﬁce for Africa. Vaccines 2023, 11, 788. https://doi.org/10.3390/ vaccines11040788 Academic Editor: Pedro Plans-Rubió Received: 30 November 2022 Revised: 3 March 2023 Accepted: 13 March 2023 Published: 3 April 2023 Citation: Mandomando, I.; Augusto Messa, Jr.; Biey, J.N.-M.; Paluku, G.; Mumba, M.; Mwenda, J.M. Lessons Learned and Future Perspectives for Rotavirus Vaccines Switch in the World Health Organization, Regional Ofﬁce for Africa. Vaccines 2023, 11, 788. https://doi.org/10.3390/ vaccines11040788 Academic Editor: Pedro Plans-Rubió Brief Report Lessons Learned and Future Perspectives for Rotavirus Vaccines Switch in the World Health Organization, Regional Ofﬁce for Africa The withdrawal of Rotateq from the African market, or cost-saving for countries that graduated or in transition from Gavi support was another reason to switch the vaccine; Conclusions: The recently WHO pre-qualiﬁed vaccines have offered the countries, opportunities to adopt these cost-effective products, particularly for countries that have graduated or transitioning from full Gavi support, to sustain the demand of vaccines products. Keywords: rotavirus; vaccine; switch; WHO AFR 1. Introduction Since the World Health Organization (WHO) recommendation for the global introduc- tion of rotavirus vaccine in high disease burden countries [1], Gavi, the Vaccine Alliance, has provided funding for the introduction of rotavirus vaccine in 53 Gavi-eligible countries globally [2]. The WHO has been providing technical guidance and support to the African countries to enable the countries make evidence based decision on the introduction of https://www.mdpi.com/journal/vaccines Vaccines 2023, 11, 788. https://doi.org/10.3390/vaccines11040788 Vaccines 2023, 11, 788 2 of 6 rotavirus vaccines [3]. In sub-Saharan Africa, 38/47 (80%) of the countries introduced the rotavirus vaccines in their National Immunization Programs [2], with Rotarix being the most adopted vaccine [4]. However, more recently, additional WHO pre-qualiﬁed rotavirus vaccines manufactured in India: Rotavac (Human neonatal strain 116E G9P [11]) and RotasIIL (Pentavalent human–bovine reassortant serotypes G (1–4) and G9) are increasingly being deployed in sub-Saharan Africa [5]. The early adopted rotavirus vaccines have shown beneﬁcial impact on reducing child- hood morbidity and -mortality due to rotavirus and cost-effective in low- and middle income countries [6–10]. However, vaccine cost, demand, manufacturer production ca- pacity and programmatic issues are, among others, the major barriers to the long-term sustainability of rotavirus vaccination, particularly for countries scheduled to graduate from Gavi ﬁnancial support. Also, local Government co-ﬁnancing is required to meet operation costs at the country level. On the other hand, cost-effectiveness analysis com- paring Rotarix, Rotateq and Rotavac showed lower price per dose for Rotavac vaccine compared to the competitor products [11], and this led countries like Palestine to switch from Rotarix to Rotavac vaccine after assuming the ﬁnancial responsibility for rotavirus vaccine procurement [12]. The decisions inﬂuencing to switching from one vaccine product to another should systematically assess multiple criteria beyond vaccine price include the health, program- matic and economic aspects. Countries should also fully evaluate all product characteristics including product presentation, number of doses per course, cold chain volume, cost of delivery, and wastage [12]. WHO recommends that the choice of rotavirus vaccine product to be used in a country, either at ﬁrst introduction or at switch, should be based on disease burden, programmatic characteristics, vaccine supply, and vaccine price including cost effectiveness analysis. Current evidence indicates that local data on circulating rotavirus strains should not drive product choice, as the WHO prequaliﬁed rotavirus vaccines have been shown to provide cross protection against heterologous strains [1]. 1. Introduction A number of countries switched from Rotateq or Rotarix to Rotavac, Rotasiil but the reason for the decisions to switch vaccines are not yet fully documented. In this evaluation, we investigated the rotavirus vaccine being used in WHO/AFRO countries and assessed the reasons for switching vaccine products at a country level. 2. Materials and Methods To assess rotavirus vaccines that are in use in WHO/AFRO countries that participate in the African Rotavirus Surveillance Network introduced in their immunization programs and whether these vaccines had changed since the introduction, we collected information by administering a questionnaire to the Program Managers, Expanded Program for Immuniza- tion of the countries that had switched or planning to switch the vaccine product. Data was also collected from literature review by searching the terms or combination of the terms “ro- tavirus vaccine switch in WHO AFRO” in the PubMed (https://pubmed.ncbi.nlm.nih.gov/, accessed on 15 February 2023), WHO (https://www.who.int/, accessed on 15 February 2023), International Vaccine Access Center [IVAC] (https://www.jhsph.edu/ivac/, ac- cessed on 15 February 2023) websites. The data collected included the type of vaccine and if the country had changed or planned to change products since introduction. In addition, the data of current status of global rotavirus introductions was accessed [2]. Data that was collected was entered in the excel sheet, checked for consistency and analysis performed using Stata version 14 (StataCorp LP, College Station, TX, USA). 3. Results As of 30 October 2022, 38 out of 47 (80%) of the countries in WHO African region had introduced rotavirus vaccines in their national immunization programs, with Nigeria being the most recent country that started phased vaccine introduction from August 2022. Of those, six (Eswatini, Botswana, Mauritius, Namibia, Seychelles and South Africa) are not- eligible for Gavi funding for vaccine introduction and three countries (Angola, Republic of Vaccines 2023, 11, 788 3 of 6 Congo and Ghana) have graduated or transitioned, and in this case, national Governments must budget and allocate funds for the procurement of vaccines and operational costs for vaccine implementation. The remaining are Gavi Phase III eligible (Table 1). Congo and Ghana) have graduated or transitioned, and in this case, national Governments must budget and allocate funds for the procurement of vaccines and operational costs for vaccine implementation. The remaining are Gavi Phase III eligible (Table 1). Table 1. Gavi eligibility and World Bank Income grouping of the countries that introduced rotavirus vaccines (initial introduction) in the WHO/AFRO. 3. Results ( ) / Country RV Introduction Date Vaccine Type Current GAVI Eligibility World Bank Income Group Angola 2014-04-28 ROTARIX (RV1) Graduated Lower middle income Benin 2019-12-01 ROTAVAC (RV1) Gavi Phase III Eligible Lower middle income Botswana 2012-07-03 ROTARIX (RV1) Not Eligible Upper middle income Burkina Faso 2013-10-31 ROTASIIL (RV5) Gavi Phase III Eligible Low income Burundi 2013-12-16 ROTARIX (RV1) Gavi Phase III Eligible Low income Cameroon 2014-03-28 ROTARIX (RV1) Gavi Phase III Eligible Lower middle income Congo 2014-04-24 ROTARIX (RV1) Graduated Lower middle income Congo, Democratic Republic of the 2019-10-01 ROTASIIL (RV5) Gavi Phase III Eligible Low income Côte d’Ivoire 2017-03-01 RotaTeq (RV5) Gavi Phase III Eligible Lower middle income Eritrea 2014-08-14 ROTARIX (RV1) Gavi Phase III Eligible Low income Eswatini 2015-05-12 ROTARIX (RV1) Not Eligible Lower middle income Ethiopia 2013-11-07 ROTARIX (RV1) Gavi Phase III Eligible Low income Gambia 2013-08-14 ROTARIX (RV1) Gavi Phase III Eligible Low income Ghana 2012-04-26 ROTAVAC (RV1) Graduated Lower middle income Guinea-Bissau 2015-11-24 ROTARIX (RV1) Gavi Phase III Eligible Low income Kenya 2014-07-01 ROTARIX (RV1) Gavi Phase III Eligible Lower middle income Lesotho 2017-12-18 ROTARIX (RV1) Gavi Phase III Eligible Lower middle income Liberia 2016-04-25 ROTARIX (RV1) Gavi Phase III Eligible Low income Madagascar 2014-05-05 ROTARIX (RV1) Gavi Phase III Eligible Low income Malawi 2012-10-29 ROTARIX (RV1) Gavi Phase III Eligible Low income Mali 2014-01-14 RotaTeq (RV5) Gavi Phase III Eligible Low income Mauritania 2014-12-06 ROTARIX (RV1) Gavi Phase III Eligible Lower middle income Mauritius 2015-05-01 ROTARIX (RV1) Not Eligible Upper middle income Mozambique 2015-09-04 ROTARIX (RV1) Gavi Phase III Eligible Low income Namibia 2014-11-11 ROTARIX (RV1) Not Eligible Upper middle income Niger 2014-08-05 ROTARIX (RV1) Gavi Phase III Eligible Low income Nigeria 2022/08/22 ROTAVAC (RV1) Graduated Lower middle income Rwanda 2012-05-25 ROTARIX (RV1) Gavi Phase III Eligible Low income Sao Tome and Principe 2016-09-22 RotaTeq (RV5) Gavi Phase III Eligible Lower middle income Senegal 2014-11-28 ROTARIX (RV1) Gavi Phase III Eligible Lower middle income Seychelles 2017-09-01 ROTARIX (RV1) Not Eligible High income Sierra Leone 2014-03-28 ROTARIX (RV1) Gavi Phase III Eligible Low income South Africa 2009-08-01 ROTARIX (RV1) Not Eligible Upper middle income United Republic of Tanzania 2012-12-06 ROTARIX (RV1) Gavi Phase III Eligible Lower middle income Togo 2014-06-19 ROTARIX (RV1) Gavi Phase III Eligible Low income Uganda 2018-06-26 ROTARIX (RV1) Gavi Phase III Eligible Low income Zambia 2013-11-26 ROTARIX (RV1) Gavi Phase III Eligible Lower middle income Zimbabwe 2014-05-01 ROTARIX (RV1) Gavi Phase III Eligible Lower middle income Of the 38 countries that introduced the rotavirus vaccine in the WHO/AFRO, three (Benin, Democratic Republic of Congo and Nigeria) were ﬁrst time introductions of the recently pre-qualiﬁed Indian manufactured vaccines, while the remaining 35 introduced Rotateq or Rotarix. 3. Results Country Vaccine Date of Introduction Date of Switch New Vaccine Reason for Switch Burkina Faso Rotateq 31 October 2013 January, 2019 RotaSIIL 1, 3 Cote d’Ivore Rotateq 1 March 2017 May, 2019 Rotarix 1, 3 The Gambia Rotateq 14 October 2013 February, 2017 Rotarix 1, 3 Ghana Rotarix 26 April 2012 January, 2020 Rotavac 2 Mali Rotateq 14 January 2014 January, 2020 RotaSIIL 1, 3 Rwanda Rotateq 25 May 2012 April, 2017 Rotarix 1, 3 São Tomé e Principe Rotateq 22 September 2016 2018/2019 Rotavac 1, 3 The Gambia, United Republic of Tanzania Rotarix 6 December 2012 October, 2022 Rotavac 4 Reason for switch code: 1 Challenges with supply after Rotateq removed from GAVI market; 2 Cost factors associated with graduation from GAVI support; 3 Challenges in cold chain; 4 Supply shortages. Table 2. Countries that have switched rotavirus vaccines in the WHO African region and the factors driven the decision. Reason for switch code: 1 Challenges with supply after Rotateq removed from GAVI market; 2 Cost factors associated with graduation from GAVI support; 3 Challenges in cold chain; 4 Supply shortages. Six countries (Rwanda, The Gambia, Burkina Faso, Cote d’Ivore, Mali, Sao Tome and Principle) switched from Rotateq to other vaccines because the manufacturer withdrew this vaccine from African market due to undisclosed reasons. Tanzania was recently forced to switch due to the supply shortage. While Ghana’s decision to switch vaccine products was due to cost as the country graduated from GAVI support and had to self-fund the vaccine procurement (Table 2). 3. Results In 2009, when rotavirus vaccination was being rolled out, only two vaccine products were available (Rotateq or Rotarix). However, from the ﬁrst 35 group of countries to introduce, 23% (n = 8) of the countries have switched the initially adopted rotavirus vaccines to an alternate, recently prequaliﬁed Indian vaccines. Among these eight countries that switched, ﬁve (63%) adopted Indian manufactured vaccines, with Rotavac being the most frequently adopted (n = 3), while only two have adopted RotaSiiL. The Vaccines 2023, 11, 788 4 of 6 rotavirus vaccines types used during initial introduction in the WHO/AFRO is shown in Table 2, including the list of countries that switched the initially adopted rotavirus vaccines and the factors driving the switch. Table 2. Countries that have switched rotavirus vaccines in the WHO African region and the factors driven the decision. Country Vaccine Date of Introduction Date of Switch New Vaccine Reason for Switch Burkina Faso Rotateq 31 October 2013 January, 2019 RotaSIIL 1, 3 Cote d’Ivore Rotateq 1 March 2017 May, 2019 Rotarix 1, 3 The Gambia Rotateq 14 October 2013 February, 2017 Rotarix 1, 3 Ghana Rotarix 26 April 2012 January, 2020 Rotavac 2 Mali Rotateq 14 January 2014 January, 2020 RotaSIIL 1, 3 Rwanda Rotateq 25 May 2012 April, 2017 Rotarix 1, 3 São Tomé e Principe Rotateq 22 September 2016 2018/2019 Rotavac 1, 3 The Gambia, United Republic of Tanzania Rotarix 6 December 2012 October, 2022 Rotavac 4 Reason for switch code: 1 Challenges with supply after Rotateq removed from GAVI market; 2 Cost factors associated with graduation from GAVI support; 3 Challenges in cold chain; 4 Supply shortages. Vaccines 2023, 11, 788 4 of 6 rotavirus vaccines types used during initial introduction in the WHO/AFRO is shown in Table 2, including the list of countries that switched the initially adopted rotavirus vaccines and the factors driving the switch. Table 2. Countries that have switched rotavirus vaccines in the WHO African region and the factors driven the decision. 4. Discussion In the last decade, we have witnessed the widespread adoption of the two second generation of WHO pre-qualiﬁed rotavirus vaccines, internationally licensed for preventing severe illness due to rotavirus diarrhea in high mortality burden countries, particularly in sub-Saharan Africa. The rate of rotavirus vaccine introduction was driven by ﬁnancial support from Gavi as almost 84% (32/38) of the countries that introduced rotavirus vaccine in their EPI in the WHO/AFRO, did so with Gavi ﬁnancial support. However, the WHO prequaliﬁcation of additional two rotavirus vaccines: ROTAVAC™(Bharat Biotech, Hyder- abad, India) and RotaSIIL (Serum Institute of India, Pune, India) in 2018 [1], offers countries that have not yet introduced the vaccines wider vaccine product choices and pricing [13]. In addition, the competitive market safeguards reasonable pricing and aids countries in ensuring long-term sustainability of their vaccine programs as countries graduate from Gavi support. These newer vaccines also provided additional options for countries that previously introduced the vaccines to consider more cost-effective vaccines [14]. In the WHO/AFRO, so far eight countries including one that have graduated from Gavi eligibility (Ghana) have switched the vaccines. The major factors that complicated this switch included the programmatic factors including (e.g., insufﬁcient cold-chain capacity); the lack of a country- speciﬁc cost effectiveness data, anticipated Gavi transition and global supply shortages (personal communication EPI Managers). Interestingly, the initial switch was driven either by manufacturer removal of Rotateq vaccine or the cost-saving on operation costs/co- ﬁnancing for countries that introduced this vaccine [15]. For example, the switch in Ghana was necessitated by anticipated Gavi transition and the recommendation was supported and endorsed by National Immunization Technical Advisory Group (NITAG) [16]; while Rwanda, The Gambia, Burkina Faso, Cote d’Ivore, Mali, Sao Tome and Principle were forced to switch because of the removal of Rotateq from the African market. The sustainability of rotavirus vaccines production is a matter of concern. Currently, some countries are facing Vaccines 2023, 11, 788 5 of 6 supply shortages of their chosen product, Tanzania was the most recent country to switch to the Indian-manufactured vaccines, Rotavac supply shortages of their chosen product, Tanzania was the most recent country to switch to the Indian-manufactured vaccines, Rotavac As many countries will transition from Gavi-eligibility in future, more countries will have to ﬁnd additional resources to procure vaccines and fund their vaccine programs potentially necessitating a switch to a lower cost product. 4. Discussion Notably, previous rotavirus vaccine switch that necessitated, the countries were informed about the decision to switch vaccine, meant country had adequate time to plan and manage the switch. More recently, ﬁve countries including Kenya, Senegal, Tanzania, Zambia and Zimbabwe have been informed by Gavi to switch from Rotarix to Indian manufactured vaccines (RotasIIL or Rotavac) abruptly due to unforeseen supply constraints. Currently, there is uncertainty in sustainable supply of early adopted rotavirus vaccines for African countries. To mitigate this supply constraints for Rotarix, Uganda notiﬁed Gavi of the intention to switch to Indian-manufactured vaccines due to unforeseen supply constraints with their current vaccine product. It is anticipated this unpredictable vaccine supply may erode the gains and documented beneﬁts of rotavirus vaccination in Africa. African governments should support the recent initiatives of vaccine manufacturing as agreed during the African ministerial conference on immunization and Addis declaration [17] to cushion against this unpredictable vaccine supply for African countries. p pp y Rotateq vaccine was completely withdrawn and no longer in use in the African region and there is eminent global supply constraint for Rotarix vaccine with ﬁve African countries currently affected. Although, the recently WHO pre-qualiﬁed vaccines offer opportunities for the countries to adopt these cost-effective products with broader availability, particularly for countries in the trajectory and transitioning from full Gavi support, these challenges emphasizes the need for future vaccines locally manufactured to sustain the local demand while the countries with high birth cohort are rolling out the vaccines (e.g., Nigeria and Democratic Republic of Congo) will sustainable supply. 5. Conclusions The recently WHO pre-qualiﬁed vaccines have offered opportunities for more options for the countries to adopt these cost-effective products, particularly for countries that have graduated or transitioning from full Gavi support. Sustained supply of vaccines should be maintained to ensure availability for all countries including those with high birth cohort to reap the full beneﬁts of these vaccination. Author Contributions: Conceptualization, I.M. and J.M.M.; Methodology, I.M., A.M.J., J.N.-M.B., G.P., M.M. and J.M.M.; Software, I.M. and A.M.J.; Validation, I.M., A.M.J., J.N.-M.B. and J.M.M.; Formal analysis, I.M., A.M.J. and J.M.M.; Investigation, I.M., A.M.J., J.N.-M.B., G.P., M.M. and J.M.M.; Data curation, I.M., A.M.J., J.N.-M.B. and J.M.M.; Writing—original draft preparation, I.M. and J.M.M.; Writing—review and editing, I.M., A.M.J., J.N.-M.B., G.P., M.M. and J.M.M.; visualization, I.M. and J.M.M.; All authors have read and agreed to the published version of the manuscript. Funding: This work was based on review of available data in the Inter-Country Support team, and no speciﬁc funding was requested for this work. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. 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Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. References MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
W1519187085.txt	https://periodicos.unemat.br/index.php/reps/article/download/9240/5089	pt		Jogos e brincadeiras na educação infantil, análise no pré II do Centro Municipal de Educação Infantil Vinícius de Moraes	Eventos Pedagógicos	2,012	cc-by	3,754	Revista Eventos Pedagógicos v.3, n.2, p. 131 - 140, Maio - Jul. 2012 JOGOS E BRINCADEIRAS NA EDUCAÇÃO INFANTIL, ANÁLISE NO PRÉ II DO CENTRO MUNICIPAL DE EDUCAÇÃO INFANTIL VINÍCIUS DE MORAES Joanil Maria da Silva Adil Antônio Alves de Oliveira RESUMO O presente artigo traz uma análise sobre jogos e brincadeiras na Educação Infantil. A pesquisa foi realizada no Centro Municipal de Educação Infantil Vinícius de Moraes, na cidade de Sinop-Mt. O objetivo da pesquisa foi analisar se como são trabalhadas as atividades com brincadeiras na educação da criança. Em dialogo com a professora e coordenação pedagógica do CMEI questionamos qual é a importância das brincadeiras no desenvolvimento processo de aprendizagem como forma de conteúdo realizada nas atividades cotidiana da instituição, e quando esta é trabalhada. Nesse contexto convidar os professores a refletir acerca do conhecimento sobre as práticas com brincadeiras que tem desenvolvido com seus alunos, a partir da bibliografia existente sobre essa temática. Para a realização deste estudo recorri aos seguintes autores Tizuko Morchida Kishimoto, Philippe Aries, Vygotsky e José Luiz Straub. Para chegar aos meus objetivos trabalhei com as observações e as entrevistas e questionário. As respostas obtidas foram analisadas com base nas leituras realizadas e foi possível verificar que as brincadeiras são as atividades preferidas das crianças. Com isso, pretendemos que, ao saber das preferências das crianças, os professores desta modalidade de ensino possam oferecer atividades que permitam um melhor aprendizado por parte delas, uma vez que o lúdico é um importante instrumento de trabalho para o professor da Educação Infantil. Palavras-chave: Educação infantil. Brincadeiras. Lúdico. Aluna do 7º semestre do curso de Pedagogia do campus universitário de Sinop UNEMAT. Pertence ao grupo de Orientação do professor Me. Adil Antônio Alves de Oliveira. Graduado em Psicologia pela Universidade Católica (UCDB) Mestrado em educação pela Universidade Federal do Rio Grande do Sul (UFRGS). 1 INTRODUÇÃO A brincadeira faz parte do universo infantil desde os tempos mais distante da História. Através dela a criança apropria-se da sua imagem, seu espaço, seu meio sociocultural, realizando inter-relações. Segundo o Referencial Curricular para a Educação Infantil, o Brincar é um precioso momento de construção pessoal e social, é permeado pelo eixo de trabalho Movimento, onde a criança movimenta-se construindo sua moralidade, afetividade perante as situações desafiadoras e significativas presentes no brincar e inerentes à produção social do conhecimento. O brincar é a atividade mais importante e clara que dá identidade à infância como o ser criança. Através do brincar de faz de conta as crianças colocam em pratica suas fantasias, alterando significados de objetos como, o cabo de vassoura, que vira cavalo, cria e recria a todo o momento e suas imaginações são super férteis. É no brincar que aparecem as diversas formas da criança realizar esse feito, seja em grupo, duplas e na maioria das vezes sozinhas. O brincar carrega tradições, nostalgias, desperta sonhos e instiga a imaginação. Brincando constrói-se conhecimento, apropria-se da cultura, aprendem-se as tradições, revivem-se memórias, combinam-se regras, inventam-se novos sentidos e significados, através de sua linguagem comum a brincadeira é expressão, representação, significação e reinterpretação da e na cultura. Com a evolução tecnológica abriram-se caminho para a era da informática e da eletrônica, o que ocasionou a diminuição dos espaços físicos, levando muitos brinquedos e brincadeiras ao esquecimento e fazendo com que as crianças deixassem de lado o que de mais precioso podiam fazer, que é o brincar. Envolvidas com a evolução tecnológica e com os muitos afazeres como natação, inglês, computador, etc. A falta do brincar ou explorar diversos espaços e materiais pode limitar o desenvolvimento das suas potencialidades e habilidades (ARIÉS, 2006). Para Macedo (2005), o brincar infantil é um processo importante na construção de conhecimentos e no desenvolvimento integral da criança, independente do local em que vive do grupo ou da cultura da qual faz parte, proporcionando a mediação entre o real e o imaginário. O brincar estimula a inteligência porque faz com que a criança solte a sua imaginação e desenvolva a criatividade, possibilitando o exercício da concentração e da atenção, levando a criança a absorver-se na atividade. Freire (1992) também afirma que existe um mundo rico e vasto de cultura infantil, repleto de movimentos, de jogos, de fantasias, quase sempre ignorados pelas instituições de Página 132 - Joanil Maria da Silva e Adil Antônio Alves de Oliveira ensino. Pelo menos até o 4° ano do ensino fundamental, a escola conta com alunos onde a maior especialidade é brincar e não toma proveito disso para ensinar. A relação que tenho com as brincadeiras é de longa data, veio da minha infância e posteriormente da Escola de Educação Infantil onde trabalhei logo que cheguei na cidade. Desta maneira sempre esteve presente em minha vida. Mas foi na Universidade, no curso de Pedagogia que percebi a real importância da temática abordada nesta pesquisa. Notei no decorrer dos estágios que as práticas pedagógicas onde o lúdico se faz presente, tornam-se contagiante não só para as crianças, mas para os adultos também, porém percebi que nem to das às pessoas envolvidas com a Educação Infantil levam a questão do brincar a sério. Depois tive a disciplina de Educação Física onde o professor desenvolveu várias atividades recreativas, que tive a certeza que esse seria o universo da minha pesquisa, pois fiquei entusiasmada para realizar as brincadeiras que aprendi com as crianças. Acredito que as brincadeiras e jogos não são somente um meio para se distrair na hora do recreio ou nos momentos vagos, mas são também muito importantes, na infância, pois toda criança precisa brincar, como também um meio que contribui para o seu processo de desenvolvimento. Para isso, procurei verificar como são trabalhados os jogos e brincadeiras com crianças da pré-escola. Diante deste contexto é que propus neste trabalho: verificar a importância de trabalhar com atividades que se valem de jogos e brincadeiras no processo de produzir e aprender conhecimentos na educação infantil, bem como analisar e mostrar como esse método de ensino contribui para o desenvolvimento das crianças. 2 REFERENCIAL TEÓRICO Sabemos que a brincadeira é uma atividade fundamental para o desenvolvimento infantil, por meio dela, a criança interage com a realidade, se socializa com as crianças e outros adultos. Dessa forma, cabe-nos discutir de que forma essa atividade vem sendo interpretada por alguns estudiosos da Psicologia, dentro dos espaços escolares, especificadamente na educação infantil. Para Kishimoto (2011), o brinquedo proporciona uma relação íntima com a criança, uma relação solta, sem regras que determinem seu uso. O brinquedo estimula a imaginação e a representação, que muitas vezes pode ser algo do cotidiano, como a representação de „mamãe e filhinha‟ com o uso de uma boneca. Brinquedos em forma de personagens, como JOGOS E BRINCADEIRAS NA EDUCAÇÃO INFANTIL, ANÁLISE... - Página 133 monstros, super-heróis e animais, incorporam o imaginário preexistente, algo criado pelos desenhos animados, filmes, seriados, contos de fadas, enfim. Porém, cada cultura possui uma forma particular de ver a criança. Assim, ao confeccionar um brinquedo o fabricante faz de acordo com o a cultura das crianças que irão brincar. Para Kishimoto (2011), brincadeira seria toda ação que a criança desempenha ao concretizar as regras do jogo, ao entrar na ação lúdica, ou seja, a brincadeira seria o lúdico em ação. Sendo assim, o brinquedo serve de estimulo material, que serve para fluir o imaginário da criança. O brinquedo está diretamente ligado à criança. Simultaneamente a brincadeira com o brinquedo é importante para o desenvolvimento da criança nos níveis social, cognitivo, afetivo e psicomotor, além propiciar a interação entre as crianças, pela brincadeira elas se comportam diferentemente do comportamento habitual, em suas praticas do dia-a-dia. 3 METODOLOGIA É importante descrever os aspectos físicos e humanos de uma escola, para situar a quem lê o campo da realidade pesquisada. Por isso Esso momento é destinado a esse objetivo. O CMEI Vinicius de Moraes possui um pátio grande coberto sendo todo cimentado, não tem um espaço gramado para recreações das crianças. Conta com um parque infantil com areia e arborizado. O CMEI é composto por quatro salas de aulas, uma secretaria, uma cozinha e dois banheiros, sendo um para uso das meninas e o outro para o uso dos meninos. Durante o período da pesquisa, que se deu no ano de 2012 o CMEI possuía 301 alunos distribuídos em 12 turmas, sendo crianças na faixa etária de quatro à cinco anos de idade no pré I, e de cinco a seis anos no pré II. A instituição está ligada a Secretaria Municipal de Educação, regida por normas e diretrizes. A Secretaria de Educação se responsabiliza pela compra de materiais pedagógicos, os quais nem sempre são suficientes para suprir as necessidades. A comunidade participa da escola sempre que é solicitada. Os eventos realizados pela escola são: datas comemorativas (páscoa, dia das mães, dia dos pais, dia das crianças e festa julina), e trabalham os temas diversificados a cada bimestre para priorizar o conhecimento da criança. Página 134 - Joanil Maria da Silva e Adil Antônio Alves de Oliveira Devido à rotina da turma, foi sugerido pela diretora do Centro Municipal de Educação Infantil, que a entrevista fosse realizada em um pequeno período depois do lanche das crianças antecedendo a ida ao parque de modo a não atrapalhar as atividades diárias dos alunos, e com a professora durante o período de hora atividade. Foi realizada com 26 (vinte e seis) crianças do Pré II e com a professora da turma, uma entrevista semi estruturada, para a coleta de dados, por ser considerada dentre todas as técnicas de interrogação a mais flexível. Assim, a entrevista foi a mais adequada para esse trabalho, pois se tratavam de crianças entre quatro e cinco anos. Segundo Lüdke e André (1986) o entrevistador deve estar atento para algumas questões relacionadas à entrevista. Durante entrevista é importante que aconteça uma interação entre o pesquisador e o pesquisado, dessa forma acontecera uma influência mútua entre quem pergunta e quem responde. Em uma entrevista bem elaborada é possível a captação imediata da informação desejada. Por meio da entrevista é possível se fazer um tratamento de assuntos de natureza pessoal e íntima do indivíduo, pois durante a mesma criase um vínculo entre o entrevistado e o entrevistador. Nas entrevistas semi-estruturadas não há a imposição de uma ordem de questões. Os entrevistados discorrem sobre o tema proposto com base nas informações que ele detém, estas questões são dirigidas aos participantes citados, uma vez que estas deixam o pesquisador situar relações entre as concepções dos entrevistados e as próprias idéias do pesquisador, num método de construções e reconstruções que descarta qualquer neutralidade. [...] privilegia a entrevista semi-estruturada porque esta, ao mesmo tempo que valoriza a presença do investigador, oferece todas as perspectivas possíveis para que o informante alcance a liberdade e a espontaneidade necessárias, enriquecendo a investigação. (TRIVIÑOS, 1997, p 146). 4 ANÁLISE DOS DADOS DA PESQUISA Como os sujeitos têm idades entre 4 e 5 anos, e que ainda não possuem domínio da escrita e que a linguagem oral ainda se mostra um tanto limitada, optamos pela entrevista semiestruturada. As respostas às perguntas foram muito parecidas possivelmente pelo fato da entrevista ter sido realizada com todos os alunos juntos, atendendo à sugestão da diretora do Centro Municipal de Educação Infantil, pois segundo ela se fosse individualmente “tomaria muito tempo das crianças e atrapalharia o horário de rotina das mesmas”. JOGOS E BRINCADEIRAS NA EDUCAÇÃO INFANTIL, ANÁLISE... - Página 135 No que se refere à primeira questão: “Você gosta de ir à escola? Porque?”, das 26 crianças entrevistadas, apenas uma não aceitou responder a questão, e uma das crianças respondeu que nem todos os dias gostava de ir para a escola, e só ia por que a mãe tinha que trabalhar, as demais, todas afirmaram que gostam de ir para escola, por que brincam, escrevem e pintam. A escola para a maioria dos alunos é importante, pois como podemos notar com base nas respostas elas gostam de estar lá. Esse fato confirma o que o RCNEI em sua introdução defende como princípios básicos deste nível de ensino: Respeito à dignidade e aos direitos das crianças, consideradas nas suas diferenças individuais, sociais, econômicas, culturais, étnicas, religiosas etc.; Direito das crianças a brincar como forma particular de expressão, pensamento, interação e comunicação infantil; O acesso das crianças aos bens socioculturais disponíveis, ampliando o desenvolvimento das capacidades relativas à expressão, à comunicação, à interação social, ao pensamento, à ética e à estética; A socialização das crianças por meio de sua participação e inserção nas mais diversificadas práticas sociais, sem discriminação de espécie alguma; O atendimento aos cuidados essenciais associados á sobrevivência e ao desenvolvimento de sua identidade (BRASIL, 1998, p. 13, vol. 1). É importante ressaltar que mesmo uma das crianças declarar que não gosta de ir para escola, o contato dela com o espaço é de suma importância para a socialização e para o desenvolvimento integral da mesma. Na segunda questão perguntamos para as crianças o que elas mais gostavam de fazer na escola, e todas responderam com unanimidade que gostam de brincar. A respeito disso Wajskop (1997), afirma que o brincar na pré escola é garantia de uma possível educação da criança em uma perspectiva, criadora voluntária e consciente. Dentro desse contexto os professores da Educação Infantil devem fazer a brincadeira presente em seu planejamento diário, almejando o desenvolvimento global de seus alunos nos aspectos psicomotores a cognitivos. A professora da turma é formada em Pedagogia, e é o primeiro ano que a mesma leciona para a Educação Infantil. Em nossas conversas informais, questionei a professora a respeito do brincar na Educação infantil, como ela contemplava tal prática em seu planejamento e como as crianças reagiam diante das propostas planejadas. A professora relatou: (01) Professora:Vejo a brincadeira como prática indispensável na socialização e desenvolvimento da criança em todas as áreas, cognitivo, motor e linguístico. O brincar é Página 136 - Joanil Maria da Silva e Adil Antônio Alves de Oliveira próprio da criança, qual criança não gosta de brincar? Percebo que quando saímos da sala para área externa as crianças parecem se sentirem livres, soltos. Brincamos todos os dias no parque de areia, realizamos também brincadeiras direcionadas no pátio, e toda sexta-feira os alunos podem trazer brinquedos de casa para a escola. Segundo Kishimoto (2011, p.189), “a brincadeira é o lúdico em ação”, assim, a brincadeira deixa de ser “coisa de criança” e passa a ser analisada como “coisa séria”, e esta pode ter como função ser um recurso didático, com capacidade de compor uma ação docente garantindo como objetivo o processo de ensino-aprendizagem que se deseja obter. Para Vygotsky (1991) ao brincar, a criança age além do seu comportamento do cotidiano habitual. A brincadeira também propicia à criança a capacidade de lidar com os limites, a atenção, a concentração e a cooperação e interação com outras crianças. Assim, a brincadeira deve ocupar um lugar central na educação infantil e o professor deve proporcionar esse contato oferecendo meios necessários para partilhar desse momento com as crianças. Referente à terceira questão: “Quando você faz isso? Você faz bastante isso?”, as crianças afirmaram que brincar no parque de areia é o que mais gostam, pois podem correr à vontade, fazer castelos de areia, bolo e etc. Levando-se em conta o local que a escola oferece (areia protegida e tratada; à sombra), o parque de areia é um recurso fundamental em qualquer espaço de educação infantil, até mesmo no quintal de casa. Quando oferecido à criança na escola, a areia molhada vira castelos, cidades, bolo, macarrão, muros e tudo o que a imaginação dela permitir, conforme as crianças relataram. A água e a areia são elementos que estimulam os sentidos, ajudam no crescimento e desenvolvimento além de trabalhar a coordenação motora. Porém, no interior do parque as crianças fazem uso de determinadas „regrinhas‟ para que as brincadeiras ocorram com segurança e sem brigas entre elas. A professora coloca que: (02) Professora: No parque as crianças são orientadas a não jogarem areia uma nas outras, pois, em situações já ocorridas crianças jogaram areia nos olhos das outras causando ardência. Não é permitido também ficar em pé nos brinquedos que devem permanecer sentados, que é o caso do balanço e da gangorra. JOGOS E BRINCADEIRAS NA EDUCAÇÃO INFANTIL, ANÁLISE... - Página 137 O tanque de areia é o local que permite a interação com outras crianças através da linguagem, enriquecendo o vocabulário, tornando-os mais criativos e promovendo a socialização. Além disso, é um lugar que favorece o brincar junto, pois o espaço físico leva as crianças a planejarem junto o que irão criar. Assim crianças tímidas conseguem trabalhar melhor e interagir com as demais sem se expor tanto. Segundo Vygotsky (1991), a imaginação é uma atividade consciente que não está presente na criança muito pequena. Como todas as funções da consciência, ela surge originalmente das ações da criança. A brincadeira que comporta uma situação imaginaria também contém regras. Não uma regra explícita, mas criada pela própria criança. A areia é um elemento natural, que auxilia ainda no trabalho com crianças agitadas, pois tranquiliza e tem o poder de fazer com que elas se concentrem na atividade. A professora da turma falou a respeito dessa euforia, agitação que é normal da criança, e que em determinados momentos ela utiliza de técnicas de relaxamento para eles se acalmarem. Com base nos relatos entendemos que o lúdico as ações vividas e sentidas, caracterizadas pela fantasia, pela imaginação e sonhos que são representados por meio de materiais simbólicos estão presentes na vida das crianças pesquisadas. E tem por característica principal o espontâneo funcional e satisfatório. Segundo Almeida (2006) na atividade lúdica, o importante é o momento vivido durante a ação. Essa atividade propicia momentos de encontro consigo e com outro, propicia momentos de fantasia e de realidade, momentos que ao mesmo tempo cuida de si e também do outro. Com a atividade lúdica no ambiente escolar, a criança aprende de maneira prazerosa, e facilita uma melhor aprendizagem e melhor aproveitamento do conteúdo. Sendo assim, o professor ao utilizar atividades lúdicas, deixa de ser o centro e o controlador, e passa a dar importância à participação do aluno na situação de ensino, dessa maneira o professor estimula o aluno a trabalhar questões como a espontaneidade e a criatividade. 5 CONCLUSÃO Diante do objetivo desse trabalho, verificar como as brincadeiras estão sendo realizadas no Pré II do CMEI Vinicius de Moraes, após as entrevistas realizadas, bem como a análise delas, concluímos que, por meio do brincar, valores, costumes, crenças, normas e regras são construídos, transmitidos e apreendidos pelas crianças, por isso é tão importante o brincar na infância. Página 138 - Joanil Maria da Silva e Adil Antônio Alves de Oliveira Por meio dessa pesquisa, pudemos perceber que as atividades preferidas das crianças do Pré II é o brincar. Percebendo isso, e sabendo da importância desse ato para o desenvolvimento das crianças sugerimos aos educadores desse nível, que ao prepararem suas aulas insiram atividades lúdicas com fundamentos pedagógicos para suas aulas. A intervenção do educador é fundamental, pois o brincar pelo brincar não deve ser uma constante, o papel mediador do adulto é que proporciona situações de aprendizagem e desenvolvimento. Esperamos que esse trabalho possa contribuir diretamente, ou indiretamente para a preparação das aulas das crianças nos CMEIs de Sinop. Também esperamos que os educadores possam refletir no ato de elaborarem suas aulas, de modo a conciliar atividades lúdicas com atividades pedagógicas. Para que, desta forma, as brincadeiras das crianças desse nível não sejam realizadas de forma “solta” e sem fundamentos. A presença das atividades lúdicas pode facilitar uma melhor aprendizagem, e também um processo de relação social, seja com as outras crianças de sala como também com o professor, pois o mesmo deve participar das brincadeiras das crianças, para que as brincadeiras aconteçam de forma organizada e com sentido pedagógico. LES JEUX ET LE JOUER DANS L'ÉDUCATION DE LA PETITE ENFANCE, ANALYSE D’UNE CLASSE D PETITS ENFANTS, NIVEAU II, AU CENTRE MUNICIPAL D’ÉDUCATION DE LA PETITE ENFANCE VINICIUS DE MORAES RÉSUMÉ1 La recherche a été menée au Centre Municipal de l'éducation de la Petite Enfance Vinicius de Moraes, dans la ville de Sinop-MT. L'objectif de cette recherche a été d‟examiner comment sont traitées activités avec du jeu dans l'éducation de l'enfant. Dans un dialogue avec la professeur et la coordination pédagogique CMEI la question posé a été quelle sur l'importance du jeu dans le processus de développement de l'apprentissage comme une forme de contenu réalisé dans les activités quotidiennes de l'institution, et quand elles sont travaillées. Dans ce contexte, en invitant les enseignants à réfléchir sur la connaissance des pratiques avec des jeux que sont fait avec leurs étudiants, à travers de la littérature existante sur ce thème. Pour cette étude on a recouru aux auteurs suivants Tizuko Morchida Kishimoto, Luiz Philippe Ariès et Joseph Straub. Pour atteindre mes objectifs, on a travaillé avec des observations, entrevues et des questionnaires. Les réponses ont été analysées en regardant des 1 Transcrição realizada pelo aluno Fernando Hélio Tavares de Barros (CRLE – Revista Eventos Pedagógicos) e revisão pelo professor Ederson Lima de Souza (CRLE – Revista Eventos Pedagógicos). JOGOS E BRINCADEIRAS NA EDUCAÇÃO INFANTIL, ANÁLISE... - Página 139 lectures prises. Il a été vérifié que les jeux sont des activités préférées des enfants. Nous avons l'intention que, connaissant les préférences des enfants, les enseignants de ce type d'enseignement peuvent offrir des activités qui permettent un meilleur apprentissage de leur part, puisque le jeu est un outil important pour l'enseignant de l'éducation de la petite enfance. Mots-clés: Éducation de la Petite Enfance. Jouer. Jeux. REFERÊNCIAS ARIÉS, Philippe. História social da criança e da família. Tradução Dora Flaksman. Rio de Janeiro: Livros Técnicos e Científicos, 1981. BRASIL. Ensino fundamental de nove anos: orientações para a inclusão da criança de seis anos de idade – Brasília: Ministério da Educação, Secretaria de Educação Básica, 2006. ______ . Constituição da República Federativa do Brasil. São Paulo: Imprensa Oficial do Estado, 1988. Disponível em www.planalto.gov.br/ccivil_03/constituicao/constituiçao.htm. Acesso em: 02 Ago 2011. ______ . Revista Criança do professor de educação infantil – Brasília: Ministério da Educação, Secretaria de Educação Básica, 2007. Disponível em <http://portal.mec.gov.br/seb/arquivos/pdf/Educinf/revista44.pdf>. Acesso em: 02 jun 2012. KISHIMOTO, Tizuko Morchida. (Org.) Jogo, brinquedo, brincadeira e a educação. 14. ed. São Paulo: Cortez, 2011. LÜDKE, Menga; ANDRÉ, Marli E. D. A. Pesquisa em educação: abordagens qualitativas. São Paulo: EPU, 1986. TRIVIÑOS, A. N. S. Introdução a Pesquisa em Ciências Sociais: a pesquisa qualitativa em educação. São Paulo: Atlas, 1997. VYGOTSKY, L.S. A formação social da mente. São Paulo: Martins, 1987. Página 140 - Joanil Maria da Silva e Adil Antônio Alves de Oliveira
https://openalex.org/W2981498720	https://www.scielo.br/j/rb/a/CqsMynPYgS4L3wg589qpX9M/?lang=en&format=pdf	English	null	The many faces of primary and secondary hepatic lymphoma: imaging manifestations and diagnostic approach	Radiologia brasileira/Radiologia Brasileira	2,019	cc-by	3,908	Aldo Maurici Araújo Alves1,a, Ulysses Santos Torres2,b, Fernanda Garozzo Velloni1,3,c, Bruno Jucá Ribeiro1,d, Dario Ariel Tiferes1,2,e, Giuseppe D’Ippolito1,2,f 1. Department of Diagnostic Imaging, Escola Paulista de Medicina da Universidade Federal de São Paulo (EPM-Unifesp), São Paulo, SP, Brazil. 2. Grupo Fleury, São Paulo, SP, Brazil. 3. Diagnósticos da América S/A, Barueri, SP, Brazil. Correspondence: Dr. Aldo Maurici Araújo Alves. EPM-Unifesp – Departamento de Diagnóstico por Imagem. Rua Napoleão de B Clementino. São Paulo, SP, Brazil, 04024-002. Email: aldomaurici@gmail.com. ://orcid.org/0000-0001-7310-5709; b. https://orcid.org/0000-0002-1911-9090; c. https://orcid.org/0000-0002-2019-7918 s://orcid.org/0000-0002-6947-1375; e. https://orcid.org/0000-0003-2339-5730; f. https://orcid.org/0000-0002-2701-1928 Received 31 January 2018. Accepted after revision 3 April 2018. How to cite this article: Alves AMA, Torres US, Velloni FG, Ribeiro BJ, Tiferes DA, D’Ippolito G. The many faces of primary and secondary hepatic lymphoma: imaging mani- festations and diagnostic approach. Radiol Bras. 2019 Set/Out;52(5):325–330. Alves AMA, Torres US, Velloni FG, Ribeiro BJ, Tiferes DA, D’Ippolito G. The many faces of primary and secondary hepatic lymphoma: imaging mani- festations and diagnostic approach. Radiol Bras. 2019 Set/Out;52(5):325–330. Abstract Hepatic lymphoma can be divided into its primary and secondary forms. To be classified as primary hepatic lymphoma, the disease should be limited to the liver and hilar lymph nodes, with no distant involvement (to the bone marrow or other sites). Primary hepatic lymphoma is quite rare, accounting for less than 1% of all cases of non-Hodgkin lymphoma, whereas secondary hepatic lymphoma affects nearly half of such cases. Familiarity with the imaging features of hepatic lymphoma is important for its early diagnosis and appropriate management. In this essay, we aim to review the roles of computed tomography and magnetic resonance imaging in identifying and monitoring hepatic lymphomas, using sample cases to describe and illustrate the wide spectrum of imaging findings in the primary and secondary forms of this disorder. Keywords: Lymphoma; Liver; Magnetic resonance imaging; Tomography, X-ray computed. Resumo O linfoma hepático pode ser dividido em primário e secundário. Para ser classificada como linfoma hepático primário, a doença deve estar limitada ao fígado e linfonodos hilares, sem acometimento de outros órgãos (incluindo a medula óssea). O linfoma he- pático primário é muito raro, correspondendo a menos de 1% de todos os linfomas não Hodgkin. Já o envolvimento secundário do fígado ocorre em quase metade dos casos de linfoma não Hodgkin. O conhecimento dos aspectos de imagem do linfoma hepático é importante para o diagnóstico precoce e manejo adequado do paciente. et al. / The many faces Pictorial Essay et al. / The many faces Pictorial Essay http://dx.doi.org/10.1590/0100-3984.2018.0013 Aldo Maurici Araújo Alves1,a, Ulysses Santos Torres2,b, Fernanda Garozzo Velloni1,3,c, Bruno Jucá Ribeiro1,d, Dario Ariel Tiferes1,2,e, Giuseppe D’Ippolito1,2,f Com base em imagens de casos, nosso objetivo é revisar o papel da imagem por tomografia computadorizada e ressonância magnética na identificação e segmento do linfoma hepático, ilustrando e descrevendo o amplo espectro de achados de imagem observados nas formas primária e secundária da doença. Unitermos: Linfoma; Fígado; Ressonância magnética; Tomografia computadorizada. Unitermos: Linfoma; Fígado; Ressonância magnética; Tomografia computadorizada. The many faces of primary and secondary hepatic lymphoma: imaging manifestations and diagnostic approach As diversas faces do linfoma hepático primário e secundário: manifestações de imagem e abordagem diagnóstica Radiol Bras. 2019 Set/Out;52(5):325–330 IMAGING FINDINGS Secondary hepatic lymphoma typically presents as diffuse infiltration (Figure 2) or multifocal homogeneous lesions along with extrahepatic disease (splenic lesions, lymphadenopathy, bone marrow infiltration, etc.). A miliary pattern is more common in secondary hepatic lymphoma (Figure 3).i Hepatic lymphoma has a wide range of imaging pre- sentations, typically being evaluated on computed tomog- raphy (CT) or magnetic resonance imaging (MRI). It can appear as a solitary (homogeneous or heterogeneous) mass or as multiple lesions, with or without a dominant lesion, or even in a miliary pattern characterized by multiple small discrete nodules. Other patterns include diffuse infiltration (with or without hepatomegaly) and a mass in the porta hepatis. Rarer forms with no defined imaging pattern can also be seen. Although the imaging findings of primary hepatic lymphoma and secondary hepatic lymphoma may over- lap, some features can facilitate the diagnostic discrimi- nation. Dominant liver lesions, which tend to be hetero- geneous (Figure 1), are characteristic of primary hepatic lymphoma and are not expected to be found in secondary hepatic lymphoma. In addition, even large secondary he- patic lymphoma lesions tend to be homogeneous before The most common manifestation of primary hepatic lymphoma is a heterogeneous solitary mass (Figure 1), whereas multiple lesions are seen in less than 33% of Figure 3. Secondary hepatic lymphoma. Axial contrast-enhanced CT image showing multiple homogeneous hypoattenuating hepatic lesions (white arrow) and multiple small hepatic and splenic nodules in a miliary pattern (black ar- rows). There was also prominent retroperitoneal lymphadenopathy (not shown). Figure 1. Axial portal venous CT image showing a solitary heterogeneous mass in the left lobe of the liver, with a rim-enhancement (target-like) pattern. The spleen and periportal nodes were not involved (not shown). The final diagnosis was primary non-Hodgkin lymphoma, as confirmed by biopsy. Figure 3. Secondary hepatic lymphoma. Axial contrast-enhanced CT image showing multiple homogeneous hypoattenuating hepatic lesions (white arrow) and multiple small hepatic and splenic nodules in a miliary pattern (black ar- rows). There was also prominent retroperitoneal lymphadenopathy (not shown). Figure 1. Axial portal venous CT image showing a solitary heterogeneous mass in the left lobe of the liver, with a rim-enhancement (target-like) pattern. The spleen and periportal nodes were not involved (not shown). The final diagnosis was primary non-Hodgkin lymphoma, as confirmed by biopsy. Figure 2. Axial contrast-enhanced CT images (A,B) showing diffuse infiltration of the liver parenchyma and hepatomegaly. INTRODUCTION Although the pathophysiology of primary hepatic lym- phoma is poorly understood, it has been associated with prolonged immunosuppression (including that caused by infection with HIV) and Epstein-Barr virus, as well as with hepatitis B and C. The most common subtype of primary hepatic lymphoma is diffuse large B-cell lymphoma(4). Hepatic lymphoma can be divided into a primary form and a secondary (metastatic) form, the two forms having different prognoses. Non-Hodgkin lymphoma predomi- nates in both forms(1). To be classified as primary hepatic lymphoma, the disease should be limited to the liver and hilar lymph nodes, with no distant involvement. Accord- ing to some authors, the diagnosis of primary hepatic lym- phoma also requires the absence of distant disease (distant lymphadenopathy, splenic infiltration or bone marrow dis- ease) within six months of diagnosis(2). The clinical manifestations of primary and second- ary hepatic lymphoma include hepatomegaly, right upper quadrant pain, jaundice, and systemic (B) symptoms (fever, weight loss, and night sweats)(1). Imaging plays an important role in the diagnosis, stag- ing, and follow-up of cases of hepatic lymphoma, being used not only to evaluate the evolution of the disease but also to guide relevant procedures. The most common extranodal site of involvement by non-Hodgkin lymphoma is the gastrointestinal tract, sec- ondary hepatic lymphoma being observed in nearly half of all patients with such involvement. However, primary he- patic lymphoma is quite rare, accounting for less than 1% of all non-Hodgkin lymphomas(3). The management and prognosis of lymphoma are re- markably different from those of other neoplasms. Hence, familiarity with the imaging features of hepatic lymphoma Radiol Bras. 2019 Set/Out;52(5):325–330 0100-3984 © Colégio Brasileiro de Radiologia e Diagnóstico por Imagem 325 Radiol Bras. 2019 Set/Out;52(5):325–330 0100-3984 © Colégio Brasileiro de Radiologia e Diagnóstico por Imagem 0100-3984 © Colégio Brasileiro de Radiologia e Diagnóstico por Imagem Alves AMA et al. / The many faces of primary and secondary hepatic lymphoma s AMA et al. / The many faces of primary and secondary hepat cases(5). The diffuse form of primary hepatic lymphoma is uncommon and is associated with a poor prognosis. More than one pattern may be present in the same patient(5). is important for its early diagnosis and appropriate man- agement. IMAGING FINDINGS Analysis of a biopsy sample indicated that the lesion was non-Hodgkin lymphoma (secondary hepatic lymphoma) A B Figure 4. A 32-year-old man with jaundice. On portal phase CT images, a hypovascular lesion is noted in the form of periportal tissue involving the portal vein (black arrow) and the bile duct, with dilatation of the intrahepatic biliary ducts (arrowheads). Splenomegaly is also seen (asterisk). Analysis of a biopsy sample indicated that the lesion was non-Hodgkin lymphoma (secondary hepatic lymphoma). A B B Figure 4. A 32-year-old man with jaundice. On portal phase CT images, a hypovascular lesion is noted in the form of periportal tissue involving the portal vein (black arrow) and the bile duct, with dilatation of the intrahepatic biliary ducts (arrowheads). Splenomegaly is also seen (asterisk). Analysis of a biopsy sample indicated that the lesion was non-Hodgkin lymphoma (secondary hepatic lymphoma). chemotherapy. Table 1 lists the imaging characteristics of the primary and secondary forms. Figure 5. Primary hepatic non-Hodgkin lymphoma. Axial venous phase CT im- age showing a solitary heterogeneous mass, with a target-like appearance, in the right lobe of the liver. Note the large nonenhancing central area (asterisk), consistent with necrosis. Another form of presentation of primary or secondary hepatic lymphoma is a periportal mass, which generally presents as an ill-defined, infiltrating, homogeneous and poorly enhancing hilar tissue (Figure 4). IMAGING FINDINGS There is encasement without occlusion of the hepatic vessels (black arrow) or celiac trunk (white arrow). Diffuse enlarged retroperitoneal lymph nodes (arrowhead) are also noted. CT-guided biopsy of the liver resulted in a diagnosis of secondary hepatic non-Hodgkin lymphoma. A B B Figure 2. Axial contrast-enhanced CT images (A,B) showing diffuse infiltration of the liver parenchyma and hepatomegaly. There is encasement without occlusion of the hepatic vessels (black arrow) or celiac trunk (white arrow). Diffuse enlarged retroperitoneal lymph nodes (arrowhead) are also noted. CT-guided biopsy of the liver resulted in a diagnosis of secondary hepatic non-Hodgkin lymphoma. Radiol Bras. 2019 Set/Out;52(5):325–330 326 Alves AMA et al. / The many faces of primary and secondary hepatic lymphoma Table 1—Imaging characteristics of primary and secondary hepatic lymphoma. Characteristic Lesion distribution Morphology Dominant mass Splenic and distant lymphadenopathy Hepatic lymphoma Primary Solitary (more common) or multiple Heterogeneous Typical Absent (by definition) Secondary Multiple (more common) or diffuse Homogenous Uncommon Present (splenic lesions in 33% of cases) Alves AMA et al. / The many faces of primary and secondary hepatic lymphoma Table 1—Imaging characteristics of primary and secondary hepatic lymphoma. Characteristic Lesion distribution Morphology Dominant mass Splenic and distant lymphadenopathy Hepatic lymphoma Primary Solitary (more common) or multiple Heterogeneous Typical Absent (by definition) Secondary Multiple (more common) or diffuse Homogenous Uncommon Present (splenic lesions in 33% of cases) Figure 4. A 32-year-old man with jaundice. On portal phase CT images, a hypovascular lesion is noted in the form of periportal tissue involving the portal vein (black arrow) and the bile duct, with dilatation of the intrahepatic biliary ducts (arrowheads). Splenomegaly is also seen (asterisk). Analysis of a biopsy sample indicated that the lesion was non-Hodgkin lymphoma (secondary hepatic lymphoma). A B Alves AMA et al. / The many faces of primary and secondary hepatic lymphoma Table 1—Imaging characteristics of primary and secondary hepatic lymphoma. Characteristic Lesion distribution Morphology Dominant mass Splenic and distant lymphadenopathy Hepatic lymphoma Primary Solitary (more common) or multiple Heterogeneous Typical Absent (by definition) Secondary Multiple (more common) or diffuse Homogenous Uncommon Present (splenic lesions in 33% of cases) Figure 4. A 32-year-old man with jaundice. On portal phase CT images, a hypovascular lesion is noted in the form of periportal tissue involving the portal vein (black arrow) and the bile duct, with dilatation of the intrahepatic biliary ducts (arrowheads). Splenomegaly is also seen (asterisk). CT and MRI features On CT, hepatic lymphoma shows soft tissue attenua- tion. Necrosis or hemorrhage can also be seen. Calcifica- tion is quite rare before treatment. The majority of hepatic lesions show minimal enhancement after the intravenous administration of a contrast agent. Rim enhancement (a target-like lesion) has also been described(6) and is more common in primary hepatic lymphoma (Figures 1 and 5). On MRI, hepatic lymphoma lesions tend to show hy- pointense or isointense signals on T1-weighted imaging (T1WI) and hyperintense signals on T2-weighted imag- ing (T2WI). Although such lesions typically show a ho- mogenous signal, necrosis or hemorrhage can cause some heterogeneity(1). A target-like appearance on T2WI, as de- picted in Figure 6, has also been described(1). Figure 5. Primary hepatic non-Hodgkin lymphoma. Axial venous phase CT im- age showing a solitary heterogeneous mass, with a target-like appearance, in the right lobe of the liver. Note the large nonenhancing central area (asterisk), consistent with necrosis. been reported(1). On diffusion-weighted imaging, hepatic lymphoma lesions usually demonstrate markedly restricted diffusion, which is explained by their highly cellular histol- ogy (Figure 7). On 18F-fluorodeoxyglucose positron emis- sion tomography/CT (FDG-PET/CT), primary and sec- ondary lesions both typically show avid hypermetabolism, been reported(1). On diffusion-weighted imaging, hepatic lymphoma lesions usually demonstrate markedly restricted diffusion, which is explained by their highly cellular histol- ogy (Figure 7). On 18F-fluorodeoxyglucose positron emis- sion tomography/CT (FDG-PET/CT), primary and sec- ondary lesions both typically show avid hypermetabolism, The pattern of enhancement in hepatic lymphoma is similar on CT and MRI. Central retention of hepatobili- ary contrast agent in the delayed (hepatobiliary) phase has 327 Radiol Bras. 2019 Set/Out;52(5):325–330 Alves AMA et al. / The many faces of primary and secondary hepatic lymphoma Alves AMA et al. / The many faces of primary and secondary hepatic lymphoma Figure 6. Primary hepatic lymphoma with a target-like appearance on MRI. Axial T2WI (A) showing a solitary mass, with a signal that is hyperintense in its center and hypointense at its periphery, in the right lobe of the liver. Axial gadolinium contrast-enhanced MRI scan (B) showing a rim-enhancement pattern. A B B Figure 6. Primary hepatic lymphoma with a target-like appearance on MRI. Axial T2WI (A) showing a solitary mass, with a signal that is hyperintense in its center and hypointense at its periphery, in the right lobe of the liver. Axial gadolinium contrast-enhanced MRI scan (B) showing a rim-enhancement pattern. Figure 7. CT and MRI features A 49-year-old man with weight loss and night sweats. MRI images show hyperintensity on T2WI (A), as well as markedly restricted diffusion on diffusion- weighted imaging with a b value of 1000 s/mm2 (B) and on an apparent diffusion coefficient map (C). The mean apparent diffusion coefficient value was 450 × 10−6 mm2/s. The final diagnosis was primary hepatic Burkitt’s lymphoma, as confirmed by biopsy and immunochemistry. A B C C C B Figure 7. A 49-year-old man with weight loss and night sweats. MRI images show hyperintensity on T2WI (A), as well as markedly restricted diffusion on diffusion- weighted imaging with a b value of 1000 s/mm2 (B) and on an apparent diffusion coefficient map (C). The mean apparent diffusion coefficient value was 450 × 10−6 mm2/s. The final diagnosis was primary hepatic Burkitt’s lymphoma, as confirmed by biopsy and immunochemistry. (Figure 8), although it is not pathognomonic, because lymph nodes in this region may also be involved second- ary to the dissemination of pelvic malignancies. FDG-PET/CT also being considered the imaging modal- ity of choice for staging and for assessing treatment re sponses(6). However, whole-body diffusion-weighted imag- ing also allows pre- and post-treatment staging, providing results comparable to those of FDG-PET/CT(7). Rarer forms of hepatic lymphoma Primary lymphoma arising from the bile duct (Fig- ure 9) is extremely rare, with imaging features overlap- ping those of cholangiocarcinoma(8). Other rare forms of hepatic lymphoma include intravascular hepatic lym- phoma(9) and hepatosplenic T-cell lymphoma(10), both of which have been scarcely reported in the literature and have no specifically defined imaging pattern. Imaging tips In addition to clinical and laboratory data favoring the diagnosis of hepatic lymphoma (e.g., a young age, B symp- toms, and abnormal bone marrow biopsy), imaging features can contribute to the diagnostic discrimination. Vascular or biliary encasement without thrombosis or ductal dilatation may occur in some cases of hepatic lymphoma (Figure 2), owing to the absence of fibrous stroma and desmoplastic tissue(6); that is, these lymphomas may be sufficiently mal- leable to mold and grow throughout the periportal space without causing substantial compression of the biliary or vascular tree. That feature may be observed in intrahepatic (diffuse or mass-forming) and periportal lesions(6). Atypical findings, mimics, and the differential diagnosis Although not typical, portal vein thrombosis (Figure 8) and bile duct obstruction may occur in hilar and periportal hepatic lymphoma (Figures 4 and 9) as well as in mass- forming intrahepatic lesions (Figure 10). Hepatic lym- phoma has a wide range of differential diagnoses and can mimic many conditions, such as intrahepatic cholangiocar- cinoma, inflammatory pseudotumor, and primary hepatic neuroendocrine tumor. In addition, because infection with hepatitis C or B virus is also a risk factor for primary hepatic The coexistence of splenic lesions and extensive (su- pra- or infra-diaphragmatic) nodal disease favors a diag- nosis of secondary hepatic lymphoma. An enlarged poste- rior iliac crest lymph node is a classic sign of lymphoma 328 Radiol Bras. 2019 Set/Out;52(5):325–330 Alves AMA et al. / The many faces of primary and secondary hepatic lymphoma Alves AMA et al. / The many faces of primary and secondary hepatic lymphoma Figure 8. A 70-year-old man with dyspepsia and weight loss. Axial contrast-enhanced CT images showing multiple homogenous hepatic nodules without a domi- nant mass. Note thrombosis of the left portal vein (black arrow in A), multiple homogeneous lesions in the gastric wall (white arrow in A), and the enlarged lymph node slightly above the left posterior iliac crest (arrowhead in B). The final diagnosis was non-Hodgkin lymphoma, as confirmed by endoscopic biopsy. B A B Figure 8. A 70-year-old man with dyspepsia and weight loss. Axial contrast-enhanced CT images showing multiple homogenous hepatic nodules without a domi- nant mass. Note thrombosis of the left portal vein (black arrow in A), multiple homogeneous lesions in the gastric wall (white arrow in A), and the enlarged lymph node slightly above the left posterior iliac crest (arrowhead in B). The final diagnosis was non-Hodgkin lymphoma, as confirmed by endoscopic biopsy. Figure 9. A 28-year-old man with jaundice. MRI showing an ill-defined, infiltrative mass extending from the porta hepatis to the parenchyma following the portal triads. Coronal T2WI (A) showing an area with a slightly hyperintense signal. Magnetic resonance cholangiopancreatography (B) showing dilatation of the segmen- tal bile ducts. These imaging characteristics are indistinguishable from those of hilar cholangiocarcinoma. The final diagnosis was diffuse large B-cell lymphoma, as confirmed by biopsy. A B B B Figure 9. A 28-year-old man with jaundice. MRI showing an ill-defined, infiltrative mass extending from the porta hepatis to the parenchyma following the portal triads. Atypical findings, mimics, and the differential diagnosis Coronal T2WI (A) showing an area with a slightly hyperintense signal. Magnetic resonance cholangiopancreatography (B) showing dilatation of the segmen- tal bile ducts. These imaging characteristics are indistinguishable from those of hilar cholangiocarcinoma. The final diagnosis was diffuse large B-cell lymphoma, as confirmed by biopsy. the rare primary biliary subtype of lymphoma and hepatic lymphoma manifesting as a mass in the porta hepatis, as depicted in Figures 4 and 9, respectively, may be indistin- guishable from cholangiocarcinoma(8). lymphoma, a solitary infiltrative lymphoma in the setting of cirrhosis may be difficult to differentiate from the dif- fuse form of hepatocellular carcinoma. Albeit uncommon, primary hepatic lymphoma can have an appearance similar to that of hepatocellular carcinoma on contrast-enhanced images. The differential diagnoses of the multifocal form of hepatic lymphoma include granulomatous diseases, fungal infections, and metastasis. Especially in immunosuppressed patients, including those with HIV infection, which is a risk factor for lymphoma (Figure 11), it can be difficult to make the distinction among those entities on the basis of imag- ing findings alone(1). The diffuse form of hepatic lymphoma may also be confused with acute hepatitis(1). On imaging, CONCLUSION CT and MRI play important roles in the management of hepatic lesions, including lymphoma. Although there are no specific imaging features for hepatic lymphoma and biopsy is nearly always necessary, familiarity with the most common imaging features can promote early suspi- cion and appropriate management, which can improve the prognosis. 329 Radiol Bras. 2019 Set/Out;52(5):325–330 Alves AMA et al. / The many faces of primary and secondary hepatic lymphoma Alves AMA et al. / The many faces of primary and secondary hepatic lymphoma Figure 10. Secondary hepatic lymphoma in a 58-year-old woman with B symptoms, presenting with the intrahepatic mass-forming and periportal forms. Severe dilatation of the bile duct in the left lobe of the liver (white arrow in A) caused by a periportal lesion (black arrow in B). The final diagnosis was Burkitt’s lymphoma, as confirmed by biopsy of the left inguinal lymph node (not shown). A B B Figure 10. Secondary hepatic lymphoma in a 58-year-old woman with B symptoms, presenting with the intrahepatic mass-forming and periportal forms. Severe dilatation of the bile duct in the left lobe of the liver (white arrow in A) caused by a periportal lesion (black arrow in B). The final diagnosis was Burkitt’s lymphoma, as confirmed by biopsy of the left inguinal lymph node (not shown). Figure 11. A 33-year-old man with HIV infection and abdominal pain. Axial contrast-enhanced CT showing hypodense hepatic lesions (A,B) and multiple hypodense nodules throughout the pancreas. The final diagnosis was diffuse large B-cell lymphoma, as confirmed by biopsy. A B Figure 11. A 33-year-old man with HIV infection and abdominal pain. Axial contrast-enhanced CT showing hypodense hepatic nodules throughout the pancreas. The final diagnosis was diffuse large B-cell lymphoma, as confirmed by biopsy. Figure 11. A 33-year-old man with HIV infection and abdominal pain. Axial contrast-enhanced CT showing hypodense hepatic lesions (A,B) and multiple hypodense nodules throughout the pancreas. The final diagnosis was diffuse large B-cell lymphoma, as confirmed by biopsy. Acknowledgment 5. Maher MM, McDermott SR, Fenlon HM, et al. Imaging of primary non-Hodgkin’s lymphoma of the liver. Clin Radiol. 2001;56:295– 301. The authors thank Dr. Henrique Simão Trad (Ribeirão Preto, SP, Brazil) for kindly having provided the case of figure 9. 6. Tomasian A, Sandrasegaran K, Elsayes KM, et al. Hematologic ma- lignancies of the liver: spectrum of disease. Radiographics. 2015;35: 71–86. REFERENCES 7. Abdulqadhr G, Molin D, Aström G, et al. Whole-body diffusion- weighted imaging compared with FDG-PET/CT in staging of lym- phoma patients. Acta Radiol. 2011;52:173–80. 1. Rajesh S, Bansal K, Sureka B, et al. The imaging conundrum of hepatic lymphoma revisited. Insights Imaging. 2015;6:679–92. 1. Rajesh S, Bansal K, Sureka B, et al. The imaging conundrum of hepatic lymphoma revisited. Insights Imaging. 2015;6:679–92. 2. Anthony PP, Sarsfield P, Clarke T. Primary lymphoma of the liver: clinical and pathological features of 10 patients. J Clin Pathol. 1990;43:1007–13. 2. Anthony PP, Sarsfield P, Clarke T. Primary lymphoma of the liver: clinical and pathological features of 10 patients. J Clin Pathol. 1990;43:1007–13. 8. Yoon MA, Lee JM, Kim SH, et al. Primary biliary lymphoma mim- icking cholangiocarcinoma: a characteristic feature of discrepant CT and direct cholangiography findings. J Korean Med Sci. 2009; 24:956–9. 3. Howell JM, Auer-Grzesiak I, Zhang J, et al. Increasing incidence rates, distribution and histological characteristics of primary gastro- intestinal non-Hodgkin lymphoma in a North American population. Can J Gastroenterol. 2012;26:452–6. 9. Abe H, Kamimura K, Kawai H, et al. Diagnostic imaging of hepatic lymphoma. Clin Res Hepatol Gastroenterol. 2015;39:435–42. 10. Ashmore P, Patel M, Vaughan J, et al. Hepatosplenic T-cell lympho- ma: a case series. Hematol Oncol Stem Cell Ther. 2015;8:78–84. 4. Chim CS, Choy C, Ooi GC, et al. Primary hepatic lymphoma. Leuk Lymphoma. 2001;40:667–70. 4. Chim CS, Choy C, Ooi GC, et al. Primary hepatic lymphoma. Leuk Lymphoma. 2001;40:667–70. Radiol Bras. 2019 Set/Out;52(5):325–330 330
https://openalex.org/W3015688288	https://bjsm.bmj.com/content/bjsports/55/3/144.full.pdf	English	null	Effects of sedentary behaviour interventions on biomarkers of cardiometabolic risk in adults: systematic review with meta-analyses	British journal of sports medicine	2,020	cc-by	15,044	Abstract living with diabetes: a figure projected to rise to 693 million (≈10% of the population) by 2045.3 Context/purpose Observational and acute laboratory intervention research has shown that excessive sedentary time is associated adversely with cardiometabolic biomarkers. This systematic review with meta-analyses synthesises results from free living interventions targeting reductions in sedentary behaviour alone or combined with increases in physical activity. The evidence tends to indicate that greater time spent in sedentary behaviour (ie, sitting/reclining at <1.5 metabolic equivalents (MET))4 is adversely associated with the risk of cardiovascular disease, type 2 diabetes and some cancers,5 6 and with levels of a range of cardiometabolic risk biomarkers.7 8 A less prolonged sedentary accumulation pattern (ie, more regular breaks, shorter sedentary bouts) has also been associated with lower body mass index (BMI).8 It has largely been acute laboratory interventions (<7 days) using structured protocols providing experimental evidence that reducing or breaking up sitting can have beneficial effects on certain cardiometabolic biomarkers.9–12 For example, compared with uninterrupted sitting time, adding short bouts of light or moderate intensity activity every 20–30 min (generally over a period of 1–5 days) results in improvements to resting blood pressure,13 14 fasting and postprandial glucose15 16 and insulin,15 17 18 and some lipids.19 For numbered affiliations see end of article. For numbered affiliations see end of article. Correspondence to Dr Paddy C Dempsey, MRC Epidemiology Unit, Institute of Metabolic Science, University of Cambridge, Cambridge Biomedical Campus, Cambridge, UK; Paddy.Dempsey@baker.edu.au NTH and EW contributed equally. Accepted 11 March 2020 Published Online First 8 April 2020 Correspondence to Dr Paddy C Dempsey, MRC Epidemiology Unit, Institute of Metabolic Science, University of Cambridge, Cambridge Biomedical Campus, Cambridge, UK; p y y Methods Six electronic databases were searched up to August 2019 for sedentary behaviour interventions in adults lasting for ≥7 days publishing cardiometabolic biomarker outcomes covering body anthropometry, blood pressure, glucose and lipid metabolism, and inflammation (54 studies). The pooled effectiveness of intervention net of control on 15 biomarker outcomes was evaluated using random effects meta-analyses in the studies with control groups not providing other relevant interventions (33 studies; 6–25 interventions analysed). Results Interventions between 2 weeks and <6 months in non-clinical populations from North America, Europe and Australia comprised much of the evidence base. on October 23, 2024 by guest. Protected by cop http://bjsm.bmj.com/ Br J Sports Med: first published as 10.1136/bjsports-2019-101154 on 8 April 2020. Downloaded from ► ►Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ bjsports-2019-101154). Abstract Pooled effects revealed small, significant (p<0.05) beneficial effects on weight (≈ −0.6 kg), waist circumference (≈ −0.7 cm), percentage body fat (≈ −0.3 %), systolic blood pressure (≈ −1.1 mm Hg), insulin (≈ −1.4 pM) and high-density lipoprotein cholesterol (≈ 0.04 mM). Pooled effects on the other biomarkers (p>0.05) were also small, and beneficial in direction except for fat-free mass (≈ 0.0 kg). Heterogeneity ranged widely (I2=0.0–72.9). NTH and EW contributed equally. Accepted 11 March 2020 Published Online First 8 April 2020 In recognition of the aforementioned evidence, several countries now, in addition to having guide- lines concerning physical activity, include guidelines to reduce the quantity of sedentary behaviour and/ or break it up.20–22 A variety of intervention strat- egies have been trialled to reduce adults’ levels of sedentary behaviour, particularly in the workplace setting.23 24 Reviews indicate these interventions are often effective for reducing sedentary behaviour, especially workplace interventions incorporating environmental modification, ideally as part of a multicomponent intervention.23 25–27 What is lacking, however, is an understanding of the nature and extent of health improvements that might be obtained when intervening to reduce sedentary behaviour over longer periods and under free-living conditions. A preliminary evaluation explored this topic (in workplace interventions only) but, having occurred prior to the emergence of several large trials of sedentary behaviour interventions, did not present any meta-analyses and could draw no firm conclusions.28 Conclusions Our review of interventions targeting sedentary behaviour reductions alone, or combined with increases in physical activity, found evidence of effectiveness for improving some cardiometabolic risk biomarkers to a small degree. There was insufficient evidence to evaluate inflammation or vascular function. Key limitations to the underlying evidence base include a paucity of high-quality studies, interventions lasting for ≥12 months, sensitive biomarkers and clinical study populations (eg, type 2 diabetes). guest. Protected by copyright. PROSPERO trial registration number CRD42016041742 PROSPERO trial registration number CRD42016041742 We therefore conducted a systematic review with meta-analyses aiming to synthesise the body of evidence that examined the effectiveness on biomarkers of cardiometabolic risk of ≥7 days interventions that targetted sedentary behaviour (alone or in combination with physical activity) in free-living conditions. We reviewed the evidence on Review Review on October 23, 2024 http://bjsm.bmj.com/ Br J Sports Med: first published as 10.1136/bjsports-2019-101154 on 8 April 2020. Downloaded from Nyssa T Hadgraft,1,2,3 Elisabeth Winkler,3 Rachel E Climie,2 Megan S Grace,2 Lorena Romero,4 Neville Owen,1,2,3 David Dunstan,2,5,6,7,8 Genevieve Healy,2,3,9 Paddy C Dempsey ‍ ‍ 1,2,10 ► ►Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ bjsports-2019-101154). Accepted 11 March 2020 Published Online First 8 April 2020 Hadgraft NT, et al. Br J Sports Med 2021;55:144–154. doi:10.1136/bjsports-2019-101154 Effects of sedentary behaviour interventions on biomarkers of cardiometabolic risk in adults: systematic review with meta-analyses Nyssa T Hadgraft,1,2,3 Elisabeth Winkler,3 Rachel E Climie,2 Megan S Grace,2 Lorena Romero,4 Neville Owen,1,2,3 David Dunstan,2,5,6,7,8 Genevieve Healy,2,3,9 Paddy C Dempsey ‍ ‍ 1,2,10 Systematic review Study inclusion Figure 1 shows the PRISMA flow diagram. In total, 23 976 arti- cles were identified. Most were rejected at abstract screening, with 267 articles screened as full text. The criteria mostly excluded studies on the basis they were not ≥7 days sedentary behaviour interventions (181/218). Fifty-four studies (55 arti- cles) were included in the systematic review.33–87 The population, design and intervention characteristics of the studies are reported in online supplementary table S3 and aggregated in table 1. Collectively, the 54 studies involved 6330 participants (48% women) with sample sizes usually <100 (k=36) and occasionally >200 (k=8), ranging between 12 and 1113. Study populations were recruited from developed nations, mostly English-speaking (table 1) usually from North America (k=20, predominantly the USA), Europe (k=19), Australia (k=10) and occasionally from Asia (k=4) or Africa (k=1). From what little and inconsistent data was reported on ethnicity, plus the study locations, we infer that most study participants were likely Caucasian or ‘white’ (variously defined) with a smaller number identifying as African American or African, Hispanic and Asian ethnicities. Study mean ages ranged from 23 years74 to 71 years.51 Typically, the studies recruited participants from the general population (k=26) or a population with a chronic disease risk factor (k=17) (generally overweight and/or obesity, occasionally in conjunction with another risk factor). Clinical conditions36 37 39 44 48 59 60 71 72 81 84 were seldom targeted for uest. Protected by copyright. Search strategy and study selection gy y Six electronic databases (Ovid Medline, Ovid Embase, EBM Reviews Cochrane Central, CINAHL, Scopus, Web of Science) were searched systematically from database inception to 27 August 2019 (7 March 2017; 16 February 2018 and, 27 August 2019). A research librarian (LR) conducted an initial search for studies in Medline and Embase and used an analysis of text words and subject terms to develop the search strategies. The final searches were then executed using the appropriate speci- fications of each database (LR; see online supplementary table S1). Using reference management software (Endnote, Clarivate Analytics, Philadelphia, USA), records were compiled, dupli- cates were removed, and two authors (NTH and PCD or REC and MSG) performed title and abstract screening and reviewed each full-text article was reviewed against the inclusion criteria. Discrepancies were resolved in consultation with an indepen- dent third reviewer (EW). on October 23, 2024 by guest. Pro http://bjsm.bmj.com/ 54 on 8 April 2020. Downloaded from Methods Analyses were performed in STATA V.16 (StataCorp). Signifi- cance was set at p<0.05 (two tailed). Pooled effects were esti- mated based on intervention effects (mean between-groups difference, in units) for the end-of-intervention endpoint extracted from each intervention, with the standard errors multi- plied by ‍ √( n + 1 ) /2‍ whenever there were n>1 eligible seden- tary behaviour intervention arms.32 Pooled effects were primarily estimated from random effects (Der Simonian Laird) meta- analysis models, with fixed effects results also reported, along with heterogeneity estimates (I2 and Cochrane’s Q test) in the forest plots. A range of sensitivity analyses were also performed. Since Begg’s test for publication bias can be underpowered, we also reported bias-corrected estimates from Tweedie and Duval’s trim and fill method. Leave-one-out sensitivity analyses were performed to consider how dependent conclusions were to any individual study. Meta-regression models, which explored possible sources of heterogeneity, are reported in the manuscript whenever heterogeneity was significant (p<0.05) or substantial (I2 >0.25), otherwise in online supplementary material. Charac- teristics considered were: mean participant age, mean outcome biomarker levels at baseline, degree of intervention effectiveness for sedentary behaviour (intervention effect on overall sedentary time in h/day), intervention duration (≤3 months/3–6 months />6 months) and study quality (RoB scores). Unadjusted and age-adjusted models were reported. This study followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)29 and the Meta-analysis of Observational Studies in Epidemiology30 reporting guidelines. Review supplementary table S2. Study quality was assessed using the risk of bias (RoB) V.2.0 tool.31 body anthropometry, indicators of blood pressure and related haemodynamics, biomarkers relevant to the metabolism of blood glucose and lipids, and inflammatory biomarkers. Introduction Globally, cardiovascular diseases are the leading cause of death and a major cause of disability and lost productivity in adults.1 2 In addition, estimates from 2017 indicate that 451 million people are Hadgraft NT, et al. Br J Sports Med 2021;55:144–154. doi:10.1136/bjsports-2019-101154 1 of 13 Inclusion and exclusion criteria l l d h Inclusion criteria, applied hierarchically, were: (1) reported intervening on sedentary behaviour for ≥7 days; (2) human study; (3) participants all aged ≥18 years; (4) English language; (5) full-length publication; (6) reported as an outcome at least one biomarker of cardiometabolic health, specifically concerning body anthropometry, glucose metabolism, lipid metabolism, blood pressure and related haemodynamics or inflammation (see online supplementary table S2); and, (7) used an intervention study design (single-group preintervention and postintervention, parallel-group design or crossover). To meet criterion (1), the intervention needed to target sedentary behaviour directly or indirectly with replacement of sedentary activity with an alter- native (eg, treadmill desks), increasing ‘whole-of-day’ activity (which includes sedentary) or increasing ‘light intensity’ activity (which is almost the inverse of sedentary) or similar. Studies that only mentioned intervening on ‘physical activity’ or exercise could increase these activities at the expense of either sedentary behaviour or light activity, and therefore did not meet criterion (1). Further inclusion criteria for the meta-analyses were: (1) a no-intervention comparison arm (usual care/conditions; atten- tion control) and (2) no other intervention that was likely to provide an appreciable impact on cardiometabolic biomarkers (eg, diet). Physical activity interventions were permitted, since reducing sedentary time very likely increases some form of phys- ical activity as a replacement. Achievement of successful seden- tary behavioural change was not considered a requirement for inclusion in the meta-analyses (to avoid potentially overstating effectiveness). Meta-analyses were conducted for each biomarker reported in at least five studies. Data extraction recruitment; of these only some conditions were pertinent to cardiovascular health. Many studies (k=25) used screening to recruit participants at risk for high sedentary behaviour based on their job (eg, office or desk-based work) and/or their reported All data were extracted, checked and discrepancies resolved by the review team (NTH, PCD, REC and EW), using standardised rules created a priori. The rules used for regarding extraction, contacting authors for missing or questionable data are in online 2 of 13 Hadgraft NT, et al. Br J Sports Med 2021;55:144–154. doi:10.1136/bjsports-2019-101154 2 of 13 on October 23, 2024 by g http://bjsm.bmj.com/ Br J Sports Med: first published as 10.1136/bjsports-2019-101154 on 8 April 2020. Downloaded from Review Figure 1 PRISMA diagram of the literature search results. a Exclusion criteria were applied in the following order: (1) not sitting intervention ≥1 week, (2) not a human study, (3) not English language, (4) not full-length publication, (5) not reporting ≥1 biomarker, (6) not randomised, quasi- randomised or pre–post trial, (7) not adults ≥18 years. b Two articles were identified for one study (Balducci et al,47 Balducci et al72). c k=32 for anthropometry measures; k=25 for blood pressure measures; k=22 for glucose measures; k=24 lipid measures; k=4 inflammatory measures. PRISMA, Preferred Reporting Items for Systematic Reviews and Meta-Analyses. behaviours, while 20 studies screened based on physical activity, and 11 studies screened on both behaviours. supplementary table S3). Workplace interventions were about half multicomponent (k=13) and half single component (k=14), on October 23, 2024 by guest. Protec http://bjsm.bmj.com/ Br J Sports Med: first published as 10.1136/bjsports-2019-101154 on 8 April 2020. Downloaded from Figure 1 PRISMA diagram of the literature search results. a Exclusion criteria were applied in the following order: (1) not sitting intervention ≥1 week, (2) not a human study, (3) not English language, (4) not full-length publication, (5) not reporting ≥1 biomarker, (6) not randomised, quasi- randomised or pre–post trial, (7) not adults ≥18 years. b Two articles were identified for one study (Balducci et al,47 Balducci et al72). c k=32 for anthropometry measures; k=25 for blood pressure measures; k=22 for glucose measures; k=24 lipid measures; k=4 inflammatory measures. PRISMA, Preferred Reporting Items for Systematic Reviews and Meta-Analyses. behaviours, while 20 studies screened based on physical activity, and 11 studies screened on both behaviours. supplementary table S3). Data extraction Workplace interventions were about half multicomponent (k=13) and half single component (k=14), while interventions in the remaining non-workplace settings were mostly multicomponent (k=21, 72%). Workplace inter- ventions almost all used environmental modification (k=26), commonly used counselling/education (k=13), sometimes used device self-monitoring (k=5), device-based social comparison (k=3), prompting via devices or SMS (k=6) and structured Interventions Table 1 Continued Review f Table 1 Summary of the study population, design and intervention characteristics of adult sedentary behaviour interventions ≥7 days with biomarker outcomes Characteristic Count* Detail Population / study characteristics (54 studies) Sample size Median n / study 57 Lowest=12, highest=1113 Total n 5217 Female n=3065 (48.4%), male n=3265 (51.6%) Location (continent and country: ISO 2-digit country codes) North America 20 USA 16, CA 4 Europe 19 UK 5, DK 4, SE 3, ES 2, NL 1, DK & GL 1, FI 1, IT 1, UK & NL & NO & PT 1 Australia 10 Asia 4 TW 2, CN 1, JP 1 Africa 1 ZA 1 Ethnicity >50% Caucasian / ‘white’ 18 <50% Caucasian / ‘white’ 2 Not reported 33 Clinical population Clinical condition 11 T2D 4, cancer 2, rheumatoid arthritis 2, obstructive sleep apnoea 1, intellectual disability 1, coronary artery disease 1 Clinical risk factors only 17 Adiposity 14, chronic disease risk (+adiposity) 3 Healthy / general 26 Screening Sedentary job / behaviour 25 11 also screened for PA, 14 did not Physical activity (PA) 30 11 also screened for PA, 19 did not Study design Randomised controlled trial 39 8 cluster / 31 individually randomised 34 parallel group / 3 crossover / 1 other † Non-randomised controlled trial 5 1 cluster / 4 individually allocated 5 parallel group / 0 crossover Multiarm (no controls) 5 1 cluster randomised / 4 individually randomised Pre–post (single arm) 5 Primary outcomes Includes sedentary 22 Includes biomarker(s) 10 Includes both 1 Includes neither 21 PA 8 / PA and diet 1, unstated 6, feasibility 5, fitness 1 Intervention characteristics (56 interventions) Duration 3 months or less 28 >3 to 6 months 16 >6 months 12 Setting Workplace 27 Community 18 Other 11 Hospital 5, primary care 4, domestic 1, education 1 N components Multicomponent 34 Workplace 13, community / other 21 Single component 22 Workplace 14, community / other 8 Continued on October 23, 2024 by guest. Protected by copyright. http://bjsm.bmj.com/ ed: first published as 10.1136/bjsports-2019-101154 on 8 April 2020. Downloaded from T2D, type 2 diabetes. activity sessions (k=2). By contrast, non-workplace interven- tions almost always used some form of counselling/education (k=29), commonly used device self-monitoring (k=18) and occasionally used environmental modification (k=8), prompting (k=6), structured activity (k=4) and financial incentives (k=1). Interventions The 54 studies delivered 56 sedentary behaviour interven- tions, mostly in the workplace (k=27) or community (k=18) settings, with healthcare (k=9), domestic (k=1) and educational settings (k=1) being less common (table 1, details in online 3 of 13 Hadgraft NT, et al. Br J Sports Med 2021;55:144–154. doi:10.1136/bjsports-2019-101154 Hadgraft NT, et al. Br J Sports Med 2021;55:144–154. doi:10.1136/bjsports-2019-101154 Characteristic Count* Detail Components‡ Counselling / education 41 Workplace 13, community / other 28 Environmental modification 34 Workplace 26, community / other 8 Prompting 12 Workplace 6, community / other 6 Structured 'activity' 5 Workplace 2, community / other 4 Device self-monitoring 23 Workplace 5, community / other 18 Device social comparison 7 Workplace 3, community / other 4 Financial incentives 2 Workplace 1, community / other 1 Sedentary targets / messaging‡ Domain specific message 29 Accumulation 21 Quantitative volume target 14 Count out of 54 studies or 56 interventions as indicated in the table unless other statistic is mentioned (eg, median). †Almost a randomised controlled trial (parallel groups) except re-enrolled some controls into the intervention on completion. ‡Not mutually exclusive (interventions can have multiple components, multiple messages). T2D, type 2 diabetes. Interventions The extent of education or counselling was also highly variable, ranging from brief advice to theoretically grounded behavioural counselling. The interventions varied in how they considered sedentary behaviour. Diverse behaviours were promoted as replacements for sedentary behaviour: primarily standing, walking or other stepping, but also sometimes pedalling, ‘incidental’ exercise (likely predominantly ‘light’ activities), activities of moderate or greater intensity and sometimes resistance exercise (online supplementary table S3). Sedentary behaviour targets seldom were domain specific, referenced accumulation patterns or set quantitative guidelines on sedentary time (table 1). Primary outcomes included sedentary behaviour in 23 studies, biomarkers in 11, and included neither in 20, instead being unstated (k=6), focused on feasibility (k=5) or involving physical activity with or without other outcomes (k=9). Evaluation of biomarker indicators of cardiovascular health The biomarkers selected for review are shown in table 2. Biomarker outcomes nearly always included indicators of body anthropometry (k=52 studies), and often included indi- cators of blood pressure (k=37), lipid metabolism (k=33) and glucose metabolism (k=31). Four studies reported on C reactive protein.72 73 75 87 Other inflammatory markers, such as tumour necrosis factor α or interleukin-6, were not found among the reported outcomes. guest. Protected by copyright. Of the anthropometric indicators, the most commonly reported were weight (k=45) or BMI (k=39), followed by waist circumference (k=37). These were almost always collected 4 of 13 Hadgraft NT, et al. Br J Sports Med 2021;55:144–154. Interventions doi:10.1136/bjsports-2019-101154 Review Table 2 Biomarkers reported as outcomes in 54 studies of adult sedentary behaviour interventions ≥7 days Outcomes Studies Detail Quality factors Body anthropometry 52 Body weight 45 45 wt, 39 body mass index BMI Objective† / self-report: 44/1 Waist circumference* 37 37 circumference, 2 waist:hip ratio Objective† / self-report: 36/1 Other body measurements 9 7 hip circumference, 1 neck circumference, 2 sagittal abdominal diameter Body composition 25 BIA: 12 DXA: 5 BIS: 2 BADP: 3 Skinfold(s): 2 Unreported: 1 Total fat 25 20 percentage of body weight, 11 mass* Total fat-free or lean 13 12 percentage of body weight, 1 mass, Other 5 fat mass or % (4 truncal, 1 arm, 1 leg, 1 android %, 1 gynoid %); fat-free mass or % (1 arm, 1 leg); 1 skeletal muscle mass; 1 visceral fat area Blood pressure (BP) regulation 37 Resting BP 37 37 systolic, 36 diastolic, two mean arterial BP Objective† / self-report: 36/1 Ambulatory BP 0 – – Heart rate 5 3 resting, 2 non-resting Objective† / self-report: 5/0 Detailed vascular health measures 3 1 flow mediated dilation, 1 carotid intima media thickness, 1 aortic augmentation index, 1 subendocardial variability, 1 pulse wave velocity Objective† / self-report: 3/0 Glucose metabolism 31 Fasting glucose* 27 Venous / capillary: 20/7 Fasting insulin* 13 HOMA / HOMA-2 7 6 HOMA-IR, 2 HOMA-%B, 1 HOMA2-%B, 1 HOMA2-%S Postprandial glucose / insulin 4 4 postprandial glucose, 1 postprandial insulin, 1 insulin AUC, 1 glucose AUC, 1 C-ISI Venous / capillary: 4/0 Duration: all 2 hours test C-peptide 0 – HbA1c* 17 15 HbA1c, 2 'estimated average glucose' reported as HbA1c Venous / capillary: 15/2 Lipid metabolism 33 Cholesterol levels or ratios 33 29 total, 28 HDL, 24 LDL, 1 VLDL, 1 non-LDL, 5 total/HDL, 2 LDL/HDL Venous / capillary: 25/8 fasted / insufficient / non-fasted state: 25/1/7 Triglycerides 32 Other 3 1 cholesterol diameter; 1 lipoprotein lipase; 3 apolipoproteins (APO): 3 APO-A1, 3 APO-B, 2 APO-A1/APO-B Inflammation 4 C reactive protein (CRP) 4 2 CRP; two high-sensitivity CRP Venous / capillary: 4/0 fasted / insufficient / non-fasted state: 4/0/0 Other: TNF-α, IL-6 0 – Data were extracted from the earlier paper related to this study Balducci 2017 when it was not reported in the 2019 paper (body fat percentage; fat-free mass; BMI; fasting insulin; HOMA). Hadgraft NT, et al. Br J Sports Med 2021;55:144–154. doi:10.1136/bjsports-2019-101154 on October 23, 2024 by guest. P http://bjsm.bmj.com/ Br J Sports Med: first published as 10.1136/bjsports-2019-101154 on 8 April 2020. Downloaded from on October 23, 2024 by guest. Protected by copyright. http://bjsm.bmj.com/ Med: first published as 10.1136/bjsports-2019-101154 on 8 April 2020. Downloaded from on October 23, 2024 by guest. Protect http://bjsm.bmj.com/ 4 on 8 April 2020. Downloaded from j y y AUC, area under the curve; BADP, body air displacement plethysmography; BIA, multifrequency bioimpedance analysis; BIS, bioelectrical impedance spectroscopy; BMI, body mass index; C-ISI, composite insulin sensitivity index; DXA, dual X-ray absorptiometry; HDL, high-density lipoprotein; HOMA-2, revised homeostatic model assessment; HOMA, homeostatic model assessment; IL-6, interleukin 6; LDL, low-density lipoprotein; TNF-α, tumour necrosis factor α; VLDL, very-low-density lipoprotein. reported on flow mediated dilatation, carotid artery intima media thickness, aortic augmentation index and sub endocardial vari- ability.53 Resting heart rate was collected in three studies.62 69 73 objectively by staff. Body composition outcomes were collected mostly using multifrequency bioimpedance analysis (k=12) or reference-grade standards: dual X-ray absorptiometry (k=5) or body air displacement plethysmography (k=3). Occasionally, other methods were used (k=4). Studies typically reported on body fat (k=25) (most commonly as percentage of body weight), and occasionally fat-free, lean or muscle mass (k=13). Thus, fewer studies were able to assess changes to specific tissues (eg, fat, lean tissue) or anatomical sites (eg, truncal fat, measured in four studies). 23, 2024 by guest. Protected by copyright. Of the glucose metabolism indicators (table 2), most (k=27) reported on fasting glucose, with only 13 reporting fasting insulin, and 7 reporting composite indicators of beta-cell func- tion or insulin resistance (ie, measures from homeostatic model assessment, HOMA or HOMA-2). Seventeen studies reported on overall glucose control (HbA1c expressed in various forms), while four studies reported effects on postprandial glucose and/ or insulin54 58 71 83 and none reported on c-peptide. While venous bl d d h f ll i f i l (k Of the glucose metabolism indicators (table 2), most (k=27) reported on fasting glucose, with only 13 reporting fasting insulin, and 7 reporting composite indicators of beta-cell func- tion or insulin resistance (ie, measures from homeostatic model assessment, HOMA or HOMA-2). Seventeen studies reported on overall glucose control (HbA1c expressed in various forms), while four studies reported effects on postprandial glucose and/ or insulin54 58 71 83 and none reported on c-peptide. While venous blood draws were the norm for collecting fasting values (k=20 studies), lower quality fingerstick capillary measures were occa- sionally used (k=7). None of the studies reported outcomes from continuous glucose monitoring. uest. Interventions Outcome included in the meta-analyses: was reported in >5 of the 33 studies eligible for the effectiveness meta-analyses (had control arm, no additional relevant intervention provided apart from active behaviours). †Measured objectively by research staff. AUC, area under the curve; BADP, body air displacement plethysmography; BIA, multifrequency bioimpedance analysis; BIS, bioelectrical impedance spectroscopy; BMI, body mass index; C-ISI, composite insulin sensitivity index; DXA, dual X-ray absorptiometry; HDL, high-density lipoprotein; HOMA-2, revised homeostatic model assessment; HOMA, homeostatic model assessment; IL-6, interleukin 6; LDL, low-density lipoprotein; TNF-α, tumour necrosis factor α; VLDL, very-low-density lipoprotein. o http://bjsm.bmj.com/ Br J Sports Med: first published as 10.1136/bjsports-2019-101154 on 8 April 2020. Downloaded from y Study inclusion Of the 44 controlled intervention studies, 33 studies (34 inter- ventions) were eligible for the meta-analyses (11 studies had provided diet intervention). For the 15 biomarkers that met the inclusion criteria (table 2), the number of studies providing data and able to be included ranged from 6 for fat mass to 25 for body weight and blood pressure, and these studies collectively represented anywhere between 724 and 2076 participants. y y Body weight and body fat percentage showed little evidence of heterogeneity (I2 <25%; p≥0.05) with slightly more substan- tial (but non-significant) heterogeneity seen for fat mass and significant heterogeneity seen for waist circumference and fat- free mass. The heterogeneity in effects on fat-free mass was completely attenuated (I2=0.0, p=0.790) by omitting a single study.45 Omission of this same study partially attenuated hetero- geneity in effects on waist circumference (I2=19.9%, p=0.217). Further exploration of the heterogeneity via meta-regression (table 5) did not show any significant predictors of effects on waist circumference. The largest effects and the smallest residual heterogeneity were seen for RoB scores (residual I2=22.6%, p=0.192), with effects stronger by just over 1 cm in studies with high versus low RoB. Meta-regression results for the outcomes not displaying substantial or significant heterogeneity are shown in online supplementary table S8. Review cholesterol (k=24) (table 2). These markers are reported widely in the context of cardiovascular risk. Studies occasionally reported VLDL cholesterol,69 non-LDL cholesterol40 or choles- terol ratios.40 42 43 60 70 Three studies reported on apolipopro- teins (APOA1, APOB and their ratio38 70 73) and one reported on the diameter of various types of cholesterol.38 None of the studies mentioned performing detailed profiling of lipid classes or subclasses. <15 min/day). Effects ranged from +11.3 to −132 min/day (see online supplementary table S4). Table 3 shows the pooled effects on biomarkers for the main analyses and sensitivity analyses. Begg’s tests were all p≥0.05 (online supplementary table S7). on October 23, 2024 by guest. Protected by copyright. http://bjsm.bmj.com/ Sports Med: first published as 10.1136/bjsports-2019-101154 on 8 April 2020. Downloaded from Risk of bias RoB overall is reported in online supplementary table S6. To simplify reporting, criteria scored for groups of outcomes with similar concerns underlying their bias risk (eg, missing data, measurement): anthropometric and blood pressure outcomes, glucose metabolism outcomes and lipid metabolism outcomes. Overall RoB was high (≥1 criteria was ‘high’ risk) in 10 studies (30%), unclear (ie, 0 ‘high’ risk and ≥1 ‘unclear’ risk) in 17 (52%) studies and ‘low’ (ie, all ‘low’ risk) in 6 studies (18%). The most common contributor to a ‘high’ RoB rating related to the randomisation process (k=6, 18%),58 64 68 76 79 87 (ie, use RoB overall is reported in online supplementary table S6. To simplify reporting, criteria scored for groups of outcomes with similar concerns underlying their bias risk (eg, missing data, measurement): anthropometric and blood pressure outcomes, glucose metabolism outcomes and lipid metabolism outcomes. Overall RoB was high (≥1 criteria was ‘high’ risk) in 10 studies (30%), unclear (ie, 0 ‘high’ risk and ≥1 ‘unclear’ risk) in 17 (52%) studies and ‘low’ (ie, all ‘low’ risk) in 6 studies (18%). The most common contributor to a ‘high’ RoB rating related to the randomisation process (k=6, 18%),58 64 68 76 79 87 (ie, use of non-random methods). Four studies were also rated as ‘high’ RoB due to deviations from intended interventions (data not analysed according to intention-to-treat principles40 58 74 79) and missing outcome data.76 79 83 87 An unclear risk level was typi- cally assigned based on inadequate reporting of randomisation (k=12), concerns with missing outcome data (k=11) and/or bias in measurement of the outcome (k=9). Low risk was still permitted with lack of blinding, given the context (behavioural intervention) in which allocation is impossible to conceal from participants and is generally known to staff, and in which outcomes are collected objectively. of non-random methods). Four studies were also rated as ‘high’ RoB due to deviations from intended interventions (data not analysed according to intention-to-treat principles40 58 74 79) and missing outcome data.76 79 83 87 An unclear risk level was typi- cally assigned based on inadequate reporting of randomisation (k=12), concerns with missing outcome data (k=11) and/or bias in measurement of the outcome (k=9). Low risk was still permitted with lack of blinding, given the context (behavioural intervention) in which allocation is impossible to conceal from participants and is generally known to staff, and in which outcomes are collected objectively. Blood pressure P i i Prior to intervention, participants had a weighted mean (±pooled SD) blood pressure of 110.0±10.5 mm Hg systolic and 78.4±7.1 mm Hg diastolic, indicating typically healthy levels, though with some studies attracting samples with average systolic blood pressure as high as 140 mm Hg or higher55 70 (table 3 and online supplementary table S4). Pooled effects showed a small significant reduction in systolic blood pressure (−1.05 mm Hg, 95% CI -2.08 to 0.02) and a smaller non-significant reduction in diastolic blood pressure (−0.69 mm Hg, 95% CI −1.69 to 0.32; table 3). Forest plots are shown in online supplemen- tary figure S7 and online supplementary figure 8. Corrections for small-study effects had no effect on the results and pooled effects consistently reflected tendencies towards reduced blood pressure in the leave-one-out sensitivity analyses. Heterogeneity guest. Protected by copyright. Body weight and body composition Consistent with the studies’ selection criteria, prior to interven- tion, participants had a weighted mean (±pooled SD) BMI of 25.4±3.2 kg/m2, with study means ranging from 22.1 kg/m2 in a workplace intervention with no weight screening criteria68 to 35.9 kg/m2 in a treadmill intervention for overweight/obese office workers.56 Baseline anthropometric values are summarised in table 4 (detail in online supplementary table S4). Pooled effects showed that the sedentary behaviour interventions tended to provide small improvements (net of control) in body anthro- pometry outcomes (table 3). Significant pooled effects in favour of intervention were seen regarding body weight (−0.56 kg, 95% CI -0.94 to 0.17), waist circumference (−0.72 cm, 95% CI -1.21 to 0.22), body fat percentage (−0.26%, 95% CI -0.50% to 0.02%), with a tendency towards reduced fat mass (−0.33 kg, 95% CI −0.74 to 0.08) and no large or significant effect on fat-free mass (0.00 kg, 95% CI −0.52 to 0.53). Effects on BMI were in a similar direction to those for body weight, but not statistically significant (−0.07 kg/m2, 95% CI −0.16 to 0.03). Forest plots for body weight and body composition are shown in online supplementary figure 1–6. Small-study effects did not lead to overstated findings, as the original findings were no more favourable than the trimmed and filled results. Also, no single study seemed to overly influence the conclusions, as improve- ments observed were always still present to some degree in the leave-one-out sensitivity analyses. Study designs y g Very few studies (k=5) used a single-group pre–post study design (table 1)46 51 63 69 78; most used two or more groups (k=49). Usually the additional group (or groups) facilitated testing effectiveness against a no-intervention or attention control comparison arm (k=44, with 39 randomised) or occa- sionally only allowed for comparison of alternate interventions (k=5, with five randomised).34 44 50 67 84 Most studies (k=42) intervened for 6 months or less (shortest=2 weeks) while few (k=10) intervened for 12 months or longer43 44 46 59 60 63 72 73 76 87 intervened for 6 months or less (shortest=2 weeks) while few (k=10) intervened for 12 months or longer43 44 46 59 60 63 72 73 76 87 (longest=36 months). Only nine studies referred to evaluation of maintenance of effects following withdrawal of intervention or intervention contact. on October 23, 2024 by guest. P http://bjsm.bmj.com/ Br J Sports Med: first published as 10.1136/bjsports-2019-101154 on 8 April 2020. Downloaded from Protected by copyright. Blood pressure was generally assessed with resting blood pres- sure (k=37), which was typically reported separately as systolic (k=37) and/or diastolic blood pressure (k=36) and as mean arte- rial pressure in two studies57 82 (table 2). Usually, staff measured blood pressure, with participants reporting values from home monitors in one study.46 Ambulatory blood pressure was not reported. Detailed biomarkers of vascular health (eg, endothelial dysfunction, arterial stiffness) were seldom collected. One study The most commonly reported lipid markers were: triglycerides (k=32); total cholesterol (k=29); high-density lipoprotein (HDL) cholesterol (k=28) and, low-density lipoprotein (LDL) Hadgraft NT, et al. Br J Sports Med 2021;55:144–154. doi:10.1136/bjsports-2019-101154 5 of 13 5 of 13 Review Effectiveness of sedentary behaviour interventions for biomarker outcomes Boldface indicates pooled effect is p<0.05. Omitted Healy et al (2017). †Omitted Ashe et al (2015). ‡Omitted Maylor et al (2018). §Omitted Pesola et al (2017). ¶Heterogeneity no longer p<0.05 in leave-one-out sensitivity analysis. Omitted Danquah et al (2017). ††Omitted Puig-Ribera et al (2015). ‡‡Omitted Healy et al (2013). §§Omitted Kallings et al (2017). ¶¶Omitted Butler et al (2018). Omitted Mantzari et al (2018). †††Omitted Lin et al (2017). ‡‡‡Omitted Taylor et al (2016) (Computer intervention). §§§Omitted Balducci (2019). ¶¶¶Omitted Biddle (2015). **Omitted Thomsen et al (2017). ††††Omitted Aadahl et al (2014). n/a, not applicable. Br J Sports Med: first published as 10.1136/bjsports-2019-101154 on 8 April 2020. Effectiveness of sedentary behaviour interventions for biomarker outcomes Downloaded fro Table 3 Pooled intervention effects on biomarkers: controlled trials of 34 adult sedentary behaviour interventions ≥7 days Outcome Main findings Publication-bias corrected Leave-one-out sensitivity analysis k n All studies Most benefit Least benefit I2, P value Pooled effect (95% CI) P value Pooled (95% CI) Pooled effect (95% CI) Pooled effect (95% CI) Weight, kg 25 1839 23.6%, p=0.142 −0.56 (−0.94 to −0.17) 0.005 n/a −0.63 (−1.03 to −0.23) −0.47 (−0.75 to −0.18)† Body mass index, kg/m2 24 1843 0.0%, p=0.804 −0.07 (−0.16 to 0.03) 0.167 −0.08 (−0.17 to 0.02) −0.10 (−0.20 to 0.01)‡ −0.04 (−0.14 to 0.06)§ Waist circumference, cm 19 2076 45.8%, p=0.016 −0.72 (−1.21 to −0.22) 0.004 −1.00 (−1.51 to −0.49) −0.95 (−1.38 to −0.51)¶ −0.61 (−1.20 to −0.01)¶†† Body fat, % 16 1618 5.5%, p=0.390 −0.26 (−0.50 to −0.02) 0.034 −0.37 (−0.65 to −0.10) −0.37 (−0.61 to −0.12)‡ −0.17 (−0.43 to 0.09) Fat mass, kg 6 724 26.6%, p=0.235 −0.33 (−0.74 to 0.08) 0.116 n/a −0.42 (−0.73 to −0.10)‡‡ −0.23 (−0.63 to 0.16)§§ Fat-free mass, kg 7 1011 72.7%, p=0.001 0.00 (−0.52 to 0.53) 0.992 0.48 (−0.02 to 0.98) 0.12 (−0.40 to 0.65)‡ −0.25 (−0.57 to 0.06)¶ Blood pressure, mm Hg Systolic 25 1932 8.6%, p=0.341 −1.05 (−2.08 to −0.02) 0.045 n/a −1.42 (−2.38 to −0.45)‡ −0.75 (−1.81 to 0.31)¶¶ Diastolic 25 1932 52.6%, p=0.001 −0.69 (−1.69 to 0.32) 0.180 n/a −0.92 (−1.86 to 0.02)* −0.36 (−1.28 to 0.56)††† Glucose, mM 19 1518 45.5%, p=0.017 −0.03 (−0.11 to 0.05) 0.526 −0.04 (−0.13 to 0.05) −0.05 (−0.11 to 0.02)¶‡‡‡ −0.01 (−0.11 to 0.09)¶¶ Insulin, pM 10 1102 64.0%, p=0.003 −1.42 (−2.82 to -0.02) 0.047 −1.03 (−2.48 to 0.42) −4.13 (−7.48 to -0.78)§§§ −0.45 (−1.60 to 0.69)¶* HbA1c, % 9 892 72.9%, p=0.000 −0.10 (−0.22 to 0.03) 0.129 −0.03 (−0.16 to 0.09) −0.14 (−0.29 to 0.01)¶¶¶ −0.05 (−0.17 to 0.07)§§ Cholesterol, mM Total 23 1798 54.1%, p=0.001 −0.06 (−0.16 to 0.04) 0.213 −0.10 (−0.20 to −0.00) −0.08 (−0.18 to 0.02)‡‡‡ −0.03 (−0.11 to 0.05)¶**** High-density lipoprotein 22 1760 22.5%, p=0.168 0.04 (0.02 to 0.07) <0.001 0.05 (0.02 to 0.07) 0.05 (0.03 to 0.07)§ 0.03 (0.01 to 0.06)¶¶ Low-density lipoprotein 20 1660 0.0%, p=0.690 −0.02 (−0.07 to 0.04) 0.562 −0.01 (−0.07 to 0.05) −0.03 (−0.09 to 0.03)‡‡‡ −0.00 (−0.06 to 0.06)†††† Triglycerides, mM 23 1742 49.0%, p=0.005 −0.02 (−0.09 to 0.04) 0.496 −0.06 (−0.13 to 0.01) −0.04 (−0.10 to 0.03)‡‡ −0.01 (−0.07 to 0.06)§§ k, n = number of interventions, number of individuals (sum of n analysed in each included study). Hadgraft NT, et al. Br J Sports Med 2021;55:144–154. doi:10.1136/bjsports-2019-101154 Effectiveness of sedentary behaviour interventions for biomarker outcomes Effects on biomarkers were evaluated in the context of interven- tions that had displayed overall sedentary time improvements net of control that were mostly moderate (k=12, 30 to <60 min/ day), otherwise strong (k=9, ≥60 min/day) or small (15 to <30 min/day, k=8) or occasionally almost zero (k=3, –15 to 6 of 13 Hadgraft NT, et al. Br J Sports Med 2021;55:144–154. Effectiveness of sedentary behaviour interventions for biomarker outcomes doi:10.1136/bjsports-2019-101154 6 of 13 Review Review Table 3 Pooled intervention effects on biomarkers: controlled trials of 34 adult sedentary behaviour interventions ≥7 days Outcome Main findings Publication-bias corrected Leave-one-out sensitivity analysis k n All studies Most benefit Least benefit I2, P value Pooled effect (95% CI) P value Pooled (95% CI) Pooled effect (95% CI) Pooled effect (95% CI) Weight, kg 25 1839 23.6%, p=0.142 −0.56 (−0.94 to −0.17) 0.005 n/a −0.63 (−1.03 to −0.23)* −0.47 (−0.75 to −0.18)† Body mass index, kg/m2 24 1843 0.0%, p=0.804 −0.07 (−0.16 to 0.03) 0.167 −0.08 (−0.17 to 0.02) −0.10 (−0.20 to 0.01)‡ −0.04 (−0.14 to 0.06)§ Waist circumference, cm 19 2076 45.8%, p=0.016 −0.72 (−1.21 to −0.22) 0.004 −1.00 (−1.51 to −0.49) −0.95 (−1.38 to −0.51)¶ −0.61 (−1.20 to −0.01)¶†† Body fat, % 16 1618 5.5%, p=0.390 −0.26 (−0.50 to −0.02) 0.034 −0.37 (−0.65 to −0.10) −0.37 (−0.61 to −0.12)‡ −0.17 (−0.43 to 0.09) Fat mass, kg 6 724 26.6%, p=0.235 −0.33 (−0.74 to 0.08) 0.116 n/a −0.42 (−0.73 to −0.10)‡‡ −0.23 (−0.63 to 0.16)§§ Fat-free mass, kg 7 1011 72.7%, p=0.001 0.00 (−0.52 to 0.53) 0.992 0.48 (−0.02 to 0.98) 0.12 (−0.40 to 0.65)‡ −0.25 (−0.57 to 0.06)¶ Blood pressure, mm Hg Systolic 25 1932 8.6%, p=0.341 −1.05 (−2.08 to −0.02) 0.045 n/a −1.42 (−2.38 to −0.45)‡ −0.75 (−1.81 to 0.31)¶¶ Diastolic 25 1932 52.6%, p=0.001 −0.69 (−1.69 to 0.32) 0.180 n/a −0.92 (−1.86 to 0.02)* −0.36 (−1.28 to 0.56)††† Glucose, mM 19 1518 45.5%, p=0.017 −0.03 (−0.11 to 0.05) 0.526 −0.04 (−0.13 to 0.05) −0.05 (−0.11 to 0.02)¶‡‡‡ −0.01 (−0.11 to 0.09)¶¶ Insulin, pM 10 1102 64.0%, p=0.003 −1.42 (−2.82 to -0.02) 0.047 −1.03 (−2.48 to 0.42) −4.13 (−7.48 to -0.78)§§§ −0.45 (−1.60 to 0.69)¶* HbA1c, % 9 892 72.9%, p=0.000 −0.10 (−0.22 to 0.03) 0.129 −0.03 (−0.16 to 0.09) −0.14 (−0.29 to 0.01)¶¶¶ −0.05 (−0.17 to 0.07)§§ Cholesterol, mM Total 23 1798 54.1%, p=0.001 −0.06 (−0.16 to 0.04) 0.213 −0.10 (−0.20 to −0.00) −0.08 (−0.18 to 0.02)‡‡‡ −0.03 (−0.11 to 0.05)¶**** High-density lipoprotein 22 1760 22.5%, p=0.168 0.04 (0.02 to 0.07) <0.001 0.05 (0.02 to 0.07) 0.05 (0.03 to 0.07)§ 0.03 (0.01 to 0.06)¶¶ Low-density lipoprotein 20 1660 0.0%, p=0.690 −0.02 (−0.07 to 0.04) 0.562 −0.01 (−0.07 to 0.05) −0.03 (−0.09 to 0.03)‡‡‡ −0.00 (−0.06 to 0.06)†††† Triglycerides, mM 23 1742 49.0%, p=0.005 −0.02 (−0.09 to 0.04) 0.496 −0.06 (−0.13 to 0.01) −0.04 (−0.10 to 0.03)‡‡ −0.01 (−0.07 to 0.06)§§ k, n = number of interventions, number of individuals (sum of n analysed in each included study). Effectiveness of sedentary behaviour interventions for biomarker outcomes Boldface indicates pooled effect is p<0.05. Omitted Healy et al (2017) on October 23, 2024 by guest. Protected by copyright. http://bjsm.bmj.com/ t published as 10.1136/bjsports-2019-101154 on 8 April 2020. Downloaded from was minimal for systolic blood pressure (I2=8.6, p=0.341) but extensive for diastolic blood pressure (I2=52.6, p=0.001), and not explained by any single study. None of the variables in was minimal for systolic blood pressure (I2=8.6, p=0.341) but extensive for diastolic blood pressure (I2=52.6, p=0.001), and not explained by any single study. None of the variables in the meta-regressions (table 5) had significant associations with diastolic blood pressure or reduced the heterogeneity appre- ciably (residual I2 >50). 7 f 13 H d f NT l B J S M d 2021 55 144 154 d i 10 1136/bj 2019 101154 Table 4 Average biomarker characteristics prior to intervention in controlled trials of 34 adult sedentary behaviour interventions ≥7 days with biomarker outcomes k n Weighted mean±pooled SD Study means (min–max) Study with lowest mean Study with highest mean Weight, kg 29 2456 71.3±10.6 62.2–99.6 Alkhajah et al68 MacEwen et al40 Body mass index, kg/m2 34 3186 25.4±3.2 22.1–35.9 Alkhajah et al68 Schuna et al56 Waist circumference, cm 21 2630 83.9±7.7 74.4–111.4 Butler et al74 MacEwen et al40 Body fat, % 18 2050 28.1±5.4 24.5–45.5 Dunning et al75 Kozey Keadle et al58 Fat mass, kg 7 753 25.3±7.7 18.4–32.3 Alkhajah et al68 Kallings et al70 Fat-free mass, kg 8 1252 40.0±6.7 44.1–56.5 Alkhajah et al68 Balducci et al72 Systolic BP, mm Hg 25 2461 110.0±10.5 109–142 Dunning et al75 Maxwell-Smith et al Diastolic BP, mm Hg 25 2457 68.4±6.5 69–86 Peterman et al83 MacEwen et al40; Maxwell-Smith et al 40 Glucose, mM 19 1975 4.7±1.0 4.1–7.6 Peterman et al83 Balducci et al72 Insulin, pM 11 1495 51.5±44.1 37.1–133.0 Dunning et al75 Kozey Keadle et al58 HbA1c, % 11 1308 4.4±0.6 4.9–7.4 Kallings et al70 Balducci et al72 Total cholesterol, mM 24 2292 4.3±0.6 4.0–5.5 Peterman et al83 Kallings et al70 HDL cholesterol, mM 22 2232 1.2±0.4 1.1–1.8 Peterman et al83 Pesola et al38 LDL cholesterol, mM 21 2142 2.5±0.8 2.5–3.3 Peterman et al83 Kallings et al70 Triglycerides, mM 23 2202 1.1±0.5 0.9–1.9 Alkhajah et al68 Kozey Keadle et al58 BP, blood pressure; HDL, high-density lipoprotein; k, number of interventions; LDL, low-density lipoprotein; n, number of participants. on October 23, 2024 by guest. Protected by copyright. http://bjsm.bmj.com/ Br J Sports Med: first published as 10.1136/bjsports-2019-101154 on 8 April 2020. Downloaded from Review Review Table 5 Associations of study characteristics with intervention effects on cardiometabolic biomarkers (meta-regression) Unadjusted Age adjusted k, n b (95% CI) P value I2, p value k, n b (95% CI) P value I2, p value Waist circumference, cm 19, 2076 45.8%, p=0.016 19, 2076 45.8%, p=0.016 Mean baseline age, per 10 y 18, 2050 −0.50 (−1.18 to 0.17) 0.144 51.6%, p=0.007 – – – Mean baseline BMI, kg/m2 19, 2076 −0.04 (−0.25 to 0.16) 0.672 48.6%, p=0.011 18, 2050 −0.01 (−0.24 to 0.22) 0.910 54.5%, p=0.005 Mean baseline level 19, 2076 0.01 (−0.08 to 0.09) 0.856 44.9%, p=0.021 18, 2050 0.07 (−0.03 to 0.18) 0.182 45.9%, p=0.023 Sedentary effectiveness, h/day 19, 2076 −0.62 (−1.42 to 0.18) 0.127 38.7%, p=0.048 18, 2050 −1.05 (−1.96 to −0.14) 0.023 40.3%, p=0.049 Duration (vs ≤3 months) 19, 2076 0.896 51.8%, p=0.007 18, 2050 0.307 57.7%, p=0.003 3–6 months 0.15 (−1.08 to 1.37) 0.816 0.92 (−0.69 to 2.52) 0.262 >6 months −0.36 (−2.28 to 1.57) 0.717 −0.25 (−2.28 to 1.78) 0.809 Risk of bias (vs high risk) 19, 2076 0.098 22.6%, p=0.192 18, 2050 0.109 25.4%, p=0.174 Some concerns −0.70 (−2.50 to 1.09) 0.441 −0.52 (−2.39 to 1.34) 0.582 Low risk −1.47 (−3.10 to 0.16) 0.077 −1.36 (−3.03 to 0.31) 0.110 Fat-free mass, kg 7, 1011 72.7%, p=0.001 7, 1011 72.7%, p=0.001 Mean baseline age, per 10 years 7, 1011 0.14 (−0.60 to 0.88) 0.714 74.5%, p=0.001 – – – Mean baseline BMI, kg/m2 7, 1011 0.07 (−0.18 to 0.32) 0.575 76.8%, p<0.001 7, 1011 0.07 (−0.24 to 0.38) 0.660 79.6%, p=<0.001 Mean baseline level 7, 1011 0.04 (−0.10 to 0.18) 0.540 75.0%, p=0.001 7, 1011 0.06 (−0.16 to 0.27) 0.618 79.4%, p=<0.001 Sedentary effectiveness, hour/day* 7, 1011 0.23 (−0.98 to 1.44) 0.711 77.1%, p<0.001 7, 1011 0.21 (−1.02 to 1.44) 0.738 79.4%, p=<0.001 Duration (>6 vs ≤3 months)† 7, 1011 0.06 (1.23 to 1.36) 0.922 77.1%, p<0.001 7, 1011 −0.14 (−1.86 to 1.58) 0.875 77.6%, p=0.001 Risk of bias (vs high risk) 7, 1011 0.490 69.9%, p=0.010 7, 1011 0.619 74.5%, p=0.008 Some concerns 0.72 (−0.53 to 1.98) 0.258 1.23 (−0.73 to 3.20) 0.219 Low risk 0.12 (−1.10 to 1.34) 0.847 0.37 (−1.07 to 1.81) 0.615 Glucose, mM 19, 1518 45.5%, p=0.017 19, 1518 45.5%, p=0.017 Mean baseline age, per 10 years 19, 1518 −0.01 (−0.08 to 0.07) 0.891 47.9%, p=0.012 – – – Mean baseline BMI, kg/m2 19, 1518 0.01 (−0.01 to 0.03) 0.391 46.0%, p=0.017 19, 1518 0.01 (−0.01 to 0.04) 0.330 48.8%, p=0.012 Mean baseline level 18, 1497 −0.01 (−0.23 to 0.20) 0.908 45.8%, p=0.021 18, 1497 0.03 (−0.23 to 0.29) 0.819 46.0%, p=0.023 Sedentary effectiveness, h/day* 18, 1497 −0.02 (−0.21 to 0.16) 0.804 45.6%, p=0.021 18, 1497 −0.05 (−0.25 to 0.15) 0.632 46.0%, p=0.023 Duration (vs ≤3 months) 19, 1518 0.611 51.5%, p=0.007 19, 1518 0.732 54.0%, p=0.005 3–6 months −0.00 (−0.19 to 0.19) 0.980 0.04 (−0.24 to 0.32) 0.769 >6 months −0.12 (−0.37 to 0.13) 0.345 −0.14 (−0.43 to 0.15) 0.355 Risk of bias (vs high risk) 19, 1518 0.424 48.3%, p=0.014 19, 1518 0.543 49.7%, p=0.013 Some concerns 0.18 (−0.13 to 0.48) 0.256 0.21 (−0.12 to 0.54) 0.212 Low risk 0.12 (−0.09 to 0.33) 0.267 0.17 (−0.09 to 0.42) 0.202 Insulin, pM 10, 1102 64.0%, p=0.003 10, 1102 64.0%, p=0.003 Mean baseline age, per 10 years 10, 1102 0.76 (−2.63 to 4.15) 0.660 68.0%, p=0.002 – – – Mean baseline BMI, kg/m2 10, 1102 0.20 (−1.09 to 1.48) 0.764 67.8%, p=0.002 10, 1102 0.02 (−1.60 to 1.65) 0.980 68.8%, p=0.002 Mean baseline level 10, 1102 0.15 (0.03 to 0.27) 0.018 61.1%, p=0.008 10, 1102 0.21 (0.12 to 0.31) <0.001 0.0%, p=0.641 Sedentary effectiveness, hour/day* 10, 1102 1.65 (−6.15 to 9.45) 0.678 66.5%, p=0.002 10, 1102 3.51 (−6.34 to 13.35) 0.485 45.4%, p=0.077 Duration (vs ≤3 months) 10, 1102 0.014 50.8%, p=0.047 10, 1102 0.268 57.8%, p=0.027 3–6 months 2.14 (−7.20 to 11.48) 0.654 1.87 (−8.64 to 12.38) 0.728 >6 months 7.87 (−0.21 to 15.95) 0.056 6.84 (−2.33 to 16.01) 0.144 Risk of bias (vs high risk) 10, 1102 <0.001 11.9%, p=0.338 10, 1102 0.211 22.8%, p=0.255 Some concerns −0.24 (−1.42 to 0.94) 0.692 −3.10 (−11.81 to 5.62) 0.486 Low risk −4.64 (−6.95 to −2.32) <0.001 −5.60 (−11.33 to 0.14) 0.056 HbA1c, % 9, 892 72.9%, p=0.000 9, 892 72.9%, p=0.000 Mean baseline age, per 10 years 9, 892 −0.10 (−0.18 to −0.02) 0.011 49.5%, p=0.054 – – – Mean baseline BMI, kg/m2 9, 892 −0.01 (−0.05 to 0.03) 0.599 76.3%, p<0.001 9, 892 −0.03 (0.05 to –0.01) 0.004 0.0%, p=0.454 Mean baseline level 9, 892 −0.15 (−0.34 to 0.04) 0.127 76.3%, p<0.001 9, 892 −0.16 (−0.32 to 0.00) 0.055 49.1%, p=0.067 Sedentary effectiveness, hour/day* 9, 892 0.08 (−0.10 to 0.26) 0.379 69.5%, p=0.002 9, 892 −0.02 (−0.20 to 0.17) 0.862 56.4%, p=0.032 Duration (vs ≤3 months) 9, 892 0.994 78.0%, p<0.001 9, 892 0.002 24.7%, p=0.249 3–6 months −0.02 (−0.35 to 0.32) 0.919 0.04 (−0.14 to 0.23) 0.641 >6 months −0.02 (−0.38 to 0.35) 0.932 −0.24 (−0.50 to 0.02) 0.069 Risk of bias (vs high risk) 9, 892 0.090 75.4%, p<0.001 9, 892 0.037 59.4%, p=0.031 Some concerns 0.30 (−0.25 to 0.85) 0.287 0.28 (−0.23 to 0.79) 0.286 Low risk 0.50 (−0.02 to 1.02) 0.058 0.40 (−0.09 to 0.89) 0.113 Diastolic blood pressure, mm Hg 25, 1932 52.6%, p=0.001 25, 1932 52.6%, p=0.001 Mean baseline age, per 10 years 25, 1932 −0.38 (–1.30 to 0.54) 0.421 54.4%, p<0.001 – – – Mean baseline BMI, kg/m2 24, 1903 −0.01 (−0.32 to 0.30) 0.946 53.2%, p=0.001 24, 1903 0.03 (–0.31 to 0.36) 0.885 55.2%, p =<0.001 Continued on October 23, 2024 by guest. Effectiveness of sedentary behaviour interventions for biomarker outcomes Table 4 Average biomarker characteristics prior to intervention in controlled trials of 34 adult sedentary behaviour interventions ≥7 days with biomarker outcomes teristics prior to intervention in controlled trials of 34 adult sedentary behaviour interventions ≥7 days with 7 of 13 on October 23, 2024 by guest. Protected by copyright. http://bjsm.bmj.com/ Br J Sports Med: first published as 10.1136/bjsports-2019-101154 on 8 April 2020. Downloaded from Protected by copyrigh http://bjsm.bmj.com/ Br J Sports Med: first published as 10.1136/bjsports-2019-101154 on 8 April 2020. Downloaded from Hadgraft NT, et al. Br J Sports Med 2021;55:144–154. on October 23, 2024 by guest. Protected by copyright. http://bjsm.bmj.com/ Br J Sports Med: first published as 10.1136/bjsports-2019-101154 on 8 April 2020. Downloaded from doi:10.1136/bjsports-2019-101154 8 of 13 Review Unadjusted Age adjusted k, n b (95% CI) P value I2, p value k, n b (95% CI) P value I2, p value Mean baseline level 24, 1911 −0.16 (−0.44 to 0.12) 0.250 52.0%, p=0.002 24, 1911 −0.11 (−0.42 to 0.20) 0.492 52.5%, p=0.002 Sedentary effectiveness, hour/day* 23, 1882 −1.07 (−2.83 to 0.69) 0.232 52.9%, p=0.002 23, 1882 −1.69 (−3.40 to 0.02) 0.053 43.2%, p=0.019 Duration (vs ≤3 months) 25, 1932 0.655 56.2%, p<0.001 25, 1932 0.712 57.9%, p =<0.001 3–6 months 0.22 (−2.50 to 2.94) 0.874 0.40 (−2.64 to 3.43) 0.798 >6 months −1.20 (−3.97 to 1.57) 0.397 −1.14 (−4.19 to 1.92) 0.465 Risk of bias (vs high risk) 25, 1932 0.699 55.1%, p<0.001 25, 1932 0.629 57.1%, p =<0.001 Some concerns 1.20 −2.05 to 4.45) 0.468 1.82 (1.90 to 5.53) 0.338 Low risk 1.03 (−1.58 to 3.64) 0.439 1.35 (1.56 to 4.25) 0.363 Total cholesterol, mM 23, 1798 54.1%, p=0.001 23, 1798 54.1%, p=0.001 Mean baseline age, per 10 years 23, 1798 −0.14 (−0.22 to -0.07) <0.001 17.1%, p=0.233 – – – Mean baseline BMI, kg/m2 23, 1798 0.01 (−0.02 to 0.04) 0.664 54.6%, p=0.001 23, 1798 0.01 (−0.01 to 0.03) 0.398 17.4%, p=0.233 Mean baseline level 23, 1798 −0.24 (−0.50 to 0.02) 0.066 45.7%, p=0.011 23, 1798 −0.05 (−0.30 to 0.21) 0.713 20.6%, p=0.195 Sedentary effectiveness, hour/day* 23, 1798 0.08 (−0.04 to 0.20) 0.206 40.3%, p=0.027 23, 1798 0.01 (−0.10 to 0.12) 0.812 19.5%, p=0.208 Duration (vs ≤3 months) 23, 1798 0.573 54.6%, p=0.001 23, 1798 0.003 20.4%, p=0.202 3–6 months 0.07 (−0.17 to 0.31) 0.577 0.06 (−0.12 to 0.24) 0.493 >6 months 0.13 (−0.12 to 0.39) 0.306 0.11 (−0.08 to 0.30) 0.259 Risk of bias (vs high risk) 23, 1798 0.044 47.2%, p=0.009 23, 1798 <0.001 13.7%, p=0.284 Some concerns −0.34 (−0.65 to –0.04) 0.028 −0.21 (−0.48 to 0.06) 0.134 Low risk −0.11 (−0.39 to 0.16) 0.419 −0.05 (−0.29 to 0.19) 0.689 Triglycerides, mM 23, 1742 49.0%, p=0.005 23, 1742 49.0%, p=0.005 Mean baseline age, per 10 y 23, 1742 −0.05 (−0.11 to 0.02) 0.149 51.3%, p=0.003 – – – Mean baseline BMI, kg/m2 23, 1742 0.00 (−0.02 to 0.02) 0.962 50.3%, p=0.004 23, 1742 0.01 (−0.02 to 0.03) 0.667 52.4%, p=0.003 Mean baseline level 22, 1721 −0.20 (−0.63 to 0.22) 0.350 45.1%, p=0.014 22, 1721 −0.13 (−0.53 to 0.26) 0.508 31.7%, p=0.087 Sedentary effectiveness, h/day* 22, 1721 −0.06 (−0.17 to 0.05) 0.279 45.9%, p=0.012 22, 1721 −0.13 (−0.21 to –0.05) 0.001 0.0%, p=0.507 Duration (vs ≤3 months) 23, 1742 0.645 53.4%, p=0.002 23, 1742 0.447 55.7%, p=0.001 3–6 months −0.07 (−0.24 to 0.10) 0.402 −0.05 (−0.24 to 0.14) 0.639 >6 months −0.06 (−0.25 to 0.13) 0.516 −0.07 (−0.28 to 0.13) 0.481 Risk of bias (vs high risk) 23, 1742 0.396 53.6%, p=0.002 23, 1742 0.485 55.7%, p=0.001 Some concerns −0.13 (−0.33 to 0.07) 0.218 −0.07 (−0.34 to 0.19) 0.587 Low risk −0.10 (−0.28 to 0.07) 0.245 −0.06 (−0.28 to 0.16) 0.573 Table presents unstandardised regression coefficient (b) and 95% CI and p value from meta-regression of controlled trials of adult sedentary behaviour interventions ≥7 days. on October 23, 2024 by guest. Pro http://bjsm.bmj.com/ Br J Sports Med: first published as 10.1136/bjsports-2019-101154 on 8 April 2020. Downloaded from on October 23, 2024 by guest. Pro http://bjsm.bmj.com/ 154 on 8 April 2020. Downloaded from on October 23, 2024 by guest. Protected by copyright. bjsm.bmj.com/ Glucose metabolism Insulin outcomes were significantly beneficially associated with lower baseline levels, shorter intervention duration and higher RoB, with limited residual heterogeneity after accounting for RoB (I2=11.9, p=0.338); however, only the association with baseline level remained significant accounting for age (residual I2=0.0, p=0.641). Higher participant age significantly predicted enhanced HbA1c outcomes, and led to lower heterogeneity (residual I2=49.5, p=0.054) while in age-adjusted models, effects were significantly beneficially associated with higher BMI, longer intervention duration and lower RoB, and a border- line association with higher baseline levels. The model with age and BMI had no residual heterogeneity (I2=0.0, p=0.454). Prior to intervention, fasting glucose averaged 4.7±1.0 mM, indicating levels consistent with healthy metabolism or predi- abetes rather than diabetes. However, the studies covered a diverse spectrum from 4.1 mM in a study of healthy adults83 to 7.6 mM in a study of type 2 diabetes patients aged 40–80 years.72 Baseline insulin and HbA1c levels averaged 51.5±44.1 pM and 4.4%±0.6% were also quite variable across studies (table 3 and online supplementary table S5). Pooled effects pointed to small benefits to glucose metabolism, which were statistically significant only for fasting insulin (−1.42 pM, 95% CI -2.82 to 0.02) and small non-significant tendencies towards lower fasting glucose (−0.03 mM, 95% CI −0.11 to 0.05) and HbA1c (−0.10%, 95% CI −0.22% to 0.03%). Forest plots are shown in online supplementary figure 9–11. Small-study effects may have overstated effects on insulin and HbA1c. ober 23, 2024 by guest. Protected by copyright. y yp p g y Baseline insulin and HbA1c levels averaged 51.5±44.1 pM and 4.4%±0.6% were also quite variable across studies (table 3 and online supplementary table S5). Pooled effects pointed to small benefits to glucose metabolism, which were statistically significant only for fasting insulin (−1.42 pM, 95% CI -2.82 to 0.02) and small non-significant tendencies towards lower fasting glucose (−0.03 mM, 95% CI −0.11 to 0.05) and HbA1c (−0.10%, 95% CI −0.22% to 0.03%). Forest plots are shown in online supplementary figure 9–11. Small-study effects may have overstated effects on insulin and HbA1c. guest. Protected by copyright. Hadgraft NT, et al. Br J Sports Med 2021;55:144–154. doi:10.1136/bjsports-2019-101154 on October 23, 2024 by guest. Protected by copyright. http://bjsm.bmj.com/ Br J Sports Med: first published as 10.1136/bjsports-2019-101154 on 8 April 2020. Downloaded from Italics indicates overall p value (omnibus test). k=total number of interventions included and n=total number of individuals analysed in the included interventions, in the meta-regressions or main meta-analysis (boldface). Residual heterogeneity (I2 and p from Cochrane’s Q test) with overall heterogeneity in the main meta-analysis shown in boldface. *Estimated effectiveness of intervention on overall sedentary time (net of control). †No studies in the 3–6 months duration category. BMI, body mass index. Table 5 Continued http://bjsm. Br J Sports Med: first published as 10.1136/bjsports-2019-101154 on 8 April 2020. Downloaded from Lipid metabolism Sometimes a single study devi- ating from the general pattern appeared to be the issue, while other key factors (different for each outcome) tended to be due to participant age and BMI, study duration and RoB. There were very few studies with each characteristic; consequently, the CIs around effects were quite wide, and findings should not be taken to indicate non-significant predictors in the meta- regressions were unimportant. The low number of studies was also the reason stratified analyses were not performed to inform the effectiveness of specific types of interventions, for specific populations (eg, men, women, older adults and those with clinical conditions such as type 2 diabetes). Some potential success factors not able to be explored were ethnicity (poorly reported), sex, behaviour settings and dose response. Prior findings have sometimes suggested the biological responses to sedentary time may vary depending on the setting or context in which it occurs,88 89 by ethnicity,90–92 by sex18 91 93 94 and by the activity replacing sedentary time.95–98 There was limited heterogeneity in outcomes concerning HDL and LDL cholesterol (I2 <25, p≥0.05) and more substantial and significant heterogeneity in total cholesterol (I2=54.1, p=0.001) and triglycerides (I2=49.0, p=0.005). Removing one study36 markedly lowered the total cholesterol heterogeneity (I2=21.1, p=0.183) while the same was not the case for triglycerides. Meta-regressions (table 5) showed significantly greater reductions in total cholesterol were seen with higher age, and higher RoB, with limited residual hetero- geneity left after accounting for age (I2=17.1, p=0.233) while in age-adjusted models, significant predictors of greater reduc- tions were shorter study duration and higher RoB. It appears multiple factors may have contributed to the heterogeneity in triglyceride outcomes. None of the variables significantly predicted effects on triglycerides and residual heterogeneity remained high in all models (residual I2=45.1–53.6). In age- adjusted models, less effectiveness in improving sedentary behaviour outcomes significantly predicted greater reductions in triglycerides (−0.13 mM, 95% CI -0.21 to 0.05), with very limited heterogeneity left when considering both these factors simultaneously (I2=0.0, p=0.507), which also involved excluding one study74 due to missing data. The systematic review showed some key considerations for interpreting the effectiveness findings. The sedentary behaviour interventions performed were highly varied in terms of their setting, use of behavioural change components, and the degree of emphasis on sedentary behaviour; thus, the heterogeneous outcomes were not highly surprising. Discussion S l i Several reviews have reported on sedentary behaviour inter- ventions in relation to sedentary behaviour outcomes23 26 27 and found them to be effective, to varying degrees. These reviews indicated success seemed to vary depending on factors including the focus on sedentary behaviour (alone vs in combination with other lifestyle behaviours) and the type of intervention (with multicomponent workplace interventions being particularly successful). The current systematic review with meta-analyses considered these interventions in the context of their effect on biomarkers of cardiometabolic health, finding a small body of evidence. In total, 54 studies were identified, with 33 eligible for the meta-analyses, and with 6–25 controlled interventions ultimately included in meta-analyses concerning body anthro- pometry, blood pressure and haemodynamics, glucose metabo- lism and lipid metabolism. To overcome the limitations of the current evidence base the next logical step would be individual patient data meta- analysis, with interventions collecting ‘dose’ data regarding sedentary behaviour and the activities that may replace it in the most harmonisable way possible, even if this is only possible in a subsample of participants. Ideally, the measure- ment should allow both calculation of some total dose (eg, in MET hours), as well as partial out time spent sedentary and in various alternative behaviours, delineated by intensity, posture and accumulation method (eg, sedentary/sitting, standing, light movement, moderate movement, vigorous movement and bouted vs non-bouted forms of the relevant behaviours). Such an approach may help to determine the populations for which each intervention may be effective, as well as ascertain which specific behaviours (if any) may achieve the greatest biomarker improvements. Broadly, the meta-analyses provided some support for small improvements in selected indicators of body anthropometry, blood pressure, glucose metabolism and lipid metabolism with intervention, with none of the outcomes tending to worsen with intervention. Specifically, significant improvements were seen in body weight, waist circumference, percentage body fat, systolic blood pressure, insulin and HDL cholesterol. For some outcomes, findings varied widely from study to study, while for others they were quite consistent, with heterogeneity ranging widely (I2=0.0–72.9). It may be the case that some types of interventions are effective (and others ineffective), and/or the interventions may be effective in some populations but not others. The sensitivity analyses and meta-regressions guest. Protected by copyright. Other key features identified within the current evidence base are the type, reporting (or lack thereof) and specificity/sensi- tivity of biomarker outcomes collected. Lipid metabolism Prior to intervention, baseline levels averaged 4.3±0.6 mM total cholesterol, 1.2±0.4 mM HDL, 2.5±0.8 mM LDL and 1.1±0.5 mM triglycerides, with comparatively limited varia- tion across studies relative to other biomarkers (table 4, online supplementary table S5). Small significant improvements in response to sedentary behaviour interventions were seen in HDL cholesterol (0.04 mM, 95% CI 0.02 to 0.07) alongside Glucose, insulin and HbA1c all showed substantial hetero- geneity (I2=45.5 for glucose to I2=72.9 for insulin; p<0.05), which remained present in all the leave-one-out sensitivity anal- yses, except for glucose, where removing a single workplace study49 that had failed to elicit changes in sedentary behaviour markedly attenuated the heterogeneity (I2=28.4, p=0.126). Hadgraft NT, et al. Br J Sports Med 2021;55:144–154. doi:10.1136/bjsports-2019-101154 9 of 13 9 of 13 Review Review on October 23, 2024 http://bjsm.bmj.com/ Br J Sports Med: first published as 10.1136/bjsports-2019-101154 on 8 April 2020. Downloaded from on October 23, 2024 by guest. Protected by co http://bjsm.bmj.com/ Br J Sports Med: first published as 10.1136/bjsports-2019-101154 on 8 April 2020. Downloaded from a small, non-significant improvement in total cholesterol (−0.06 mM, 95% CI −0.16 to 0.04) and very small, non- significant effects on LDL cholesterol (−0.02 mM, 95% CI −0.07 to 0.04), and triglycerides (−0.02 mM, 95% CI −0.09 to 0.04). Forest plots for cholesterol and triglycerides are shown in online supplementary figure S12 and online supple- mentary figure 13–15. Small-study effects if anything limited the effects seen for lipid metabolism, with trimmed-and-filled estimates all either larger or virtually unchanged, and with a significant effect on total cholesterol emerging (−0.10 mM, 95% CI -0.20 to 0.00). a small, non-significant improvement in total cholesterol (−0.06 mM, 95% CI −0.16 to 0.04) and very small, non- significant effects on LDL cholesterol (−0.02 mM, 95% CI −0.07 to 0.04), and triglycerides (−0.02 mM, 95% CI −0.09 to 0.04). Forest plots for cholesterol and triglycerides are shown in online supplementary figure S12 and online supple- mentary figure 13–15. Small-study effects if anything limited the effects seen for lipid metabolism, with trimmed-and-filled estimates all either larger or virtually unchanged, and with a significant effect on total cholesterol emerging (−0.10 mM, 95% CI -0.20 to 0.00). provided some insight into potential factors underlying some of the heterogeneous results. Lipid metabolism Also, some caution should be exerted in extrapolating findings to groups with limited or no representation in the evidence base. Evidence has mostly been collected from studies of Caucasian or ‘white’ populations (variously defined) of working age, often with overweight/obese BMI or waist circumference, with very limited representation of those with clinical conditions pertinent to cardiovascular health, such as type 2 diabetes. The short duration of most interventions may have influenced the degree of effectiveness observed in the meta-analyses; there was a paucity of studies intervening ≥12 months and including maintenance evaluations from which to consider sustainability or determine what may happen in the longer term. Previously, it has been reported that biomarker results have been more promising at 12 months compared with 3 months, despite sitting reduction being greatest at 3 months.43 Hadgraft NT, et al. Br J Sports Med 2021;55:144–154. doi:10.1136/bjsports-2019-101154 10 of 13 Conclusions This systematic review with meta-analyses synthesised the body of work concerning the effectiveness of sedentary behaviour interventions on biomarkers of cardiometabolic risk, specifi- cally: body anthropometry; blood pressure and related haemody- namics; glucose metabolism; lipid metabolism and inflammation. Consistent with evidence from prior observational research and acute laboratory-based experiments (<7 days) linking seden- tary behaviour with cardiometabolic health,8 11 12 the evidence from ≥7 days interventions in free-living conditions showed small improvements in some cardiometabolic biomarkers. These biomarker improvements definitively occurred in response to interventions targeting sedentary behaviour (alone or alongside physical activity), but how they occurred in response to seden- tary reductions and increases in various forms of physical activity remains unclear. Our review indicated that studies in clinical populations, ethnicities other than Caucasian or ‘white’ in predominantly Western countries, and evaluation of biomarkers of inflammation and postprandial metabolism are key areas for future research. There were some caveats regarding the overall quality of the evidence. Trimmed-and-filled results mostly suggested publica- tion bias did not affect findings, but the insulin finding may be overstated and some of the lipid findings understated. Inferences were sometimes made from a very small number of studies (espe- cially regarding biomarkers of glucose and lipid metabolism), which is especially concerning with the findings varying so much between studies. The paucity of ‘low’ RoB studies is a limitation, though importantly most studies had an ‘unclear’ rather than a ‘high’ RoB and the meta-regressions did not usually show high RoB equated to the most promising results (if anything, findings showed the opposite). Correction notice This article has been corrected since it published Online First. The data and formatting in the tables have been updated. Correction notice This article has been corrected since it published Online First. The data and formatting in the tables have been updated. ► ►Observational and experimental research (mostly acute laboratory-based work) links both high volumes and prolonged periods of sedentary behaviour (sitting) with adverse health outcomes. Twitter Paddy C Dempsey @PC_Dempsey Twitter Paddy C Dempsey @PC_Dempsey Contributors All authors reviewed the systematic review strategy. LR executed the searches. PCD, NTH, REC, MSG and EW conducted the review and screened the initial results using standardised rules created a priori. PCD, NTH, REC and EW appraised the studies and extracted data from the primary studies and EW analysed the penultimate results. PCD, NTH and EW drafted the manuscript and all authors contributed to the critical revision of the manuscript and approved the final revised version. PCD is the guarantor. ► ►However, less is known about the nature and extent of health effects with sedentary behaviour interventions over longer periods and under free-living conditions. Funding PCD is supported by a National Health and Medical Research Council (NHMRC) of Australia Fellowship (#1142685) and the UK Medical Research Council [MC_UU_12015/3]. NO, DD and GH are supported by NHMRC of Australia Fellowships (#1003960, #1078360 & #1086029). Funding PCD is supported by a National Health and Medical Research Council (NHMRC) of Australia Fellowship (#1142685) and the UK Medical Research Council [MC_UU_12015/3]. NO, DD and GH are supported by NHMRC of Australia Fellowships (#1003960, #1078360 & #1086029). What is already known 8School of Sport Science, Exercise and Health, The University of Western Australia, Perth, WA, Australia 9School of Physiotherapy and Exercise Science, Curtin University, Perth, WA, Australia 10MRC Epidemiology Unit, Institute of Metabolic Science, University of Cambridge, Cambridge Biomedical Campus, Cambridge, UK 9School of Physiotherapy and Exercise Science, Curtin University, Perth, WA, Australia 10MRC Epidemiology Unit, Institute of Metabolic Science, University of Cambridge, Cambridge Biomedical Campus, Cambridge, UK ► ►Recent reviews have demonstrated sedentary-reduction interventions are effective at modifying behaviour (reducing sitting). Correction notice This article has been corrected since it published Online First. The data and formatting in the tables have been updated. Author affiliations 1C f U b T i 1Centre for Urban Transitions, Swinburne University of Technology, Melbourne, VIC, Australia 2 1Centre for Urban Transitions, Swinburne University of Technology, Melbourne, VIC, Australia 2 on October 23, 2024 by guest. Prot http://bjsm.bmj.com/ 154 on 8 April 2020. Downloaded from 2Baker Heart and Diabetes Institute, Melbourne, VIC, Australia 2Baker Heart and Diabetes Institute, Melbourne, VIC, Australia 3 3School of Public Health, The University of Queensland, Brisbane, QLD, Australia 4The Alfred Hospital, Melbourne, VIC, Australia p 5Central Clinical School/Department of Epidemiology and Preventive Medicine, Faculty of Medicine, Nursing and Health Sciences, Monash University, Melbourne, VIC, Australia 6 6Institute of Physical Activity and Nutrition Research, School of Exercise and Nutrition Sciences, Deakin University, Melbourne, VIC, Australia y 7Mary MacKillop Institute of Health Research, Australian Catholic University, Melbourne, VIC, Australia 8 Discussion S l i For example, most of the biomarkers collected (eg, blood glucose, insulin, triglycerides and blood pressure) are subject to homeostatic regulation but were only measured in fasted or resting states. It is important to also evaluate how some sensitive biomarkers (without these limitations) that have fairly consistently responded beneficially Hadgraft NT, et al. Br J Sports Med 2021;55:144–154. doi:10.1136/bjsports-2019-101154 10 of 13 Review in acute laboratory interventions lasting <7 days9 11 12 99 respond over longer intervention timeframes. Specifically, postprandial glucose, insulin, triglycerides and ambulatory blood pressure should be measured. Other understudied outcomes that are potentially useful to measure are: detailed markers of vascular haemodynamics and structure (eg, cardiovascular and cere- brovascular blood flow, flow-mediated dilatation and arterial stiffness)99 100; C-peptide; continuous glucose monitoring; postprandial lipids; lipid subclasses101 102; site-specific tissue samples (eg, muscle, adipose tissue) and additional interme- diate biomarkers (such as those related to systemic metabolic/ oxidative stress and inflammation).9 100 These outcomes could be collected in all participants or in subsamples, as they represent opportunities to detect changes that might otherwise be missed, and improve our understanding of shared risk factors and poten- tial mechanistic pathways. Hadgraft NT, et al. Br J Sports Med 2021;55:144–154. doi:10.1136/bjsports-2019-101154 What are the new findings ► ►This review evaluated the evidence regarding the impact that interventions to reduce sedentary behaviour, alone or in combination with physical activity increases, may have on important indicators of cardiometabolic risk, when intervening for ≥7 days under free-living conditions. Competing interests None declared. Competing interests None declared. Competing interests None declared. Patient consent for publication Not required. Patient consent for publication Not required. Provenance and peer review Not commissioned; externally peer reviewed. Open access This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/ licenses/by/4.0/. g y g ► ►Available evidence for different outcomes ranged from 6 to 25 controlled trials. On average, these interventions led to modest improvements in selected indicators of body anthropometry, glucose and lipid metabolism, and blood pressure regulation, with no adverse effects observed. p g ► ►Potential improvements for future research were noted: more high-quality studies and interventions > 12 months; more population diversity (based on ethnicity, age, and clinical factors); more sensitive biological indicators; and, more studies evaluating vascular function and inflammation. p g ► ►Potential improvements for future research were noted: more high-quality studies and interventions > 12 months; more population diversity (based on ethnicity, age, and clinical factors); more sensitive biological indicators; and, more studies evaluating vascular function and inflammation. 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https://openalex.org/W4390954737	https://bmcmedgenomics.biomedcentral.com/counter/pdf/10.1186/s12920-023-01782-7	English	null	Nine dietary habits and risk of colorectal cancer: a Mendelian randomization study	BMC medical genomics	2,024	cc-by	8,090	†Mengyang He and Luyao Huan shared first authors. Correspondence: Yingyi Fan fan38898901@126.com Jinchang Huang zryhhuang@163.com 1Graduate School, Beijing University of Chinese Medicine, Beijing, China 2Xiyuan Hospital of China Academy of Chinese Medical Sciences, Beijing, Chinafi 3Beijing University of Chinese Medicine Third Affiliated Hospital, No. 51 Anwai Xiaoguan Street, Chaoyang District, Beijing 100029, China © The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Nine dietary habits and risk of colorectal cancer: a Mendelian randomization study Mengyang He1†, Luyao Huan2†, Xuan Wang1, Yingyi Fan3 and Jinchang Huang3* BMC Medical Genomics BMC Medical Genomics He et al. BMC Medical Genomics (2024) 17:21 https://doi.org/10.1186/s12920-023-01782-7 Open Access Abstract Background Epidemiological studies have provided evidence that there is an association between diet and colorectal cancer. However, the causal relationship between dietary habits and colorectal cancer remains unknown. Methods The UK Biobank provided summary-level genome-wide association study data for nine dietary habits, including alcohol consumption (n = 549,703), instant coffee consumption (n = 250,308), fruit consumption (n = 210,947), meat consumption (n = 210,947), full cream milk consumption (n = 41,306), sweets consumption (n = 25,521), tea consumption (n = 501,494), vegetable consumption (n = 210,947), and yogurt/ice cream consumption (n = 210,947). Additionally, data on colorectal cancer were collected, consisting of 5,567 cases and 372,016 controls. The MR analysis employed inverse variance weighted, weighted median, MR-Egger regression, and MR multivariate residuals tests. Results In the predominantly European population, a positive association was observed between vegetables (OR = 1.014, 95% CI = 1.000-1.029, p = 0.048) and an increased risk of colorectal cancer. The results for vegetable did not survive correction for multiple comparisons. However, no strong evidence was found for other dietary factors, such as alcohol (OR = 1.012, 95% CI = 0.974–1.051, p = 0.556), fruit (OR = 1.007, 95% CI = 0.986–1.029, p = 0.512), meat (OR = 1.000, 95% CI = 0.987–1.026, p = 0.968), full cream milk (OR = 1.019, 95% CI = 0.979–1.061, p = 0.357), sweets (OR = 0.998, 95% CI = 0.991–1.004, p = 0.524), and tea (OR = 1.002, 95% CI = 0.994–1.009, p = 0.672), with regards to colorectal cancer risk in the European population. Conclusions Our study highlights the need for a more nuanced approach to dietary recommendations for CRC prevention, with greater emphasis adherence to the Mediterranean dietary pattern. Keywords Causality, Dietary, Mendelian randomization, Colorectal cancer †Mengyang He and Luyao Huan shared first authors. Methods Colorectal cancer (CRC) is a prevalent malignant tumor globally, ranking third in frequency and second in mor tality after lung cancer. In 2020, more than 1.9 million new cases and 935,000 deaths were reported [1], and if the current trend persists, the burden of CRC will surge by 60% to over 2.2 million cases and 1.1 million deaths annually by 2030 [2]. Although lifestyle factors and meta bolic conditions like smoking, physical inactivity, seden tary behavior, and diabetes mellitus have been linked to an increased incidence and mortality of CRC, the exact cause of the disease remains unclear [3–8]. Consequently, CRC is a multifactorial condition involving many poten tial etiological factors, emphasizing the need for identify ing risk factors to aid in its prevention. Genetic variants associated with 9 dietary habits This MR study is reported according to the reporting guidelines for enhancing observational epidemiological studies using MR (STROBE-MR). The data for this study were obtained from UK Biobank (https://www.nealelab. is/uk-biobank) and are publicly available without access restrictions. To increase the number of single nucleo tide polymorphisms (SNPs) related to dietary habits, a more relaxed threshold (p < 5 × 10− 6) was used, and the chain imbalance was set to LD (r2 < 0.001) to ensure study robustness. An F-statistic for SNPs greater than the con ventional value of 10 was used to assess the potential of the tool to predict instrumental variable [20].h The exposure instrumental variable was assessed using nine genome-wide association studies (GWAS) based on UK Biobank data, which examined the association between SNPs and vegetable consumption (n = 210,947), vegetables (female), vegetables (male) consumption, fruit consumption (n = 210,947), beef intake consumption (n = 69,687), tea consumption (n = 501,494), meat con sumption (n = 210,947), sweets consumption (n = 25,521), decaffeinated coffee consumption (n = 88,784), ground coffee consumption (n = 115,952), instant coffee con sumption (n = 250,308), other type of coffee consump tion (n = 8,754), full cream milk consumption (n = 41,603), skimmed milk consumption (n = 119,480), semi-skimmed milk consumption (n = 382,990), soya milk consumption (n = 22,717), other type of milk consumption (n = 9,933) and yogurt/ice cream consumption (n = 210,947). Over the past few decades, numerous epidemiological studies have established a correlation between specific dietary patterns and the risk of colorectal cancer (CRC). Page 2 of 11 He et al. BMC Medical Genomics (2024) 17:21 Page 2 of 11 He et al. BMC Medical Genomics (2024) 17:21 He et al. BMC Medical Genomics Methods According to the Global Burden of Disease study (GBD) 2019, dietary factors are considered to be one of the most critical factors impacting the prognosis of CRC. Dietary compounds have the potential to influence CRC in vari ous ways [9, 10]. For example, a meta-analysis of 13 pro spective cohort studies conducted by Zhong et al. [11] in 2020 revealed that compliance with a Mediterranean diet was associated with a 10% reduction in CRC inci dence. Similarly, Bradbury et al. (2019) [12] found that individuals who consumed an average of 76 g of red and processed meats daily had a 20% higher risk of CRC than those who consumed only 21 g daily. While some studies have reported positive results for a vegetarian diet in pre venting and treating CRC [13, 14], others have produced conflicting results [15–17]. Therefore, it remains unclear whether there are any causal associations between dietary habits and CRC risk. To provide a more detailed analysis of the types of alco hol consumption, we included ten phenotypes related to alcohol consumption, including alcohol (female), alcohol (male), alcohol intake frequency (n = 501,494), alcohol drinker status (current) (n = 549,703), alcohol drinker sta tus (previous) (n = 21,317), alcohol drinker status (never) (n = 25,396), red wine consumption (n = 60,887), rose wine consumption (n = 11,404), white wine consumption (n = 48,877), and fortified wine consumption (n = 7,585). Using a touch-screen tablet, the participants filled in the prepared relevant questionnaire, from which the instru mental variables were obtained. This resource may be accessed at: https://biobank.ndph.ox.ac.uk/showcase/ ukb/docs/TouchscreenQuestionsMainFinal.pdf. Mendelian randomization (MR) is a genetically informed methodology that utilizes single-nucleotide polymorphisms (SNPs) as instrumental variables (IVs) for risk factors of interest. This approach provides a valid way to assess causality free from confounding or reverse causality bias [18]. Unlike randomized controlled trials (RCTs) [19], MR allows investigation of many exposures that cannot be studied using RCTs. However, to date, no MR studies have explored the potential causal relation ships between dietary habits and colorectal cancer (CRC) risk.h Statistical analyses f b After obtaining GWAS summary data for different dietary habits and CRC from UK Biobank, we employed various MR methods to determine MR estimates for the different dietary habits in CRC, including inverse vari ance weighted (IVW), weighted median, and MR-Egger. Since these methods have different underlying assump tions regarding horizontal pleiotropy, using multiple methods helped increase the robustness of our results. Our main result was based on an inverse variance- weighted meta-analysis of Wald ratios for individual SNPs, assuming that the instrument could only influence the results through exposure of interest and not through any alternative pathway [23]. g g We found no potential causal association between vegetables (female) (OR = 1.003, 95% CI = 0.981–1.026, p = 0.788), vegetables (male) (OR = 1.006, 95% CI = 0.995– 1.017, p = 0.320), fruit (OR = 1.007, 95% CI = 0.986–1.029, p = 0.512), red beef intake (OR = 1.002, 95% CI = 0.992– 1.013, p = 0.692), tea (OR = 1.002, 95% CI = 0.994–1.009, p = 0.672), meat (OR = 1.000, 95% CI = 0.987–1.013, p = 0.968), sweets (OR = 0.998, 95% CI = 0.991–1.004, p = 0.524) (Fig. 2A) on CRC risk. And there was no het erogeneity in the causal relationship between these expo sure dietary factors and CRC risk (Table 1). The results of MR regression analysis, funnel plots and leave-one- out sensitivity analysis can be found in Supplementary Figs. 1–7. To complement our IVW estimates, we also used MR- Egger and weighted median methods. While these meth ods are less efficient and have wider confidence intervals, they can provide more robust estimates across a wider range of scenarios. Sensitivity analysis played a crucial role in our MR study, allowing us to detect potential pleiotropy. We used a heterogeneity marker (Cochran Q-derived p < 0.05) from the IVW method to indi cate potential horizontal pleiotropy, while the intercept obtained from MR-Egger regression indicated directional pleiotropy (p < 0.05 was considered evidence of direc tional pleiotropy) [24]. We also used the Bonferroni mul tiple correction method. Statistical analyses f b We also found no potential causal effect on CRC risk for alcohol drinker status (current) (OR = 1.012, 95% CI = 0.974–1.051, p = 0.556), alcohol drinker status (never) (OR = 1.01., 95% CI = 0.957–1.067, p = 0.715), alco hol drinker status (previous) (OR = 1.001, 95% CI = 0.935– 1.072, p = 0.971), alcohol (female) (OR = 1.004, 95% CI = 0.998–1.011, p = 0.184), alcohol (male) (OR = 1.001, 95% CI = 0.993–1.008, p = 0.870), alcohol intake frequency (OR = 0.999, 95% CI = 0.997–1.001, p = 0.154), white wine (OR = 1.001, 95% CI = 0.997–1.005, p = 0.708), fortified wine (OR = 0.997, 95% CI = 0.981–1.014, p = 0.764), and red wine (OR = 1.000, 95% CI = 0.997–1.002, p = 0.893) (Fig. 2B). And there was no heterogeneity in the causal relationship between these exposure dietary factors and CRC risk (Table 1). The results of MR regression analysis, funnel plots, and leave-one-out sensitivity analysis can be found in Supplementary Figs. 8–16.i To assess whether MR estimates were driven or biased by individual SNPs, we performed leave-one-out analy sis. All analyses were performed using Two-Sample MR in R (version 4.2.2) The study frame chart is presented in Fig. 1. GWAS summary data for CRC UK Biobank Cohort Study’s GWAS yielded overall can cer risk data for 5,567 cases and 372,016 controls [21]. Briefly, cancer cases were categorized according to ICD-9 (http://www.icd9data.com/2007/Volume1/default. htm) and ICD-10 (https://icd.who.int/browse10/2016/ en), with data completed through September 2019, and controls were defined as individuals without any cancer code (ICD10 or ICD2) and without a self-reported cancer The aim of this study is to examine the potential causal associations between nine dietary habits (vegetable consumers, alcohol consumption, instant coffee con sumption, tea consumption, milk consumption, yoghurt consumption, meat consumption, fruit consumption, and sweets consumption) and CRC risk using MR analyses. He et al. BMC Medical Genomics (2024) 17:21 Page 3 of 11 He et al. BMC Medical Genomics for multiple comparisons (The corrected p-value was 0.0028). diagnosis. More information on estimation and quality control measures can be found in other topics [22]. We retrieved the data from the IEU OPEN GWAS PROJ ECT and extracted the single nucleotide polymorphisms (SNPs) associated with various dietary habits, along with their effect sizes and standard errors. Any SNPs with intermediate allele frequency were removed from the analysis. Detailed information on the SNPs associated with each dietary habit and their association with CRC can be found in Supplementary Table 1. The p-value of Cochran Q test for MR-Egger was 6.13E-01 and for IVW was 6.39E-01. That is, there was no heterogeneity in the causal association between vegeta bles and CRC. Figure 3A shows the MR regression slope and individual causal estimates for each of the 12 SNPs. In addition, there was no evidence of significant intercep tion (intercept = 1.95E-04, SE = 2.41E-04, P = 4.38E-01), indicating that no directional pleiotropy was observed. In addition, the funnel plot was symmetrical, suggesting no pleiotropy (Fig. 3B). In the leave-one-out sensitivity anal ysis, no single SNP strongly violated the overall effect of vegetables on CRC (Fig. 3C). Resultsf Causal effect from 9 dietary habits to CRCf Our findings suggest no potential causal link between coffee (Fig. 2C) and milk (Fig. 2D) and CRC. And there was no heterogeneity and pleiotropy in the causal rela tionship between these exposure factors and CRC (Table 1). The results of MR regression analysis, funnel plots and leave-one-out sensitivity analysis can be found in Supplementary Figs. 17–25. Causal effect from 9 dietary habits to CRCf Using an IVW random effects model with 12 SNPs asso ciated with vegetable consumers, we found a poten tially causal effect of vegetable consumers on CRC risk that was significant (OR = 1.014, 95% CI = 1.000-1.029, p = 0.048) (Fig. 2A). Meanwhile, similar risk estimates were obtained using the weighted median (WM) method (OR = 1.019, 95% CI = 1.000-1.038, p = 0.045) (Table 1). The results for vegetable did not survive correction Due to the large directional pleiotropy (P < 0.05) for the two instrumental variables of rose wine yogurt/ice cream, Page 4 of 11 He et al. BMC Medical Genomics (2024) 17:21 He et al. Causal effect from 9 dietary habits to CRCf BMC Medical Genomics Table 1 Mendelian randomization estimates of the associations between dietary habits and colorectal cancer Exposure Outcome nSNP IVW-derived P value OR (95%CI) MR-Egger- derived P value OR (95%CI) WM- derived P value OR (95%CI) Egger in tercept- derived P value Vegetables Colorectal cancer 12 0.048 1.014 (1.000-1.029) 0.819 1.004 (0.974-1.034) 0.045 1.019 (1.000-1.038) 0.438 Vegetables (Female) Colorectal cancer 6 0.788 1.003 (0.981-1.026) 0.702 1.013 (0.953-1.076) 0.644 1.005 (0.984-1.027) 0.75 Vegetables (Male) Colorectal cancer 9 0.32 1.006 (0.995-1.017) 0.978 1.001 (0.962-1.041) 0.916 0.999 (0.986-1.013) 0.804 Fruit Colorectal cancer 8 0.512 1.007(0.986-1.029) 0.95 1.002 (0.932-1.078) 0.327 1.019 (0.984-1.055) 0.896 Beef intake Colorectal cancer 5 0.692 1.002 (0.992-1.013) 0.731 1.004 (0.985-1.022) 0.821 1.002 (0.988-1.015) 0.865 Tea Colorectal cancer 23 0.672 1.002 (0.994-1.009) 0.418 1.008 (0.990-1.026) 0.963 1.000 (0.991-1.010) 0.482 Meat Colorectal cancer 9 0.968 1.000 (0.987-1.013) 0.683 0.993 (0.961-1.026) 0.982 1.000 (0.984-1.017) 0.645 Sweets Colorectal cancer 18 0.524 0.998 (0.991-1.004) 0.891 1.001 (0.989-1.013) 0.59 0.998 (0.990-1.006) 0.547 Alcohol Alcohol drinker status (current) Colorectal cancer 27 0.556 1.012 (0.974-1.051) 0.427 0.961 (0.873-1.058) 0.726 1.009 (0.960-1.061) 0.268 Alcohol drinker status (Never) Colorectal cancer 23 0.715 1.010 (0.957-1.067) 0.543 0.965 (0.862-1.080) 0.73 0.987 (0.915-1.065) 0.374 Alcohol drinker status (Previous) Colorectal cancer 20 0.971 1.001 (0.935-1.072) 0.058 0.877 (0.773-0.996) 0.806 1.011 (0.929-1.100) 0.312 Alcohol (Female) Colorectal cancer 12 0.184 1.004 (0.998-1.011) 0.824 1.002 (0.984-1.020) 0.257 1.004 (0.997-1.011) 0.798 Alcohol (Male) Colorectal cancer 9 0.87 1.001 (0.993-1.008) 0.912 1.001 (0.983-1.020) 0.085 1.006 (0.999-1.012) 0.958 Alcohol intake frequency Colorectal cancer 184 0.154 0.999 (0.997-1.001) 0.178 0.997 (0.992-1.001) 0.12 0.997 (0.993-1.001) 0.404 White wine Colorectal cancer 20 0.708 1.001 (0.997-1.005) 0.38 1.004 (0.996-1.012) 0.617 1.001 (0.996-1.006) 0.422 Fortified wine Colorectal cancer 16 0.764 0.997 (0.981-1.014) 0.949 0.999 (0.971-1.028) 0.747 0.996 (0.975-1.018) 0.894 Red wine Colorectal cancer 14 0.893 1.000 (0.997-1.002) 0.803 1.000 (0.997-1.003) 0.897 1.000 (0.997-1.003) 0.804 Coffee Decaffeinated coffee Colorectal cancer 14 0.104 1.025 (0.995-1.005) 0.671 0.985 (0.921-1.054) 0.109 1.033 (0.993-1.075) 0.227 Ground coffee Colorectal cancer 91 0.266 0.994 (0.983-1.005) 0.677 1.009 (0.969-1.050) 0.35 0.993 (0.978-1.0008) 0.456 Instant coffee Colorectal cancer 33 0.435 0.994 (0.978-1.010) 0.666 1.012 (0.960-1.067) 0.338 0.990 (0.970-1.010) 0.484 Other type of coffee Colorectal cancer 5 0.997 1.000 (0.869-1.150) 0.25 1.212 (0.930-1.579) 0.838 1.019 (0.850-1.220) 0.192 Milk Full cream milk Colorectal cancer 22 0.357 1.019 (0.979-1.061) 0.086 1.117 (0.991-1.259) 0.809 1.007 (0.950-1.067) 0.127 Skimmed milk Colorectal cancer 29 0.608 0.994 (0.970-1.018) 0.674 0.982 (0.903-1.068) 0.679 0.994 (0.964-1.024) 0.775 Semi-skimmed milk Colorectal cancer 12 0.662 0.994 (0.965-1.023) 0.995 1.000 (0.922-1.083) 0.974 1.001 (0.9639-1.03) 0.876 Soya milk Colorectal cancer 23 0.597 0.983 (0.923-1.047) 0.886 0.987 (0.832-1.172) 0.329 0.960 (0.886-1.042) 0.956 Other type of milk Colorectal cancer 12 0.126 0.893 (0.773-1.032) 0.459 0.894 (0.671-1.190) 0.414 0.925 (0.768-1.115) 0.998 Rose wine Colorectal cancer 23 – – – – – – 0.013 Yogurt/Ice-cream Colorectal cancer 14 – – – – – – 0.031 Abbreviation: SNP, single-nucleotide polymorphism; IVW, inverse variance weighted; WM, weighted median. Causal effect from 9 dietary habits to CRCf Due to the large directional pleiotropy (P<0.05) for the instrumental variables of yogurt/ice cream and rose wine, we considered further analysis of the causal association with CRC to be of little significance Page 5 of 11 He et al. BMC Medical Genomics (2024) 17:21 He et al. BMC Medical Genomics Page 5 of 11 He et al. BMC Medical Genomics (2024) 17:21 we deemed it of little significance to further analyze the causal association with CRC. Causal effects of different dietary habits on potential CRC risk factors Our study aimed to investigate whether the association between 9 genetically determined dietary habits and CRC is influenced by pleiotropic pathways related to CRC. To achieve this, we employed the IVW approach to analyze the association between these dietary habits and vari ous CRC risk factors, including family history of diges tive organ malignancies, history of tobacco, diabetes, and inflammatory bowel disease. Our analysis revealed no Fig. 1 Study flame chart of the Mendelian randomization study revealing the causal relationship between dietary habits and colorectal cancer Fig. 1 Study flame chart of the Mendelian randomization study revealing the causal relationship between dietary habits and colorectal cancer Fig. 1 Study flame chart of the Mendelian randomization study revealing the causal relationship between dietary habits and colorectal cancer the Mendelian randomization study revealing the causal relationship between dietary habits and colorectal cancer Fig. 1 Study flame chart of the Mendelian randomization study revealing the causal relationship between dietary habit we deemed it of little significance to further analyze the causal association with CRC. we deemed it of little significance to further analyze the causal association with CRC. is influenced by pleiotropic pathways related to CRC. To achieve this, we employed the IVW approach to analyze the association between these dietary habits and vari ous CRC risk factors, including family history of diges tive organ malignancies, history of tobacco, diabetes, and inflammatory bowel disease. Our analysis revealed no Causal effects of different dietary habits on potential CRC risk factors Our study aimed to investigate whether the association between 9 genetically determined dietary habits and CRC He et al. BMC Medical Genomics (2024) 17:21 He et al. BMC Medical Genomics Page 6 of 11 Fig. 2 (A) Odds ratio plot for dietary habits and colorectal cancer. (B) Odds ratio plot for dietary habits of alcohol and colorectal cancer. (C) Odds ratio plot for dietary habits of coffee and colorectal cancer. (D) Odds ratio plot for dietary habits of milk and colorectal cancer Fig. 2 (A) Odds ratio plot for dietary habits and colorectal cancer. (B) Odds ratio plot for dietary habits of alcohol and colorectal cancer. (C) Odds ratio plot for dietary habits of coffee and colorectal cancer. (D) Odds ratio plot for dietary habits of milk and colorectal cancer causal effect of the nine dietary habits on these potential risk factors for CRC, as presented in Table 2. A long-term cohort study by Gilsing et al. showed that vegetarians did not have a significantly lower risk of colorectal cancer compared to 6–7 days/week meat consumers [14]. Interestingly, our findings also suggest a positive causal link between vegetables and CRC. There fore, we disagree that eliminating animal protein sources from the diet is beneficial to human health [15]. It is rec ommended following the Mediterranean dietary pattern for CRC prevention, which involves a high intake of olive oil and plant foods (fruits, vegetables, legumes, nuts, and whole grains), moderate consumption of fish, poultry, dairy products, and alcohol, and a low intake of red meat, processed foods, and confectionery [27, 28]. Discussion We employed a multi-sample MR approach to compre hensively evaluate the potential causal effect of various dietary habits on the incidence of CRC. According to our findings, no conclusive evidence supports a causal rela tionship between the genetic prediction of certain food habits, such as tea and coffee, and CRC risk. We did observe a causal effect of the genetic prediction of veg etables on CRC risk. However, the results for vegetable did not survive correction for multiple comparisons.i The causal relationship between dietary habits, particu larly alcohol consumption, and CRC has attracted grow ing attention. A MR analysis conducted on a Japanese population suggests a potential causal link between alco hol consumption and CRC risk in Asians [29]. Another MR analysis examining alcohol consumption and CRC risk found that genetically predicted alcohol use and con sumption is a risk factor for CRC, while genetically pre dicted coffee consumption is protective [30].However, these studies on alcohol consumption were conducted on Asian populations. Interestingly, epidemiological evi dence suggests that increased alcohol consumption is not It is widely accepted that dietary fiber have chemother apeutic potential for treating cancer through direct action in the gastrointestinal tract, such as by reducing trans port time and contact of carcinogens with the colonic mucosa, increasing carcinogen binding, and production of short-chain fatty acids [25]. However, previous cohort studies and meta-analyses have shown no significant association between vegetable consumers and reduced risk of CRC [14, 15]. Interestingly, the large cohort study of the European Prospective Investigation into Cancer and Nutrition (EPIC-Oxford) showed a higher incidence of CRC in vegetarians than in meat-eaters [26]. He et al. BMC Medical Genomics (2024) 17:21 Page 7 of 11 He et al. BMC Medical Genomics Fig. 3 (A). Scatter plot of SNPs associated with vegetable and their risk of colorectal cancer. (B). Funnel plot of SNPs associated with vegetable and their risk of colorectal cancer. (C). Leave one-out of SNPs associated with vegetable and their risk of colorectal cancer Fig. 3 (A). Scatter plot of SNPs associated with vegetable and their risk of colorectal cancer. (B). Funnel plot of SNPs associated with vegetable and their risk of colorectal cancer. (C). Leave one-out of SNPs associated with vegetable and their risk of colorectal cancer Fig. 3 (A). Scatter plot of SNPs associated with vegetable and their risk of colorectal cancer. (B). Discussion BMC Medical Genomics Table 2 Mendelian randomization estimates of the associations from dietary habits on common risk factors of colorectal cancer Outcome Causal effect (95% CI); p value Alcohol Coffee Fruit Meat Milk Sweets Tea Vegetables Yogurt-ice DM 1.32 (0.80-2.20); 0.278 1.05 (0.86-1.28); 0.649 2.44 (0.53-11.34); 0.255 0.95 (0.59-1.51); 0.819 1.19 (0.52-2.72); 0.673 0.79 (0.50-1.26); 0.330 1.14 (0.83- 1.57); 0.406 0.91 (0.11- 7.62); 0.931 0.98 (0.41- 2.38); 0.972 Family history 1.00 (0.99-1.00); 0.608 1.00 (0.99-1.01); 0.897 1.02 (0.99-1.06); 0.060 1.00 (0.99-1.01); 0.483 1.00 (0.99-1.01); 0.806 1.01 (0.99-1.01); 0.196 1.00 (0.99- 1.01); 0.823 0.99 (0.98- 1.01); 0.902 0.99 (0.98- 1.00); 0.140 IBD 1.22 (0.82-1.82); 0.331 1.06 (0.90-1.24); 0.491 0.69 (0.16-3.03); 0.626 1.33 (0.90-1.97); 0.150 0.77 (0.36-1.63); 0.487 1.12 (0.79-1.60); 0.524 1.02 (0.60- 1.74); 0.929 1.13 (0.40- 3.23); 0.816 1.09 (0.54- 2.18); 0.820 Tobacco 0.99 (0.20-4.99); 0.993 1.67 (0.80-3.48); 0.170 0.01 (0.02-3.36); 0.110 0.50 (0.09-2.68); 0.414 0.93 (0.03-33.61); 0.969 0.98 (0.13-7.14); 0.982 2.67 (0.63- 11.28); 0.182 0.17 (0.01- 9.68); 0.397 0.18 (0.01- 136.28); 0.286 Table 2 Mendelian randomization estimates of the associations from dietary habits on common risk factors of colorectal cancer Outcome Causal effect (95% CI); p value Alcohol Coffee Fruit Meat Milk Sweets Tea Vegetables Yogurt-ice DM 1.32 (0.80-2.20); 0.278 1.05 (0.86-1.28); 0.649 2.44 (0.53-11.34); 0.255 0.95 (0.59-1.51); 0.819 1.19 (0.52-2.72); 0.673 0.79 (0.50-1.26); 0.330 1.14 (0.83- 1.57); 0.406 0.91 (0.11- 7.62); 0.931 0.98 (0.41- 2.38); 0.972 Family history 1.00 (0.99-1.00); 0.608 1.00 (0.99-1.01); 0.897 1.02 (0.99-1.06); 0.060 1.00 (0.99-1.01); 0.483 1.00 (0.99-1.01); 0.806 1.01 (0.99-1.01); 0.196 1.00 (0.99- 1.01); 0.823 0.99 (0.98- 1.01); 0.902 0.99 (0.98- 1.00); 0.140 IBD 1.22 (0.82-1.82); 0.331 1.06 (0.90-1.24); 0.491 0.69 (0.16-3.03); 0.626 1.33 (0.90-1.97); 0.150 0.77 (0.36-1.63); 0.487 1.12 (0.79-1.60); 0.524 1.02 (0.60- 1.74); 0.929 1.13 (0.40- 3.23); 0.816 1.09 (0.54- 2.18); 0.820 Tobacco 0.99 (0.20-4.99); 0.993 1.67 (0.80-3.48); 0.170 0.01 (0.02-3.36); 0.110 0.50 (0.09-2.68); 0.414 0.93 (0.03-33.61); 0.969 0.98 (0.13-7.14); 0.982 2.67 (0.63- 11.28); 0.182 0.17 (0.01- 9.68); 0.397 0.18 (0.01- 136.28); 0.286l no evidence supported an association between alcohol consumption and overall or site-specific cancer risk [33]. Nevertheless, further larger MR studies are required to confirm the genetically predicted association between high-dose, frequent alcohol consumption and CRC. While high consumption of fruits has been suggested to reduce the risk of colon cancer, our study does not support this claim. Discussion Funnel plot of SNPs associated with vegetable and their risk of colorectal cancer. (C). Leave one-out of SNPs associated with vegetable and their risk of colorectal cancer Fig. 3 (A). Scatter plot of SNPs associated with vegetable and their risk of colorectal cancer. (B). Funnel plot of SNPs associa risk of colorectal cancer. (C). Leave one-out of SNPs associated with vegetable and their risk of colorectal cancer oxidative metabolite acetaldehyde, which is a known human carcinogen [34]. However, there may be other mechanisms through which alcohol consumption can reduce cancer risk, such as increased insulin sensitivity through increased lipocalin levels [35]. In particular, red wine contains flavonoids and polyphenolic compounds. Furthermore, it has been demonstrated that these com pounds have chemotherapeutic potential for treating cancer and inflammation [36, 37]. Moderate alcohol con sumption has been empirically shown to reduce inflam matory markers and C-peptides [38–40], and a basic study on alcohol rat models found that moderate alcohol consumption does not increase biological risk factors for CRC development and may even provide beneficial effects by reducing inflammation and decreasing DNA damage [41]. Interestingly, a large meta-analysis showed a protective association for light/moderate alcohol con sumption at proximal colon, distal colon, and rectal can cer sites [42]. However, the results of the current MR study are consistent with previous MR studies in which significantly associated with CRC risk in the UK Dietary Cohort Consortium [31]. A cohort study by Song-Yi Park et al. [32] indicates that not all alcohol is associated with CRC and that the relationship between alcohol and CRC varies by race/ethnicity. Another MR study [33] involving participants from the UK Biobank and the International Genetic Alliance found no evidence to support a causal association between alcohol consumption and site-spe cific cancers (lung, breast, ovarian, and prostate).h i Thus, we believe that the causal association between alcohol consumption and other dietary habits and the risk of CRC in European populations requires further investigation. To address this issue, we conducted a MR estimation using patterns of consumption of dif ferent genders and 10 alcohol subtypes in predominant European populations. The results revealed no causal association between alcohol consumption and CRC in predominant European populations. In general, alcohol consumption is believed to poten tially increase cancer risk through the production of its Page 8 of 11 Page 8 of 11 He et al. BMC Medical Genomics (2024) 17:21 He et al. Data availability All data used in the current study are publicly available GWAS summary data (https://gwas.mrcieu.ac.uk/) and (https://www.nealelab.is/uk-biobank). Supplementary Information Th li i i l pp y The online version contains supplementary material available at https://doi. org/10.1186/s12920-023-01782-7. Supplementary Material 1 (STables): Harmonized SNP data related with dietary habits and colorectal cancer Supplementary Material 1 (STables): Harmonized SNP data related with dietary habits and colorectal cancer The use of MR in our study allowed us to minimize the effects of confounding bias and reverse causation. The random assignment of SNPs at conception adds strength to our findings, making them more compelling than those of observational studies. Our results emphasize the importance of establishing causal relationships between dietary habits and CRC in order to inform public health policies for early prevention and timely intervention. Supplementary Material 2 (SFigures): The results of MR regression analysis, funnel plots, and leave-one-out sensitivity analysis for other dietary habits Supplementary Material 2 (SFigures): The results of MR regression analysis, funnel plots, and leave-one-out sensitivity analysis for other dietary habits Author contributions Conceived and designed the experiments: JC.H. and MY.H.Performed the experiments: LY.H, MY.H and X.W.Analyzed the data: LY.H and MY.H.Prepared figures: LY.H, MY.H, YY.F and X.W.Prepared tables: MY.H and X.W.Wrote the paper: LY.H and MY.H.All authors reviewed the manuscript. CRC arises through three major pathways, including the adenoma-carcinoma sequence, the serrated path way, and the inflammatory pathway. It is an etiologically heterogeneous disease based on the anatomical location of the tumor or the overall molecular subtype alteration [64]. Genetic factors have an etiologic role in predispos ing individuals to CRC. However, the majority of CRC is disseminated and is primarily attributable to a range of modifiable environmental risk factors (e.g., obesity, phys ical inactivity, and smoking). This confounding may have contributed to the negative causal association between dietary factors and CRC based on the patients with CRC included in our study. Discussion Given the marginal association between vegetable con sumption and CRC, gender-specific subgroup analyses Page 9 of 11 He et al. BMC Medical Genomics (2024) 17:21 (2024) 17:21 He et al. BMC Medical Genomics were conducted. However, the gender subgroup analy sis did not support this conclusion. Based on this, we believe that future validation will require additional quantitative data. Existing studies provide evidence of a dose-response relationship between different dietary habits and CRC. For example, the meta-analysis by Wu et al. [61]showed a non-linear dose-response relation ship between only citrus intake and CRC risk, with the risk being minimized when intake reached 120 g/d (OR = 0.85) and no significant dose-response relationship was observed with continued increases in intake. Chen et al. [62]showed that the linear curve of red and processed meat and colorectal cancer approached its plateau at high intakes of up to about 100 g/day. Ken Horisaki et al. [63] found that the higher the coffee consumption, the higher the value of the relative risk of CRC, although there was no statistically significant. results. Thirdly, the source population of our data is pre dominantly European, and further research is needed to determine the generalizability of our findings to other populations. Fourthly, MR analysis has its own limita tions, such as statistical power. In MR studies, statistical power is determined by the frequency of genetic vari ables used, the magnitude of the effect of the variables on risk factors, and the study sample size. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Consent for publication Not applicable. However, our study has several limitations. First, the genetic information in this study’s data was insufficient to determine whether CRC is a germline or somatic mutation. The confounding effect of the two mutations may compromise the results. Second, our study data lacked specific data on the high and low doses and fre quency of dietary habits, which may have affected the Discussion This conclusion aligns with a 10-year follow-up cohort study conducted in the European pop ulation, which also found that fruit consumption alone did not provide protection against CRC [43]. Similarly, a cohort study of Asian populations found that fruit intake was not associated with CRC morbidity and mortality in either sex [44, 45]. A recent MR analysis study demonstrated that pro cessed meat intake increases the risk of CRC, whereas no causal association was found between red and white meat intake and CRC [46]. In contrast, our study did not establish any causal association between meat consump tion and CRC, including red meat. The ratio of red meat to white meat in the meat instrumental variable in this study could not be determined. Therefore, this conclu sion needs to be further confirmed in the future. i Tea is one of the most commonly consumed beverages worldwide, and drinking tea has been hypothesized to reduce the risk of CRC. Antioxidants, such as polyphe nols, in tea protect colon epithelial cells from oxidative DNA damage caused by free radicals [47, 48]. However, we are skeptical about the conclusion. most cohort stud ies and Meta-analyses do not support the conclusion that tea consumption reduces CRC risk [49–54]. Second, our MR analysis similarly found no evidence for a negative causal association between tea consumption (both black and green tea) and CRC. Our explanation for this finding is that, first, tea has been reported to have mutagenic and genotoxic compounds, such as tannins and caffeine [55, 56], which may increase the risk of colon cancer. Second, the brewing method and type of tea may also affect the amount of tea polyphenols [57], which cannot be fully captured using tea addition alone. Our analysis also indicates no causal link between cof fee and CRC. Complex compounds in coffee with oppos ing effects may account for the observed results. Coffee consumption may increase colonic peristalsis, reducing the exposure of colonic epithelial cells to potential carcin ogens [47]. It may also reduce the synthesis and secretion of bile acids, which are potential colon carcinogens [58]. In contrast, caffeine in high concentrations has genotoxic and mutagenic properties, which may increase the risk of colon cancer [47]. In addition, caffeine has been shown to decrease insulin sensitivity, which may increase the risk of CRC [59, 60]. Conclusionsi Our findings suggest that there is no causal associa tion between genetically predicted alcohol, meat, milk, sweets, tea and fruit consumption and CRC. However, we did observe a positive causal association between veg etable and CRC. The identification of causal relationships between dietary habits and CRC is crucial for designing effective preventive strategies. Our study highlights the need for a more nuanced approach to dietary recommen dations for CRC prevention, with greater emphasis on adherence to the Mediterranean dietary pattern. i Based on the current relevant literature, the relation ship between different dietary habits and CRC is likelier to exhibit a non-linear relationship, which may only exist at a certain dose interval. The lack of consideration of the dose-response relationship in the MR analysis may be one of the reasons of our negative results. In the mean time, this conclusion needs to be further verified with MR analysis, in the future, with the existence of data on relevant dose SNPs. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Received: 19 April 2023 / Accepted: 16 December 2023 Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. 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W3096095075.txt	null	de		Inklusive Pädagogik und Intersektionalitätsforschung. Vergleich zweier Konzeptionen aus Sicht der feministischen Frauenforschung über Geschlecht und Behinderung	Gender	2,020	cc-by-sa	6,334	www.ssoar.info Inklusive Pädagogik und Intersektionalitätsforschung: Vergleich zweier Konzeptionen aus Sicht der feministischen Frauenforschung über Geschlecht und Behinderung Schildmann, Ulrike; Schramme, Sabrina Veröffentlichungsversion / Published Version Zeitschriftenartikel / journal article Zur Verfügung gestellt in Kooperation mit / provided in cooperation with: Verlag Barbara Budrich Empfohlene Zitierung / Suggested Citation: Schildmann, U., & Schramme, S. (2020). Inklusive Pädagogik und Intersektionalitätsforschung: Vergleich zweier Konzeptionen aus Sicht der feministischen Frauenforschung über Geschlecht und Behinderung. GENDER - Zeitschrift für Geschlecht, Kultur und Gesellschaft, 12(3), 11-26. https://doi.org/10.3224/gender.v12i3.02 Nutzungsbedingungen: Dieser Text wird unter einer CC BY-SA Lizenz (NamensnennungWeitergabe unter gleichen Bedingungen) zur Verfügung gestellt. Nähere Auskünfte zu den CC-Lizenzen finden Sie hier: https://creativecommons.org/licenses/by-sa/4.0/deed.de Terms of use: This document is made available under a CC BY-SA Licence (Attribution-ShareAlike). For more Information see: https://creativecommons.org/licenses/by-sa/4.0 Diese Version ist zitierbar unter / This version is citable under: https://nbn-resolving.org/urn:nbn:de:0168-ssoar-71329-0 Schwerpunkt Ulrike Schildmann, Sabrina Schramme Inklusive Pädagogik und Intersektionalitäts forschung. Vergleich zweier Konzeptionen aus Sicht der feministischen Frauenforschung über Geschlecht und Behinderung Zusammenfassung Summary Wenn unter Inklusiver Pädagogik eine ge meinsame Erziehung aller Kinder und Ju gendlichen zu verstehen ist, dann kann es nicht nur darum gehen, dass behinderte Kin der gemeinsam mit ihren nicht behinderten Peers erzogen werden. Es geht auch um ei nen reflektierten Umgang mit der Heteroge nität aller Beteiligten: Die Inklusive Pädago gik muss einerseits auf die individuellen Aus gangslagen und Bedürfnisse aller Lernenden eingehen, andererseits auf deren strukturel le Lebensbedingungen. Hier kommt die Inter sektionalitätsforschung ins Spiel, deren Anlie gen darin besteht, einzelne soziale Ungleich heitslagen – Geschlecht, Klasse/Schicht, Al ter, Ethnizität und Behinderung – und deren mögliche Wechselwirkungen zu analysieren, mit denen die Kinder und Jugendlichen kon frontiert sind. Aus Sicht der feministischen Forschung über Geschlecht und Behinderung – vor allem be zogen auf das Feld der Integrativen/Inklusi ven Pädagogik – wird in diesem Beitrag un tersucht, ob und wie Inklusive Pädagogik und Intersektionalitätsforschung, die in jeweils unterschiedlichen wissenschaftlichen Tradi tionen stehen, voneinander profitieren kön nen. Inclusive education and intersectionality re search. A comparison of two concepts from the perspective of feminist women’s studies on gender and disability Inclusive education not only reflects on the in dividual requirements of each child but also on different aspects of social inequality be tween groups of pupils/students based on class, gender, race and (dis)ability. The article examines whether the theoretical approach of intersectionality, which concentrates on in equality, dominance and discrimination, could be useful for broadening the perspec tives of inclusive education. It focuses on dif ferent backgrounds, trends and on the poten tials which might result if the two theoretical approaches were to cooperate. Keywords inclusive education, intersectionality, social inequality, gender, (dis)ability Schlüsselwörter Inklusive Pädagogik, Intersektionalitätsfor schung, Umgang mit Heterogenität, Ge schlecht, Behinderung https://doi.org/10.3224/gender.v12i3.02 2-Gender3-20_Schildmann.indd 11 GENDER Heft 3 \| 2020, S. 11–26 26.08.2020 15:41:41 12 Ulrike Schildmann, Sabrina Schramme 1 Ausgangspunkt: feministische Forschung über Geschlecht und Behinderung unter besonderer Berücksichtigung der beiden genannten Strukturkategorien sowie der Integrationspädagogik als Vorläuferin der Inklusiven Pädagogik 1.1 Anfänge der feministischen Forschung über Geschlecht und Behinderung Seit Beginn der 2010er-Jahre beschäftigt sich das genannte Forschungsfeld (vom Netzwerk Frauen- und Geschlechterforschung NRW in den 1990er-Jahren als „Frauenforschung in der Behindertenpädagogik“ institutionell verankert; Schildmann 2018) gezielt mit den Wechselwirkungen zwischen einzelnen, für die Inklusive Pädagogik relevanten gesellschaftlichen Strukturkategorien im Sinne der Intersektionalitätsforschung (Schildmann 2010, 2012, 2019a; Schildmann/Schramme 2017a, 2017b, 2018a, 2018b; Schramme 2019a, 2019b). Die Vorgeschichte dieses wissenschaftlichen Diskurses, der sich als einer unter mehreren möglichen wissenschaftlichen Perspektiven versteht (vgl. die vorliegenden Beiträge dieses Heftes), geht bis in die 1970er-Jahre zurück und soll hier in Kürze nachgezeichnet werden: „Das Interesse an der Erforschung von Verhältnissen zwischen Geschlecht und Behinderung entstand in einer Zeit der sozialen Bewegungen: Studentenbewegung, Kinderladenbewegung, Frauenbewegung und Behindertenbewegung […]. Vor allem an der um 1970 entstandenen Frauenbewegung und der Mitte der 70er Jahre begonnenen feministischen Frauenforschung orientiert, entstand ein Diskurs über Zusammenhänge zwischen (weiblichem) Geschlecht und Behinderung, an dem sich sowohl nicht be hinderte als auch behinderte Frauen beteiligten“ (Schildmann 2018: 17f.). Es waren im Wesentlichen drei Diskursstränge, die Ende der 1970er-Jahre die Forschung über Geschlecht und Behinderung begründeten: Analysen über die Lebensbedingungen behinderter Frauen in Anlehnung an feministisch orientierte Forschungsansätze über allgemeine weibliche Lebenszusammenhänge (Schildmann 1983); Kritik der Koedukation – Lehrerinnen und Schülerinnen an Sonderschulen (Rohr 1980; Prengel 1984); politische (Selbst-)Reflexionen behinderter Frauen im Zuge der „Krüppelbewegung“ (Behinderten- und Selbstbestimmt-Leben-Bewegung) und des Internationalen UNO-Jahres der Behinderten 1981 (Ewinkel et al. 1985), der zu Beginn der 2000erJahre in die Disability Studies einmündete (Waldschmidt 2015), die sich vor allem aus Betroffenenperspektive u. a. mit dem Feld des „Gendering Disability“ beschäftigen (Jacob/Köbsell/Wollrad 2010). 1.2 Integrationspädagogik, von Beginn an unter Berücksichtigung von Zusammenhängen zwischen Behinderung und Geschlecht Schon in den 1970er-Jahren entstanden erste Ansätze der Integrationspädagogik, die eine Nichtaussonderung behinderter Kinder aus regulären pädagogischen Einrichtungen und damit deren ‚Integration von Anfang an‘ forderten und sich zunächst im Rahmen GENDER 3 \| 2020 2-Gender3-20_Schildmann.indd 12 26.08.2020 15:41:41 Inklusive Pädagogik und Intersektionalitätsforschung 13 der frühkindlichen Erziehung innerhalb der Kinderladenbewegung und alternativen Pädagogik (vor allem in Anlehnung an Maria Montessori) etablieren konnten. Als sich dort die gemeinsame Erziehung behinderter mit nicht behinderten Kindern als erfolgversprechend erwiesen hatte, folgten entsprechende Modellversuche an Grund- und später weiterführenden Schulen. Sie setzten sich zum Ziel, die starre Abgrenzung zwischen Regel- und Sonderschulen zu überwinden. Von Anfang an getragen von Eltern, vor allem Müttern, behinderter und nicht behinderter Kinder sowie von Erzieherinnen in Kindertagesstätten und später auch von (vor allem weiblichen) Lehrerinnen an Grund- und weiterführenden Schulen (vgl. Schramme 2019a, 2019b), wurde ab Mitte der 1980erund vor allem in den 1990er-Jahren die Geschlechterdimension in der Integrationspädagogik als ein relevantes, wissenschaftlich zu bearbeitendes Feld erkannt (Prengel 1993; Hinz 1993; Schildmann 1996; Merz-Atalik 2001). Damit verfügten die Integrationsbewegung und die sozialwissenschaftliche Forschung über Verhältnisse zwischen Geschlecht und Behinderung von Anfang an über eine gemeinsame Perspektive: die Aufmerksamkeit für verschiedene soziale Ungleichheitslagen der integrativ geförderten und unterrichteten Kinder und Jugendlichen, die auch heute im Rahmen der Inklusiven Pädagogik thematisiert werden (Sturm 2016; Hackbarth 2017). 1.3 Inklusive Pädagogik als Weiterentwicklung der Integrationspädagogik Als im Laufe der 1990er-Jahre die – im Allgemeinen noch stark auf die Kategorie Behinderung konzentrierte – Debatte über eine Weiterentwicklung der Integrationspä dagogik hin zu einer umfassenden Inklusiven Pädagogik begann, waren also die Grundsteine für die Berücksichtigung unterschiedlicher sozialer Ungleichheitslagen – vor allem, aber nicht nur, zwischen Geschlecht und Behinderung – in dieser pädagogischen Reformströmung bereits gelegt. Im Zuge internationaler Abkommen der Vereinten Nationen (wie die „Education for All“-Agenda 1990, die Salamanca-Erklärung der UNESCO zu „Special Needs Education“ 1994 und die Behindertenrechtskonvention/UN-BRK 2006) erfolgte seit den 1990er-Jahren – wenn auch unter den beteiligten Integrationspädagoginnen nicht unumstritten – eine schrittweise Neuorientierung von der Integrativen hin zur Inklusiven Pädagogik. Dahinter stand folgende Überzeugung, die in konkrete strukturelle Maßnahmen einfließen sollte: „Special Needs Education [...] könne nicht in Isolation fortgeführt werden und müsse folglich unter dem Vorzeichen des neuen Leitprinzips der Inklusion in Richtung der im Jahr 1990 ins Leben gerufenen globalen bildungspolitischen Education for All-Agenda umorientiert werden, ‚to ensure that Education for All effectively means FOR ALL, particular those who are most vulnerable and most in need‘ (UNESCO 1994: S. iv; Hervorh. im Original)“ (Kiuppis 2014: 131). Diese Position der Vereinten Nationen richtete sich gegen die Weiterführung der – in einigen Ländern (wie z. B. Deutschland) traditionell gewachsenen – Spaltung des Bildungswesens in Regel- und Sondereinrichtungen mit deren je eigenen Binnendifferenzierungen und entsprach damit den Forderungen nach einer, z. B. von dem Integrationspädagogen Georg Feuser seit den 1980er-Jahren geforderten, gemeinsamen Pädagogik für alle Kinder: GENDER 3 \| 2020 2-Gender3-20_Schildmann.indd 13 26.08.2020 15:41:41 14 Ulrike Schildmann, Sabrina Schramme „Integration bedarf einer Pädagogik, in der – alle Kinder – in Kooperation miteinander – auf ihrem jeweiligen Entwicklungsniveau – an und mit einem gemeinsamen Gegenstand spielen und lernen“ (Feuser 1986: 129). Die im Zusammenhang mit der Integrationspädagogik entwickelten Theorieansätze über Verhältnisse zwischen Geschlecht und Behinderung wurden im Zuge der Eta blierung einer Inklusiven Pädagogik in unterschiedliche Richtungen erweitert: So fand z. B. die Ende der 1990er-Jahre begonnene Normalismusforschung über Behinderung und Geschlecht unter besonderer Berücksichtigung des Feldes der Integrationspädagogik (Schildmann 2004) statt, und der seit der zweiten Hälfte der 2000er-Jahre etablierte Theoriediskurs über die Strukturkategorien Geschlecht, Alter, Behinderung (Schildmann 2010; Schildmann/Schramme 2017a, 2017b) brachte auch die Theorieansätze von Inklusiver Pädagogik und Intersektionalitätsforschung (Schildmann 2012) in Verbindung miteinander, um klarzustellen, dass die Inklusive Pädagogik u. a. theoretischer Grundlagen bedarf, die auf strukturelle gesellschaftliche Problemlagen eingehen: „Wenn es in einer Inklusiven Pädagogik darum gehen soll, eine gemeinsame Bildung ALLER Kinder und Jugendlichen zu etablieren, dann kann dies [...] nur gelingen, wenn neben der Berücksichtigung indi vidueller Vielfalt aller Beteiligten auch strukturell bedingte Unterschiede ernst genommen werden und Berücksichtigung finden“ (Möller-Dreischer/Schildmann 2019: 3; Hervorh. im Original). Warum hier – statt anderer Theorieansätze der sozialen Ungleichheitsforschung – gerade die Intersektionalitätsforschung ins Spiel kommt, liegt vor allem daran, dass sich diese als Weiterentwicklung der feministischen Frauen- und Geschlechterforschung versteht, in deren Rahmen die Forschung über Verhältnisse zwischen Geschlecht und Behinderung inzwischen bereits etabliert war (Schildmann 2018: 29ff.). So lautet die Ausgangsposition für die weiteren Überlegungen: „Inklusive Pädagogik bedarf einer theoretischen Grundlage, die sich mit den sozialen Ungleichheits lagen der beteiligten Kinder und Jugendlichen beschäftigt, welche im konkreten pädagogischen Ge schehen aufeinandertreffen. Hier sind Klasse/Schicht, Geschlecht, Alter, ggf. Migrationshintergrund, Behinderung u. a. zu nennen. Während die Inklusive Pädagogik ihren Fokus auf die Förderung von gleichberechtigter Teilhabe und Partizipation aller Menschen an gesellschaftlich relevanten Institutionen und Ressourcen legt, konzentriert sich die – aus der Frauen- und Geschlechterforschung heraus entwi ckelte – Intersektionalitätsforschung auf die Analyse ‚historisch gewordene(r) Diskriminierungsformen, Machtverhältnisse, Subjektpositionen sowie soziale(r) Ungleichheiten […] in ihren Interdependenzen oder Überkreuzungen (intersections)‘ (Walgenbach 2016: 212)“ (Schildmann/Schramme 2018b: 301). GENDER 3 \| 2020 2-Gender3-20_Schildmann.indd 14 26.08.2020 15:41:41 Inklusive Pädagogik und Intersektionalitätsforschung 2 15 Unterschiede: Wie trifft die – weitgehend – soziologisch orientierte Intersektionalitätsforschung auf ein pädagogisches Anwendungsfeld, die Inklusive Pädagogik? 2.1 Dominante soziale Strukturkategorien in der Intersektionalitätsforschung: Welche Rolle spielt die für die Inklusive Pädagogik relevante Kategorie Behinderung? Aus Sicht der Verfasserinnen des vorliegenden Beitrags und ihrer wissenschaftlichen Verortung in der Inklusiven Pädagogik ist die Intersektionalitätsforschung als ein überwiegend soziologischer Theorieansatz anzusehen, der sich mit der Analyse sozialer Ungleichheitslagen und den Wechselwirkungen zwischen den zentralen sozialen Strukturkategorien der heutigen westlichen Gesellschaften befasst. Das sind vor allem die Kategorien Klasse und Geschlecht (Beer 1987; vgl. auch die seit der Studentenbewegung geführten Debatten über den sogenannte Haupt- und Nebenwiderspruch: Klasse und Geschlecht). Der Ende der 1980er-Jahre in den USA von der Juristin Kimberlé Crenshaw (1989) geprägte Begriff Intersektionalität enthält jedoch noch eine dritte zentrale Perspektive: Es handelt sich um die Strukturkategorie race. Sie wurde seit den 1970er-Jahren von schwarzen Frauen in kritischer Absicht an der weitgehend an Problemlagen weißer mittelschichtangehöriger Frauen orientierten feministischen Bewegung, die andere Gruppen von Frauen und deren soziale Konstellationen übersah oder als „add-on“ betrachtete, in den feministischen Diskurs eingebracht (Winker/Degele 2009: 11f.). Es sind also die Wechselwirkungen zwischen „gender – class – race“, die der allgemeinen (nordamerikanisch geprägten) Intersektionalitätsforschung zugrunde liegen und, zunächst in den USA, zu der angesprochenen Weiterentwicklung der feministischen Frauen- und Geschlechterforschung geführt haben. Diese Struktur wurde von deutschen Wissenschaftlerinnen, auf deren Ansätze sich der vorliegende Beitrag beschränkt, übernommen, auch wenn sie sich bewusst darüber sind, dass konzeptionelle Übertragungen US-amerikanischer Problemstellungen auf europäische resp. deutsche Verhältnisse nur unter Berücksichtigung hiesiger Sozialstrukturen und Diskurse (z. B. über Antisemitismus und Rassismus) möglich sind (Knapp 2013: 342f.). Die Relevanz der Einbeziehung weiterer sozialer Strukturkategorien in die Analysen der Intersektionalitätsforschung wird unterschiedlich beurteilt. Während einzelne Forscherinnen tendenziell von einer Erweiterung des Spektrums abraten (exemplarisch Hagemann-White 2011), listen andere diverse weitere Strukturkategorien auf, die für die Intersektionalitätsforschung infrage kommen könnten (exemplarisch Lutz/Wenning 2001). Welche Rolle in diesem Kanon die Strukturkategorie Behinderung spielt, ist bislang noch weitgehend offen. Nur Gabriele Winker und Nina Degele (2009) haben sich mit einem Ansatz vorgewagt, in dem sie – neben der Trias von Klasse, Geschlecht, Rasse/Ethnizität – an vierter Position eine Superkategorie „Körper“ konstruieren, unter der sie die Kategorien Behinderung und Alter zusammenfassen, ein Versuch, der jedoch einzelnen anderen Autorinnen aus verschiedenen Gründen als kritikwürdig erscheint (Schildmann 2012; Knapp 2013; Villa 2013; Schildmann/Schramme 2017a, 2017b, 2018a, 2018b). GENDER 3 \| 2020 2-Gender3-20_Schildmann.indd 15 26.08.2020 15:41:42 16 Ulrike Schildmann, Sabrina Schramme Festgehalten werden kann an dieser Stelle, dass die Kategorie Behinderung in der allgemeinen Intersektionalitätsforschung bislang eher unterrepräsentiert ist. 2.2 Verortung der Kategorie Behinderung in der Intersektionalitäts forschung: ein exemplarischer soziologischer Ansatz Für eine wissenschaftliche Kooperation zwischen Inklusiver Pädagogik und Intersektionalitätsforschung ist jedoch die Auseinandersetzung mit den Wechselwirkungen zwischen den verschiedenen Strukturkategorien, darunter Behinderung, ausschlaggebend. Entlang des feministischen Theorieansatzes der Soziologin Regina Becker-Schmidt (2017) soll deshalb an dieser Stelle in Kürze skizziert werden, wie sich die Kategorie Behinderung in deren soziologische Gesellschaftsanalyse einfügen ließe. Ausgehend davon, dass „die verschiedenen Sektoren einer Gesellschaft – Politik, Ökonomie, Kulturindustrie, private Lebenswelten – strukturell aufeinander bezogen“ (Becker-Schmidt 2017: 309) sind, auch wenn sie weitgehend als gegeneinander abgeschottet erscheinen, identifiziert Becker-Schmidt drei Achsen sozialer Ungleichheit: „Die Scheidung von familialer und marktvermittelter Reproduktion, von Haushaltung und Geldwirt schaft geht mit einer Hierarchisierung der Geschlechter einher. Klassenbildung entsteht durch die an tagonistische Entgegensetzung von Kapital und Lohnarbeit. Die Diskriminierung von ‚Fremden‘ be ginnt, sobald sich eine Population von einer anderen Bevölkerungsgruppe absondert, weil sie sich ihr qua sozialem Status, Hautfarbe, Kultur oder Religion überlegen dünkt“ (Becker-Schmidt 2017: 310). Die erwähnte Diskriminierung von Fremden, die Becker-Schmidt im Sinne der Strukturkategorie Ethnizität versteht, kann nach Auffassung der Verfasserinnen des vorliegenden Beitrags – im Sinne von Fremdheit in der eigenen Gesellschaft – folgendermaßen auf die Kategorie Behinderung und auf die von Behinderung betroffenen Menschen bezogen werden: „Die Mehrheitsgesellschaft legt fest, was sie unter mindestens durchschnittlicher Leistungsfähigkeit versteht, während eine soziale, als behindert definierte Minderheit diesen Leistungserwartungen real oder fiktiv nicht nachkommen kann, weshalb sie auf verschiedenste Weise sozial kontrolliert, sank tioniert, besondert wird. Die diversen Formen der Ausgrenzung – nicht zuletzt strukturell angelegter institutioneller Art – machen die von Behinderung betroffenen Menschen zu Fremden in der eigenen Gesellschaft“ (Schildmann/Schramme 2018a: 51f.). Diese Konstruktion wirkt auch in die Inklusive Pädagogik hinein, in der Mädchen und Jungen verschiedener sozialer und (inter- sowie sub-)kultureller Herkunft mit unterschiedlichsten individuellen Fähigkeiten/Leistungsvoraussetzungen und Verhaltensweisen zusammentreffen. Soll nun die Intersektionalitätsforschung für die Inklusive Päda gogik nutzbar gemacht werden, dann sind diese Konstellationen auf allen relevanten Ebenen des pädagogischen Miteinanders analytisch zu durchdringen. Dass dabei die Kategorie Behinderung – im schulpolitischen Sinne als ‚sonderpädagogischer Förderbedarf‘ aufgefasst – einer ggf. besonderen Berücksichtigung bedarf, liegt vor allem daran, dass in dem hierarchisch organisierten deutschen Bildungssystem für die mit diesem Label belegten Mädchen und Jungen eigene Sondereinrichtungen – darunter vor allem nach verschiedenen Förderschwerpunkten differenzierte Schulen – ein traditionell GENDER 3 \| 2020 2-Gender3-20_Schildmann.indd 16 26.08.2020 15:41:42 Inklusive Pädagogik und Intersektionalitätsforschung 17 gewachsenes Gebilde darstellen, das ihre in sich sehr differenzierte Schülerinnenschaft bisher nur teil- bzw. schrittweise in die Regelschulen überführt: So wurden seit Inkrafttreten der UN-BRK 2009 in Deutschland Direkteinschulungen in die verschiedenen Förderschulen nur um 0,6 Prozentpunkte auf zuletzt 3 Prozent reduziert, d. h., immer noch über 21 000 Kinder – darunter fast doppelt so viele Jungen wie Mädchen – beginnen ihre Schullaufbahn nicht in einer Regelschule (Autorengruppe Bildungsbericht erstattung 2018: 83, 2016: 68). Über die gesamte Schulzeit hinweg betrachtet ist sogar folgende Entwicklung zu vermerken: „Mit einem Anstieg auf 7 % aller Schülerinnen und Schüler gab es 2016 erneut mehr Kinder und Ju gendliche mit sonderpädagogischer Förderung als in den Vorjahren. Dabei findet sonderpädagogische Förderung weiterhin überwiegend in Förderschulen statt. Bundesweit liegt der Inklusionsanteil bei nun mehr 39 %, lediglich in 4 Ländern wird die Mehrheit der Schülerinnen und Schüler mit Förderbedarf in allgemeinen Schulen unterrichtet. Das Angebot eigenständiger Förderschulstandorte hat sich in vielen Ländern und Kreisen kaum verändert“ (Autorengruppe Bildungsberichterstattung 2018: 7). Diese statistischen Daten weisen vor allem auf strukturelle Diskriminierungen im Bildungswesen hin, welche mit den Ansätzen und Methoden der Intersektionalitätsforschung analysiert werden können, um schließlich im Rahmen einer sich als inklusiv verstehenden allgemeinen Pädagogik abgebaut zu werden. 3 Verbindungslinien: Ansätze des wissenschaftlichen Diskurses zwischen Inklusiver Pädagogik und Intersektionalitätsforschung 3.1 Berücksichtigung aller für Erziehung und Bildung relevanten Strukturkategorien, darunter Behinderung Bisher vorliegende Ansätze des wissenschaftlichen Diskurses über Inklusive Pädagogik und Intersektionalitätsforschung gehen nach Auffassung der Autorinnen des vorliegenden Beitrags im Wesentlichen von der – in der Tradition der Integrationspädagogik (s. o.) mit deren besonderer Aufmerksamkeit für die gemeinsame Erziehung behinderter und nicht behinderter Kinder bestehenden – Inklusiven Pädagogik aus. Soll es jedoch um eine gemeinsame Erziehung und Bildung aller Kinder und Jugendlichen gehen, stellen sich auch alle Indikatoren für soziale Ungleichheitslagen als gleichermaßen wichtig und zu beachten heraus, wodurch auch die wissenschaftlichen Ansätze der Intersektionalitätsforschung zum Tragen kommen. 3.2 Aufmerksamkeit für die Binnenstrukturen einzelner sozialer Kategorien, exemplarisch dargestellt an Zusammenhängen von Geschlecht und Behinderung Um die Wechselwirkungen zwischen einzelnen Kategorien untersuchen zu können, müssen zunächst deren, ggf. sehr unterschiedliche, Binnenstrukturen analysiert und ge- GENDER 3 \| 2020 2-Gender3-20_Schildmann.indd 17 26.08.2020 15:41:42 18 Ulrike Schildmann, Sabrina Schramme genseitig vermittelt werden. Dies sei hier exemplarisch für die Kategorien Geschlecht und Behinderung ausgeführt: „Geschlecht ist wie andere soziale Strukturen vielschichtig; es geht nicht nur um Identität oder nur um Arbeit oder nur um Macht oder nur um Sexualität, sondern um all das gleichzeitig [...] Geschlechter arrangements werden sozial reproduziert (nicht biologisch), und zwar durch die Macht der Strukturen, die soziales Handeln prägen“ (Connell 2013: 30). Diese Definition von Geschlecht ist aus mehreren Gründen auch grundlegend für die Definition von Behinderung. Zu berücksichtigen ist dabei, dass Behinderung ein Sammelbegriff für unterschiedlichste Beeinträchtigungen ist. Durch gesellschaftliche Einstellungen und Vorurteile gegenüber körperlichen, geistigen und psychischen Beeinträchtigungen geraten die betroffenen Menschen in sozial benachteiligte Positionen gegenüber ihren nicht behinderten Mitmenschen ebenso wie in hierarchische Verhältnisse untereinander. Diejenigen, die den gesellschaftlichen Normalitätsvorstellungen am wenigsten entsprechen, sind sozialer Diskriminierung am stärksten ausgesetzt. Auch bei Behinderung geht es also um Macht (in Form institutionellen Ausschlusses bzw. institutioneller Besonderung), Arbeit (in Form der Definition minderer Arbeitsgüte), Sexualität (in Form direkter oder indirekter eugenischer Zwänge) und Identität (in Form der Übernahme gesellschaftlicher Normalitätsvorstellungen, die individuelle Abweichung hervorbringen). Behinderung resultiert, wie hier deutlich wird, u. a. aus dem, was eine jeweilige Gesellschaft unter Geschlecht versteht und welche Geschlechterarrangements sie fordert oder zulässt, womit die Orientierung an bestimmten Normalitätsvorgaben einhergeht. Die Kategorie Behinderung drückt dabei die negative Seite dessen aus, was als gesellschaftlich erwünscht und mindestens durchschnittlich angesehen wird (zur Normalismusforschung vgl. zusammenfassend Schildmann 2009). Von dieser Perspektive können die Inklusive Pädagogik und die Intersektionalitätsforschung gleichermaßen profitieren: Indem exemplarisch auch die (sich verändernden) Verhältnisse zwischen Normalität und Abweichung analysiert werden, die einen Einblick in die Strukturen des ‚dis-ableism‘ gewähren, können die Einblicke in die gesellschaftlich vorherrschenden Strukturen von ‚sexism‘ und ‚racism‘ analytisch ergänzt werden. 4 Potenziale der Inklusiven Pädagogik unter intersektionaler Perspektive Wenn, wie oben dargestellt, Behinderung u. a. aus dem hervorgeht, was die Gesellschaft unter Geschlecht versteht, dann sollten die Geschlechterarrangements noch ausführlicher betrachtet werden. Dabei spielt, wie dem o. g. Zitat von Raewyn Connell zu entnehmen ist, neben Aspekten von Macht, Arbeit und Identität auch das gesellschaftliche Feld der Sexualität eine dominante Rolle. Auf dieses geht innerhalb der feministischen Forschung ganz gezielt die Queer-Theorie ein. Mit dieser Perspektive beschäftigt sich, die Kategorie Behinderung berücksichtigend, unter theoretischen Aspekten Abschnitt 1. Abschnitt 2 verdeutlicht die Zusammenschau von Integrativer/Inklusiver Pädagogik, Intersektionalitätsforschung und Queer-Theorie anhand eines ausgewählten empirischen Beispiels. Abschnitt 3 enthält eine zusammenfassende Einschätzung darüber, welche GENDER 3 \| 2020 2-Gender3-20_Schildmann.indd 18 26.08.2020 15:41:42 Inklusive Pädagogik und Intersektionalitätsforschung 19 Potenziale die Verknüpfung der wissenschaftlichen Ansätze von Inklusiver Pädagogik und Intersektionalitätsforschung einschließlich der Queer-Theorie beinhaltet. 4.1 Theoriebezug: Queer-Theorie als Perspektiverweiterung des Zusammenhangs zwischen Intersektionalität und Inklusion Auch die Queer-Theorie ist anschlussfähig an die Diskurse über den Umgang mit Verschiedenheit in inklusiven Settings. Die drei folgenden Aspekte können als relevante Perspektivenerweiterungen angesehen werden. 4.1.1 Betonung der Untrennbarkeit von Geschlecht und sexueller Identität Gabriele Dietze, Elahe Haschemi Yekani und Beatrice Michaelis konstatieren eine weitgehende „Abwesenheit von Sexualitäten in der Theoretisierung von Intersektionalität in den Gender Studies“, wodurch „eigene heteronormative Positionen des MainstreamFeminismus“ (Dietze/Haschemi Yekani/Michaelis 2012: 1) nicht hinreichend reflektiert würden. Von queer-theoretischen Ansätzen könnte die Intersektionalitätsforschung profitieren, so die Autorinnen, weil diese „Identitätskategorien wie ‚schwul‘ und ‚lesbisch‘ (und damit auch Heterosexualität als Identität) in ihrer (vermeintlichen) Kohärenz infrage [...] stellen und Normalisierungsvorgänge sowie spezifische Herrschaftsformationen wie Heteronormativität [...] analysieren“ (Dietze/Haschemi Yekani/Michaelis 2012: 3). Die Forderung nach mehr Aufmerksamkeit für die verschiedenen Heterogenitätsdimensionen in der Inklusiven Pädagogik lässt dabei eine angemessene Berücksichtigung von geschlechtlicher Identität und sexuellem Begehren einschließlich der Kritik an Heteronormativität in mehrerer Hinsicht als sinnvoll erscheinen: So zeigt eine Untersuchung zu „Gleichstellung und Akzeptanz sexueller und geschlechtlicher Vielfalt“ (Lenz/Sabisch/Wrzesinski 2012) im Bundesland Nordrhein-Westfalen, dass z. B. Kinder aus „Regenbogenfamilien“ – d. h. „Familien, in denen mindestens ein Elternteil (sozial, rechtlich, biologisch) lesbisch oder schwul lebt“ (Lenz/Sabisch/Wrzesinski 2012: 25) – vor allem im schulischen Umfeld „ihre Familiensituation verheimlichen“ (Lenz/Sabisch/Wrzesinski 2012: 26f.), weil sie von Gleichaltrigen ausgehende Diskriminierung befürchten oder bereits erlebt haben (Lenz/Sabisch/Wrzesinski 2012: 28). Auch lesbische, schwule, bisexuelle trans1 und inter2 Jugendliche berichten davon, durch ihr Outing Freundschaften eingebüßt sowie (homo- und transnegative) Abwertungen erlebt zu haben (Lenz/Sabisch/Wrzesinski 2012: 29). Ähnlich wie die Lebenslagen von Kindern und Jugendlichen mit trans- und inter-Erfahrungen (Lenz/Sabisch/ Wrzesinski 2012: 30) sei auch die Situation von lesbisch, schwul, bisexuell, transsexuell, transgender und intersexuell (LSBTTI) lebenden Personen mit Behinderungserfahrungen bisher nur unzureichend erforscht worden, so Lenz, Sabisch und Wrzesinski (2012: 39). Jedoch lassen die Ergebnisse dieser Studie den Schluss zu, dass eine weitergehende intersektionale Analyse der hier angesprochenen Heterogenitätsdimensionen (Wech1 2 Der Begriff trans* wird hier als Begriff für Selbstbezeichnungen wie Transgender, Transsexuell, Transident, Transgeschlechtlich genutzt. Der Begriff inter* wird hier als Überbegriff für Intersexuelle, Intersex, Hermaphroditen, Zwitter, Intergender sowie inter- oder zwischengeschlechtlich verwendet. GENDER 3 \| 2020 2-Gender3-20_Schildmann.indd 19 26.08.2020 15:41:42 20 Ulrike Schildmann, Sabrina Schramme selwirkungen zwischen Behinderung, Geschlecht, Sexualität/Begehren, Klasse etc.) für die inklusive pädagogische Praxis fruchtbar wäre, um die konkreten Lebenslagen dieser Personen(gruppen) sichtbar zu machen und mit sexueller Identität und Heteronormativität verwobene Themen machtkritisch reflektieren zu können. Die Autorinnen Heike Raab (2010) und Elisabeth Tuider (2014), die sich mit Körperkonzepten jenseits heteronormativer Ordnungen befassen, beschreiben darüber hinaus eine „wechselseitige Verschränkung von Heteronormativität, Sexualität und abled bodyness“ (Tuider 2014: 108f.), die auf der Basis eines intersektionalen Forschungszugangs deutlich werde. Unter Beachtung intersektionaler Analysen kann die Kategorie Behinderung demnach auch bezogen auf queere Körperkonzepte und Strukturen des ‚ableism‘ (vgl. auch Punkt 3.2) wichtige Ergänzungen in queer-theoretische Zusammenhänge einbringen, da hier Verhältnisse zwischen Normalität und Abweichung in Bezug auf Fähigkeiten bzw. „abled bodies“ (Tuider 2014) thematisiert werden können. 4.1.2 Destabilisierung und Dekonstruktion von Kategorien Während die Intersektionalitätsforschung unterschiedliche (inter-, intra- und antikategoriale) Zugangsweisen zu ihren Forschungsgegenständen wählt, ist die Queer-Theorie eindeutig antikategorial ausgelegt. Sie fokussiert vor allem Fragen von Identität: „Fragen der Aneignung und Selbstbestimmung“ (Förster 2017: 13) und damit auch der „Bedingungen und Prozesse der Subjektwerdung“ (Förster 2017: 14), was beinhaltet, dass sich die einzelne Person selbstbemächtigend definieren bzw. benennen kann. Im Rahmen inklusiver pädagogischer Prozesse dient diese Perspektive auf Subjektwerdungsprozesse der kritischen Sensibilisierung für den Akt der Kategorisierung, die dem Anspruch auf positive Wertschätzung menschlicher Heterogenität ggf. entgegensteht. 4.1.3 Kritische Thematisierung von Privilegierung zum Zweck des Abbaus sozialer Hierarchien Auch wenn sowohl die Intersektionalitätsforschung als auch die Queer-Theorie aus der Kritik am weißen Mainstream-Feminismus bzw. an der weißen Schwulen- und Lesbenbewegung entstanden sind, weist doch vor allem Letztere auf Verhältnisse zwischen Diskriminierung und Privilegierung hin. Im Rahmen der Inklusiven Pädagogik erhält die Reflexion über soziale Privilegien gegenüber Benachteiligungen und Ausschlüssen eine besondere Relevanz: Geht es in diesem pädagogischen Reformansatz vor allem um einen positiven Umgang mit Heterogenität und die Wertschätzung der unterschiedlichen Potenziale aller Beteiligten, dann sollten nicht nur soziale Benachteiligungen der Einen kritisch reflektiert werden, sondern auch und vor allem die Privilegien der Anderen, die sich häufig hinter dem verbergen, was als gesellschaftliche ‚Normalität‘ gilt. Die Sichtbarmachung privilegierender bzw. diskriminierender Facetten kann vor allem dazu dienen, für Macht- und Herrschaftsverhältnisse im Bildungswesen zu sensibilisieren und an deren Überwindung zu arbeiten. Zum Abbau sozialer Ungleichheitslagen gehört also, so lehrt vor allem die Queer-Theorie, nicht nur und nicht zuvorderst, die Analyse sozialer Benachteiligungen, sondern im Gegenteil ebenso die Analyse sozialer Privilegierung und deren kritische Überprüfung, auch durch die privilegierten Personen selbst, GENDER 3 \| 2020 2-Gender3-20_Schildmann.indd 20 26.08.2020 15:41:42 Inklusive Pädagogik und Intersektionalitätsforschung 21 die ggf. Verzicht lernen müssen (Krasniqi/Kraut 2017: 116). Diese Aspekte sind nicht zuletzt für in inklusiven Zusammenhängen tätige Pädagoginnen im Sinne der Selbstreflexion eigener Haltungen und Positionen (sowie der damit einhergehenden Wahrnehmungsperspektive in pädagogischen Prozessen) bedeutsam. 4.2 Praxisbezug: Subjektperspektiven auf Heterogenitätsdimensionen in der Inklusiven Pädagogik Wenn die Inklusive Pädagogik, wie unter Punkt 1.3 dargestellt, u. a. einer theoretischen Grundlage bedarf, die sich mit den sozialen Ungleichheitslagen der Kinder und Jugendlichen beschäftigt, welche im konkreten pädagogischen Geschehen aufeinandertreffen, dann scheinen sich als solche Grundlagen sowohl die Intersektionalitätsforschung im Allgemeinen als auch die Queer-Theorie im Besonderen zu eignen. Das belegt, neben einigen anderen Studien (Buchner 2018; Boger 2019), auch eine Arbeit, in deren Rahmen erstmals „[b]iografische Erfahrungen mit Integration (Inklusion) in Kindergarten und Schule aus der Rückschau behinderter Frauen und Männer“ (Schramme 2019a; 36 ehemalige „Integrationskinder“ der ersten Generation, Geburtsjahrgänge 1965–1988) systematisch erhoben wurden. Sie geht, wie in anderer Weise auch die Arbeiten von Buchner (2018) und Boger (2019), gezielt auf die Subjektperspektiven der Befragten unter Einbeziehung von Verhältnissen zwischen Diskriminierung und Privilegierung ein und berücksichtigt dabei verschiedene Heterogenitätsdimensionen (insbesondere Geschlecht, soziale Klasse/Schicht, Alter, Behinderungserfahrung) wie auch zentrale gesellschaftliche Normalitätsfelder, vor allem Leistung, aber auch Sexualität. 4.2.1 Relevanz unterschiedlicher Heterogenitätsdimensionen Im Rahmen dieser Studie mit 20 weiblichen und 16 männlichen Interviewpartnerinnen spielt die Kategorie Behinderung insofern eine herausgehobene Rolle, als alle Befragten – wenn auch in unterschiedlicher Weise – von individuellen Beeinträchtigungen und Behinderungen betroffen sind. Die Kategorie Alter spielt eine wichtige Rolle, weil sich die gesellschaftlichen Definitionen von Behinderung weitgehend daran orientieren, welche Leistungsanforderungen und Verhaltensweisen in den aufeinander folgenden Lebensabschnitten, insbesondere im Kindes- und Jugendalter, für eine jeweilige Altersgruppe als durchschnittlich und damit als normal angenommen werden. Die Kategorie Geschlecht weist in der vorliegenden Untersuchung zwei unterschiedliche Aspekte aus: Erstens erfahren Mädchen und Jungen geschlechterspezifische Normalitätserwartungen, was sich auf einzelne Lebensbereiche unterschiedlich auswirkt (z. B. Leistung und Sexualität, s. u.). Zweitens waren – zumindest in den ersten Jahrzehnten – an der Durchsetzung und Etablierung der Integrationspädagogik mit Müttern, Erzieherinnen und Lehrerinnen (auch aus Sicht der Befragten) mehr Frauen als Männer beteiligt. Dass in diesen ersten Jahrzehnten in die Integrationsversuche und -modelle vor allem Kinder aus gebildeten Schichten (vor allem aus Akademikerfamilien) einbezogen wurden, weist auf die Relevanz der sozialen Klassen-/Schichtzugehörigkeit für die individuelle Berücksichtigung auf diesem reformpädagogischen Feld hin. GENDER 3 \| 2020 2-Gender3-20_Schildmann.indd 21 26.08.2020 15:41:42 22 Ulrike Schildmann, Sabrina Schramme 4.2.2 Relevanz von Diskriminierung und Privilegierung auf dem Feld der (Schul-)Leistungen Welch große Rolle der eigenen Leistungsfähigkeit zugemessen wird, zeigen die Äußerungen der behinderungserfahrenen Interviewpartnerinnen auf dem Gebiet der erwarteten bzw. erbrachten (Schul-)Leistungen, die auf einen entsprechenden Normalitätsdruck hinweisen: „Wenn ich nicht behindert gewesen wäre, hätte es die Diskussion gar nicht gegeben ..., aber der Leiter des Realschulzweigs sagte zu meinen Eltern ich würde ja sowieso nie Karriere machen und sie hätten zu wenig Schüler auf dem Realschulzweig, er sei dafür, dass ich auf die Realschule gehe, weil dann würde ich niemandem auf dem Gymnasium den Platz wegnehmen“ (B19w, 4; Schramme 2019a: 149). Die Studie belegt, dass insbesondere die befragten Frauen unter starkem Normalitätsund Leistungsdruck standen. Sie sprechen auch „von einem extrem kräftezehrenden Alltag, der oft überfordernd war, den sie aber mit Rekurs auf den Durchschnitt der Klasse (unter großem Kraftaufwand) bewältigen wollten bzw. bewältigten“ (Schramme 2019a: 208). 4.2.3 Relevanz von Diskriminierung und Privilegierung auf dem Feld der Sexualität Dem mit dem Alter zunehmenden und vor allem in der Adoleszenz sichtbar werdenden Ausschluss aus Gesprächen und Aktivitäten nicht behinderter Mitschülerinnen wurde von den Befragten der genannten Studie, ähnlich wie bei Buchner (2018) und Boger (2019), vor allem dadurch begegnet, sich in Aussehen und Verhalten „so normal wie möglich“ zu geben (vgl. Punkt 3.2). Dennoch fühlten sich die Befragten von ihren nicht behinderten Peers vor allem als (potenzielle) Sexualpartnerinnen nicht wahrgenommen, wie folgendes Beispiel belegt: „Ich war immer der schwule beste Freund, mit dem man über alles reden kann. Aber ich war halt nie der […], der in Frage kam. Und wenn ich dann einer Frau meine Liebe gestand, dann ist die immer aus allen Wolken gefallen. Und das ist total frustrierend und ich glaube, dass ich auch ein unglaublich ro mantischer/ oder gute Flirttalente habe aber selbst das reicht nicht“ (B22m, 69; Schramme 2019a: 165). Diese und ähnliche Aussagen könnten dazu dienen, allgemeine Tendenzen von Privilegierung und Diskriminierung auf dem Feld der Sexualität auszuleuchten, spezielle Vorurteile gegenüber der Sexualität behinderter Mädchen und Jungen (bzw. Frauen und Männer), die sich um Begriffe wie Asexualität, sexuelle Neutralität u. ä. ranken, auszuräumen und die Ergebnisse im Sinne einer positiven Wertschätzung von sexueller Vielfalt auf der Basis der Ansätze der Queer-Theorie für die Inklusive Pädagogik nutzbar zu machen. Insgesamt belegt die Studie die Bedeutung der Subjektperspektive für die Umsetzung von Inklusion in die pädagogische Praxis. Alle relevanten gesellschaftlichen Heterogenitätsdimensionen einschließlich Sexualität/Begehren werden angesprochen. Mit den Potenzialen der Intersektionalitätsforschung und der Queer-Theorie kann es aus Sicht der Verfasserinnen des vorliegenden Beitrags gelingen, die deutlich sichtbar werdenden Verhältnisse zwischen Privilegierung und Diskriminierung im Bildungswesen machttheoretisch zu durchleuchten und für die Überwindung sozialer Ungleichheiten einzutreten. GENDER 3 \| 2020 2-Gender3-20_Schildmann.indd 22 26.08.2020 15:41:42 Inklusive Pädagogik und Intersektionalitätsforschung 23 4.3 Schlussbetrachtung: Potenziale einer Verknüpfung von Inklusiver Pädagogik und Intersektionalitätsforschung (einschließlich QueerTheorie) Die im vorliegenden Beitrag angestellten Überlegungen zu möglichen Verknüpfungspunkten zwischen Inklusiver Pädagogik und Intersektionalitätsforschung (einschließlich der Queer-Theorie) schließen mit der Einschätzung, dass sich die genannten wissenschaftlichen Ansätze gegenseitig bereichern und voneinander profitieren können. Wenn es der Inklusiven Pädagogik darum gehen soll, dass eine gemeinsame Erziehung und Bildung aller Kinder und Jugendlichen nicht nur deren unterschiedliche individuelle Fähigkeiten und Besonderheiten berücksichtigt, sondern auch auf ihre sozialen Ungleichheitslagen eingeht, dann können dafür die wissenschaftlichen Ansätze der Intersektionalitätsforschung (einschließlich der Queer-Theorie) von analytischem Nutzen sein; denn diese geht gezielt auf die einzelnen Indikatoren sozialer Ungleichheit und auf deren mögliche Wechselwirkungen ein und kann so die unterschiedlichsten sozialen Gefüge im Bildungswesen (von vorschulischen Einrichtungen bis hin zu den unterschiedlichen Schultypen) systematisch erfassen. Umgekehrt kann die Intersektionalitätsforschung einen Gewinn daraus ziehen, dass sie mit der Inklusiven Pädagogik ein pädagogisches Praxisforschungsfeld erobert, auf dem einerseits alle gesellschaftlich relevanten Strukturkategorien (Klasse, Geschlecht, Alter, Ethnizität, Behinderung) in Wechselwirkung miteinander treten und andererseits für sie noch weitgehend unbearbeitete Strukturkategorien – wie etwa Behinderung und deren Binnendifferenzierungen – ins Blickfeld geraten. Dabei können auf diesem Feld exemplarisch auch die (sich verändernden) Verhältnisse zwischen Normalität und Behinderung analysiert werden, die einen Einblick in die Strukturen des ‚ableism‘ gewähren, durch die die gesellschaftlich vorherrschenden Strukturen von ‚sexism‘ und ‚racism‘ analytisch zu ergänzen sind. Literaturverzeichnis Autorengruppe Bildungsberichterstattung (2016). Bildung in Deutschland 2016. Bielefeld: Bertelsmann. Autorengruppe Bildungsberichterstattung (2018). Bildung in Deutschland 2018. 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Wiesbaden: VS Verlag für Sozialwissenschaften. https://doi.org/10.1007/978-3-53192555-4_11 Waldschmidt, Anne (2015). Disability Studies als interdisziplinäres Forschungsfeld. In Theresia Degener & Elke Diehl (Hrsg.), Handbuch Behindertenrechtskonvention (S. 334–344). Bonn: Bundeszentrale für politische Bildung. Winker, Gabriele & Degele, Nina (2009). Intersektionalität. Zur Analyse sozialer Ungleichheiten. Bielefeld: transcript. https://doi.org/10.14361/9783839411490 Zu den Personen Ulrike Schildmann, Prof. Dr., 1950, Univ.-Prof. i. R. der TU Dortmund, Fakultät Rehabilitationswissenschaften. Arbeitsschwerpunkte: Verhältnisse zwischen Behinderung und Geschlecht, Normalismusforschung, inklusive Pädagogik, Intersektionalitätsforschung. E-Mail: ulrike.schildmann@tu-dortmund.de Sabrina Schramme, Dr. phil., *1984, Evangelische Hochschule Rheinland-Westfalen-Lippe, Fachbereich Heilpädagogik und Pflege. 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https://openalex.org/W4394812474	https://bmcgeriatr.biomedcentral.com/counter/pdf/10.1186/s12877-024-04924-3	English	null	Exploring the knowledge and skills for effective family caregiving in elderly home care: a qualitative study	BMC geriatrics	2,024	cc-by	5,887	© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Gebrezabher Niguse Hailu1, Muntaha Abdelkader1, Feven Asfaw1 and Hailemariam Atsbeha Meles2 Gebrezabher Niguse Hailu1, Muntaha Abdelkader1, Feven Asfaw1 and Hailemariam Atsbeha Meles2 Gebrezabher Niguse Hailu1, Muntaha Abdelkader1, Feven Asfaw1 and Hailemariam Atsbeha Me BMC Geriatrics BMC Geriatrics Hailu et al. BMC Geriatrics (2024) 24:342 https://doi.org/10.1186/s12877-024-04924-3 Open Access Abstract Efforts should also be made to raise awareness about Correspondence: Gebrezabher Niguse Hailu gebrezabher.niguse@mu.edu.et *Correspondence: Gebrezabher Niguse Hailu gebrezabher.niguse@mu.edu.et Correspondence: Gebrezabher Niguse Hailu gebrezabher.niguse@mu.edu.et Full list of author information is available at the end of the article Full list of author information is available at the end of the article Full list of author information is available at the end of the article Abstract Background Family caregivers play a crucial role in providing physical, emotional, and social support to the elderly, allowing them to maintain their independence and stay in their preferred living environment. However, family caregivers face numerous challenges and require specific knowledge and skills to provide effective care. Therefore, understanding the knowledge and skills required for effective family caregiving in elderly home care is vital to support both the caregivers and the elderly recipients. Methods The research was carried out in Mekelle City, Ethiopia, utilizing the phenomenology study design and purposive sampling technique. A total of twenty-two in-depth interviews were conducted. Individuals with experience in providing care for elderly people in their homes were targeted. Data was gathered through the use of an open-ended guide, transcribed word-for-word, inputted into ATLAS.ti8 software, and translated. Codes and themes were then extracted from the transcribed data, and a thematic analysis was performed. To minimize personal biases, the collected data were coded independently by the data collection assistants and the PI. The analysis was carried out by authors who were not involved in the data collection process. The interviews were conducted in a quiet place. Results A total of 22 in-depth interviews were conducted as part of this research. The results indicated that although the participants had knowledge about common health problems experienced by older people, they were uninformed about how to manage these conditions at home and were unaware of specialized healthcare resources for the elderly. Furthermore, they had limited knowledge about suitable exercise routines, strategies to prevent falls, and home healthcare practices for older individuals. On the other hand, they exhibited a solid comprehension and awareness of abusive behaviors specifically directed at older adults. Conclusion The results emphasized the importance of enhancing education and training for family caregivers in handling elderly health issues, raising awareness about specialized healthcare services catered to the elderly, improving understanding of activities of daily living (ADLs) and fall prevention, and offering inclusive training in healthcare tasks related to elder care. Recommendation Participants should receive comprehensive education and training programs to enhance their knowledge and skills in managing these conditions. Study area and periodh The research took place in Mekelle, which is a city located in the Tigray region of northern Ethiopia. The study was conducted between December 2022 and October 2023. Mekelle is both the capital and largest city in the region, with a population of about 500,000 residents. The city is situated at an altitude of 2,084 m (6,837 feet) above sea level and is approximately 783 km (487 miles) away from the Ethiopian capital of Addis Ababa. The older adult population suffers from poverty and social isolation especially in developing country like Ethi opia since many of them are retired and have scant or no retirement plans or benefits. For example, a study on the elderly conducted in Cameron revealed that older adults are the most vulnerable and disadvantaged people in society and are reliant on family members for their fun damental requirements [7]. Source and study populations Source population All individuals who are over the age of 18 and living in Mekelle city and responsible for taking care of their elderly relatives in their homes. Study population All selected family care givers who were responsible for providing care for their elderly loved ones within their own homes. In sub-Saharan Africa, it is typical for the younger spouse, (often the wife), children, or grandkids to be responsible for caring for their elderly family members [12].These family carers are required to help their elderly family members with everyday tasks and guard against elder abuse and falls [13–15].More broadly, they aid older Study design Phenomenological Study design was used to explore the personal experiences, thoughts, feelings, and perceptions of family caregivers in the context of providing care for elderly individuals at home. Elderly family members who are unable to take care of themselves are supported by family caregivers who offer them physical, emotional, psychological, and occasionally financial support [8]. The majorities of them, however, perform this duty with little to no understanding of care, assistance, or support [9] Family caregivers who lack suf ficient understanding may unintentionally injure their loved ones and sometimes even themselves [10, 11]. © The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. (2024) 24:342 Page 2 of 7 ailu et al. BMC Geriatrics (2024) 24:342 Hailu et al. BMC Geriatrics the availability of geriatric hospitals or specialized nurses for the elderly. Participants need to be educated about suitable exercise routines for the elderly and fall prevention strategies. Healthcare skills training is also necessary for participants, focusing on activities such as wound dressing, vital sign monitoring, and establishing a specific schedule for changing positions. Keywords Family caregiving, Elderly home care, Knowledge, Skills, Qualitative study Introduction family members who are unable to care for themselves by providing physical, emotional, psychological, and occa sionally financial support [8]. Healthy aging is a constant process that improves the person’s health and quality of life [1]. i Ethiopian traditional beliefs presuppose that younger family members will care for the elderly. Families, includ ing children and relatives, are expected to care for the elderly, thus helping them is seen as a blessing and a good opportunity for kids. However, the majority of the fam ily care givers in sub-Saharan Africa, including Ethiopia carry out this function with little to no knowledge of care, aid, or support [8, 9]. Thus, exploring family care giver’s knowledge and skills for effective family caregiving in elderly home care using qualitative study to gather rich and detailed information that goes beyond numerical data is paramount important to take further action about caring of older adults at home. According to a UN estimate on the world’s population outlook, by 2050, more than 63% of the world’s popula tion—or approximately 22% of all people—will be 60 years of age or older and more than 63% will reside in Africa [2]. Caregiving becomes increasingly complicated and difficult as people age because of chronic illnesses and ongoing loss of mental and physical independence [3]. Furthermore, research indicates that more than 70% of persons 60 and older view old age as a nightmare, a time of uncertainty about the future, and a time when they must rely on others for nearly everything [4].h The family-centered care paradigm was famously cre ated in pediatrics and has now been applied to various fields, such as cancer, HIV, palliative care, and hospice care [5]. It is currently being modified for the care of vul nerable older individuals. Through information exchange, involvement in decision-making, and cooperation in the formulation of policies and programs, patient and family knowledge, values, beliefs, and cultural backgrounds are incorporated into the planning and delivery of treatment [6].hf Sample size determinationh The sample size was determined by the level of satura tion. Twenty two study participants were used for in- depth interview. Page 3 of 7 Hailu et al. BMC Geriatrics (2024) 24:342 Hailu et al. BMC Geriatrics (2024) 24:342 Hailu et al. BMC Geriatrics Results A total of twenty two individuals were participated in the study (Table 1). The results of the study were summa rized in to five thematic areas: Awareness of participants on common elderly health conditions and their home based management care, Awareness of study participants on availability of elderly care resources, Awareness on practice of ADLs and fall prevention, awareness towards practices related to simple and highly skilled healthcare activities, and awareness on abuse related practices. We used a purposive sampling technique, which is non- probability, to select participants for our study. We spe cifically targeted individuals with experience in providing care for elderly people in their homes. To find these par ticipants, we reached out to healthcare professionals like doctors, nurses, and social workers who regularly inter act with elderly patients. Community events related to elderly care and family caregiving also allowed us to con nect with potential participants. Additionally, we made use of online platforms, such as social media groups focused on family caregiving or elderly care, in order to reach a larger audience. Data quality assurance y Before starting the actual work, open-ended guiding questions were prepared and discussed with experts. Data collection assistants were trained to take notes and record using a tape recorder for one day. The use of an open-ended guiding question helped prevent dominance from participants. After each day of data collection, a debriefing session was conducted by the data collec tion assistants and the principal investigator (PI). The recorded data were read, re-read, and transcribed by both the researchers and data collection assistants sepa rately to ensure data reliability. To minimize personal biases, the collected data were coded independently by the data collection assistants and the PI. The analysis was carried out by authors who were not involved in the data collection process. The interviews were conducted in a quiet place. “…….Joint pain is a common health condition among the elderly. My mother often complains about her knees and hip joints hurting, particularly during long periods of walking or standing. While I pro vide her with exercises, I am unsure about the most appropriate type of exercise to alleviate her pain. I know also some other common elderly health con ditions like diabetes, high blood pressure and eye problems…….P1”. “I have observed instances of forgetfulness and memory loss not only in my father but also in sev eral other elderly individuals. It is quite common for him to misplace his belongings frequently or repeat edly ask the same question……… This issue poses a significant challenge for me as I am uncertain about how to effectively manage and assist him with this problem on my own. Additional support and guid ance are desperately needed in order to address this situation adequately…P7”. Awareness of participants towards common health conditions and their home based management care Concerning the awareness of participants regarding com mon health conditions in elders, participants showed a good understanding. They mentioned nearly all of the common health conditions that affect elders. However, most participants lacked knowledge about how to man age these conditions properly. Caregivers, based on their opinions, have a widespread awareness of the common health conditions that affect elderly individuals. Condi tions such as joint pain, forgetfulness and memory loss, visual impairment, hearing loss, and age-related muscle weakness were among those mentioned. However, what is particularly notable is the lack of knowledge regard ing the management of these conditions. The partici pants expressed uncertainty about selecting appropriate exercises for joint pain, effectively managing forgetful ness and memory loss, providing assistance beyond clear communication for hearing loss, and measuring blood pressure. Data collection method We developed and translated an open-ended English guide into the local language, Tigrigna, with the assis tance of experts. Two experienced research assistants helped with the data collection process as data collection assistants. One assistant used a tape recorder to record interviews, while the other took notes. The PI moderated the data collection process. In-depth interviews were conducted to gather thorough information on the experi ences, perspectives, and insights of family caregivers who provide care for elderly individuals in their homes. Data analysis and presentationh The information was gathered using a tape recorder and notes. It was later transcribed, entered into Atlas-ti8, and translated. Codes were generated and organized, eventu ally leading to the formation of five themes. The thematic analysis approach was employed, and the outcome was presented in both written form and as a table. “My father is experiencing respiratory conditions, and I am also aware that high blood pressure is com mon among older individuals……. I have noticed Page 4 of 7 Page 4 of 7 Hailu et al. BMC Geriatrics (2024) 24:342 Hailu et al. BMC Geriatrics (2024) 24:342 several instances where it remains undetected until it causes significant health problems……….Unfor tunately, we do not have a blood pressure measur ing device at home and I have no any clue how to measure it, so I am unable to check my father’s blood pressure. However, when I take him to the hospital for his other health concerns, they do measure his blood pressure during the check-up….P10”. idea if there are specific geriatric hospitals or specialized nurses for the elderly….P2”. However, they were aware of the presence of non- governmental organizations and social care services that offer assistance and support for the elderly population. f “Yes, I was aware of the existence of non-governmental organizations and social care services that offer assistance and care for the elderly. In fact, I have personally utilized some of these services for my aging parents………. These organizations provide various forms of support, such as in-home care, transportation assistance, and respite ser vices. They have been a valuable resource for my family, helping us ensure that our elderly loved ones receive the necessary care and support they need…… P4”. One participant demonstrated a good understanding and proactive approach towards managing his grandmother’s visual impairment caused by age-related eye problems. But nevertheless, the participant lacked knowledge and understanding on management of other common health conditions experienced by older adults such as joint pain, neck and back pain, memory loss, and chronic diseases. “………….Certainly, I have knowledge about the presence of non-governmental organizations and social care ser vices that offer assistance and care for the elderly. When my grandmother required more support than our family could provide on our own, we explored these services and found several reputable organizations that support elders. They offer services such as personal care, meal prepara tion, and even companionship for the elderly. Awareness on practices of ADLs and fall prevention Another one participant has also demonstrated aware ness and proactive measures in managing his father’s health condition related to age-related muscle weakness. By installing handrails and providing a walker, he has sig nificantly improved his father’s mobility and independent functioning. Study participants were actively involved in assisting their elder family members with certain activities of daily living (ADLs) such as cooking meals, washing clothes, and bathing. “……….As a family caregiver, I take care of my aging loved one by cooking meals, washing their clothes, and ensuring they receive proper assistance with bathing….P16.” “……….As a family caregiver, I take care of my aging loved one by cooking meals, washing their clothes, and ensuring they receive proper assistance with bathing….P16.” “My father has difficulties walking due to age-related muscle weakness. He often requires assistance with activities like climbing stairs or getting in and out of bed. I have installed handrails around the house and provided him with a walker, significantly improving his mobility…P15.” “I have taken on the responsibility of cooking meals, washing my elderly family member’s clothes, and helping them with bathing to ensure their comfort and well-being in their home….P1.” But nevertheless, they lacked knowledge about suitable exercise routines for the elderly and, therefore, do not provide any assistance in that aspect. But nevertheless, they lacked knowledge about suitable exercise routines for the elderly and, therefore, do not provide any assistance in that aspect. Data analysis and presentationh The avail ability of these organizations has been a great relief for families like ours, as they provide professional caregivers who contribute to the overall well-being and quality of life of the elderly population……P8”. “…………….My grandmother suffers from visual impairment caused by age-related eye problems. She experiences difficulty in reading or recognizing faces from a distance. To effectively manage this condi tion, I ensure her eyeglasses are clean and take her for regular eye check-ups. I know also many other cases which are common in older adults although, I have no clue about their management. For example: joint pain, neck and back pain, memory loss, and chronic diseases….P6”. Awareness of study participants on availability of Elderly Care resourcesh Additionally, they stress the responsibility of providing attention and care, rejecting the notion of ignoring elders. “….To be honest, I never really thought about the safety aspects of the home in relation to the elderly. I didn’t make any specific changes or modifications to prevent accidents or accommodate their needs…P13”. Awareness of study participants on availability of Elderly Care resourcesh “……I didn’t know that there were limits to the elderly’s exercise capacity. I just assumed they would let me know if something was too much for them. I never actively engaged them in exercise or encouraged any physical activities….P17.” “……I didn’t know that there were limits to the elderly’s exercise capacity. I just assumed they would let me know if something was too much for them. I never actively engaged them in exercise or encouraged any physical activities….P17.” The participants have expressed a lack of awareness regarding the availability of geriatric hospitals or special ized nurses for the elderly. They acknowledged their lim ited knowledge and stated that they had not come across any specific resources or services catering to elderly care. “……………As far as my knowledge goes, I don’t think there are geriatric hospitals. I haven’t come across any infor mation or services specifically focused on elderly care. I know there are general hospitals and nurses, but I have no The participants have expressed a lack of awareness regarding the availability of geriatric hospitals or special ized nurses for the elderly. They acknowledged their lim ited knowledge and stated that they had not come across any specific resources or services catering to elderly care. “Exercise routines?…. I guess I never thought much about it. I mean, they’re old, so I didn’t think they need much physical activity. I haven’t made any specific mea sures to ensure their safety or meet their needs in terms of exercise….P22.” “Exercise routines?…. I guess I never thought much about it. I mean, they’re old, so I didn’t think they need much physical activity. I haven’t made any specific mea sures to ensure their safety or meet their needs in terms of exercise….P22.” i “……………As far as my knowledge goes, I don’t think there are geriatric hospitals. I haven’t come across any infor mation or services specifically focused on elderly care. I know there are general hospitals and nurses, but I have no Page 5 of 7 Hailu et al. BMC Geriatrics (2024) 24:342 One has expressed with limited awareness of home arrangements with the elderly consideration. the significance of providing proper nourishment and addressing dietary needs, highlighting the harm of neglecting the elderly’s access to healthy meals. Respect ful and calm communication is advocated to avoid any emotional or mental harm to elders. Discussionhi The findings of this qualitative study highlight several important gaps in knowledge and skills among family caregivers in elderly home care. The participants dem onstrated a good understanding of common health con ditions affecting elders, indicating a basic foundation of knowledge in this area. However, they lacked knowledge about proper management of these conditions, sug gesting a need for education and training to improve their caregiving abilities. This finding aligns with previ ous research, which has also identified knowledge gaps among family caregivers in areas such as medication management and symptom control [16].i No, I am not aware of any specific healthcare activi ties related to wound dressing or changing positions frequently to prevent becoming bedridden. I only change the position when my elderly family member asks for it and change the soiled linens as soon as they get soiled.P7. One notable gap identified in this study is the lack of awareness regarding the availability of geriatric hospi tals or specialized nurses for the elderly. This suggests that participants may be unaware of the resources and support services available to them, which could impact their ability to provide optimal care for their elderly rela tives. Improving awareness of these resources is therefore crucial to ensure that family caregivers are accessing the necessary support to fulfill their caregiving responsibili ties effectively. Previous studies have also highlighted the importance of providing information and education to family caregivers about available resources and services [17]. “I don’t have the knowledge or skills to monitor vital signs or perform wound dressing at home. I do change the posi tions of my elderly family member when they request it, but I am not aware of the specific time intervals when to change….P20”. Awareness towards practices related to simple and highly skilled healthcare activitiesi “I believe that abuse towards our elders is completely unacceptable……… I have never resorted to beating my elderly parents as a means of control or discipline. Instead, I try to communicate with them calmly and find peaceful solutions to any issues that may arise….P3.” Participants in the study fulfilled their duty of look ing after their elderly family member by complying with the doctor’s instructions and ensuring that the recom mended medications were taken as instructed. Their dedication involved following the prescribed dosage and timing to ensure their family member derived the utmost advantage from the medication. Additionally, the partici pants showed their support by accompanying the elderly family member to medical appointments. “…………Refusing food to our elders is a form of neglect and abuse. I strongly believe in providing nourishment and taking care of their dietary needs. It is vital to ensure that they have access to healthy and balanced meals to maintain their well-being….P21”. “Yelling at our elders is not only disrespectful but also harmful to their mental and emotional health. I always try to maintain a calm and patient demeanor when communicating with my elders, even in challenging situations….P9”. “I always make sure to give the prescribed medications to my elderly mother member as per the doctor’s instruc tions………. I try my best to follow the dosage and timing mentioned on the prescription…P18”. “……………Taking my elderly family member for doc tor’s appointments is an important responsibility for me. I make sure to accompany them and ask relevant ques tions to the doctor regarding their health condition and medication…….P10”. “I believe it is our responsibility to provide proper atten tion and care to our elders. Ignoring them is simply not an option…………. I dedicate time every day to spend qual ity moments with them, listen to their stories, and address their concerns….P11.” However, they lacked knowledge and skills in home based healthcare activities such as wound dressing, monitoring vital signs, and establishing a specific sched ule for changing positions. While they strived to meet their family members’ requests for position changes and the changing of soiled linens, they were not necessarily adhering to a specific time interval. Awareness on abuse related practices The care givers had a strong awareness and understand ing of abuse-related practices towards elders. They condemn any form of physical abuse, such as beating, and emphasize the importance of communication and peaceful resolution of conflicts. They also recognize Hailu et al. BMC Geriatrics (2024) 24:342 Page 6 of 7 Hailu et al. BMC Geriatrics (2024) 24:342 Hailu et al. BMC Geriatrics Table 1 Socio demographic characteristics of study participants n(22) Variables Category Frequency Age 26–44 8 45–59 14 Gender Male 8 female 14 Marital status Married 19 Single 2 Divorced 1 Kebelle Ayder 7 Adishumdhun 4 05 5 Adihaki 1 Adihawsi 2 17 2 11 1 NB: Kebelle is a place where the study participants reside in or came from. Another significant gap identified in this study is the lack of awareness and knowledge about suitable exercise routines for the elderly activities on daily living (ADLs) and fall prevention. ADLs are essential for maintaining independence and quality of life among elderly individu als, and family caregivers have a crucial role in assisting with these activities. Similarly, fall prevention is vital to prevent injuries and maintain the well-being of elderly individuals. Addressing this knowledge gap is essential to ensure that family caregivers are equipped with the nec essary skills to promote independence and prevent acci dents. Previous studies have emphasized the importance of caregiver education and training in ADLs and fall pre vention [18]. Furthermore, participants in this study exhibited insuf ficient knowledge and skills in healthcare activities such as wound dressing, monitoring vital signs, and estab lishing a specific schedule for changing positions. These activities are critical for maintaining the health and well-being of elderly individuals, particularly those with chronic illnesses or physical limitations. Providing educa tion and training on these healthcare activities is crucial to enhance the caregiving skills of family caregivers and ensure the provision of adequate medical care at home. of geriatric hospitals or specialized nurses for the elderly. Moreover, there was a notable lack of awareness and knowledge regarding practices related to activities of daily living (ADLs) and fall prevention. Additionally, par ticipants exhibited insufficient knowledge and skills in healthcare activities such as wound dressing, monitor ing vital signs, and establishing a specific schedule for changing positions. Despite these gaps, most participants demonstrated a strong awareness and understanding of abuse-related practices towards older adults. Recommendation P i i h Participants should receive comprehensive education and training programs to enhance their knowledge and skills in managing these conditions. This includes learn ing proper management techniques, understanding avail able healthcare resources, and being aware of specialized care options for older adults. Efforts should also be made to raise awareness about the availability of geriatric hos pitals or specialized nurses for the elderly. Participants need to be educated about suitable exercise routines for the elderly and fall prevention strategies. Healthcare skills training is also necessary for participants, focusing on activities such as wound dressing, vital sign monitor ing, and establishing a specific schedule for changing positions. Abbreviations Abbreviations ADLs Activity of Daily living HIV Human immune virus IRB Institutional review board PI Principal investigator UN United Nation Awareness on abuse related practices Despite these knowledge and skills gaps, it is encourag ing to note that most participants demonstrated a strong awareness and understanding of abuse-related practices towards older adults. This highlights the importance of recognizing and addressing elder abuse, as family care givers are in a unique position to detect and prevent such abuse. However, further research is needed to explore the specific actions and strategies that family caregivers can employ to effectively address elder abuse. Conclusionh The results indicate that while participants showed a good understanding of common health conditions affect ing elders, they lacked knowledge about managing these conditions properly. Participants also expressed a lack of awareness regarding the availability of geriatric hospi tals or specialized nurses for the elderly. There was also a lack of awareness and knowledge about suitable exer cise routines for the elderly on activities of daily living (ADLs) and fall prevention. Furthermore, participants lacked knowledge and skills in healthcare activities such as wound dressing, monitoring vital signs, and establish ing a specific schedule for changing positions. However, most participants had a strong awareness and under standing of abuse-related practices towards elders. In conclusion, the study reveals that participants possess a solid understanding of common health conditions affect ing the elderly but lack knowledge about proper man agement of these conditions. Furthermore, participants expressed a lack of awareness regarding the availability Received: 20 November 2023 / Accepted: 27 March 2024 Received: 20 November 2023 / Accepted: 27 March 2024 18. Shah F, Rahman H, Hadjistavropoulos H. Caregiver training programs to improve elder care quality: A systematic review. Geriatric Nursing. 2019;40(1):64–77. (Shah 2019). Declarations 11. Robert MM, David A, Nicewander HQ, David JB. Improving quality and reduc ing inequities: a challenge in achieving best care. Proc Bayl Univ MedCent. 2006;19:103–18. Data availability h d d The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. •. 9. Gibson MJ, Kelly KA, Kaplan AK. Family caregiving and transitional care: a criti cal review. Family caregiver alliance. National Centre on Care Giving; 2012. 9. Gibson MJ, Kelly KA, Kaplan AK. Family caregiving and transitional care: a criti cal review. Family caregiver alliance. National Centre on Care Giving; 2012. 10. Ballard DJ, Spreadbury B, Hopkins RS. Health care quality improvement across the baylor health care system: the first century. Proc Bayl Univ Med Cent. 2004;17:277–88. 10. Ballard DJ, Spreadbury B, Hopkins RS. Health care quality improvement across the baylor health care system: the first century. Proc Bayl Univ Med Cent. 2004;17:277–88. Acknowledgements g We sincerely appreciate Mekelle University and want to express our deep gratitude to them. Furthermore, we wish to acknowledge and express our Page 7 of 7 (2024) 24:342 Hailu et al. BMC Geriatrics (2024) 24:342 Hailu et al. BMC Geriatrics 4. Eboiyehi FA. The perception of old age: it’s implication for care and support for the aged among the Esan of South-South Nigeria. J Inter Social Res. 2015;8:340. 4. Eboiyehi FA. The perception of old age: it’s implication for care and support for the aged among the Esan of South-South Nigeria. J Inter Social Res. 2015;8:340. gratitude to the data collectors and study participants for their cooperation and willingness. No funding. 8. National Alliance for Caregiving. (2010) Care for the family caregiver: a place to start: The White House Conference on Aging. Emblem health. 8. National Alliance for Caregiving. (2010) Care for the family caregiver: a place to start: The White House Conference on Aging. Emblem health. 8. National Alliance for Caregiving. (2010) Care for the family caregiv Ethics approval and consent to participate Ethical clearance has been received from Mekelle university, Intuitional review board (IRB). Informed consent was received from the study participants. Participants were fully informed about the study’s purpose, procedures, potential risks, and benefits before they decided to participate voluntarily. Participants’ personal information and data were kept confidential and only disclosed as required by legal and ethical guidelines. 12. McDaniel SH, Pisani ARI. Multiple dimensions of caregiving and disability:research, practice, policy and caregiving. New York, USA: Springer Science & Business Media; 2012. 13. Help Age International. Elder abuse in the healthcare services in Kenya. Kenya; 2001. 14. . WHO (2006) Global report on falls prevention in older age. Geneva: World Health Organisation. Consent for publication 16. Smith AE, Tyerman J. Knowledge, experience and education of family carers supporting someone living with a chronic illness or disability: a qualitative study. J Community Health. 2019;44(5):899–908. (Smith & Tyerman, 2019). Not applicable as there is no image or other confidentiality related issues. Competing interests The authors declare no competing interests. 15. Blake A. Falls by elderly people at home: prevalence and associated factors. Age Ageing. 2017;17:365–72. Author details 1 17. Reinhard SC, Given B, Petlick NH et al. Supporting Family Caregivers in Providing Care. In: Hughes RG, ed. Patient Safety and Quality: An Evidence- Based Handbook for Nurses. Rockville (MD): Agency for Healthcare Research and Quality (US); 2008. Chapter 28. https://www.ncbi.nlm.nih.gov/books/ NBK2664/ (Reinhard 2008). 17. Reinhard SC, Given B, Petlick NH et al. Supporting Family Caregivers in Providing Care. In: Hughes RG, ed. Patient Safety and Quality: An Evidence- Based Handbook for Nurses. Rockville (MD): Agency for Healthcare Research and Quality (US); 2008. Chapter 28. https://www.ncbi.nlm.nih.gov/books/ NBK2664/ (Reinhard 2008). 1School of Nursing, Mekelle University, Mekelle, Ethiopia 2School of Public Health, Department of Health Informatics, Mekelle University, Mekelle, Ethiopia Author contributions 5. Kovacs PJ, Bellin MH, Fauri DP. Family-Centered Care: A Resource of Social Work in End-of-Life and Palliative Care. Journal of Social Work in End-of-Life & Palliative Care. Vol. 2 (1), 2006. 5. Kovacs PJ, Bellin MH, Fauri DP. Family-Centered Care: A Resource of Social Work in End-of-Life and Palliative Care. Journal of Social Work in End-of-Life & Palliative Care. Vol. 2 (1), 2006. Gebrezabher Niguse Hailu: Supervision, Transcription and translation, inception and design; Muntaha Abdelkader: Tape recording, transcription; Feven Asfaw: Taking note, transcription, translation, design; Hailemariam Atsbeha: Transcription, translation, analysis, inception and design. 6. Institute for Patient- and Family-Centered Care. Frequently-Asked Questions Accessed April 2, 2012. 7. Bassah N, Ubenoh US, Palle JN. An exploratory study of the knowledge and practices of family caregivers in the care of the elderly at home in the Buea Health District, Cameroon. J Gerontol Geriatr Res. 2018;7(473):2. 3. Davis LL, Gilliss CL, Harper MS. (2011) Family caregiving: implications for rural practice, policy, education, and research. Rural Caregiving in the United States pp: 19–31. References 1. Joseph LM. Aging in place: interprofessional approaches to Empower Infor mal caregivers. J Nurse Practitioners. 2023;19(1):104412. 2. WHO. (2015) GSAP Age Health Draft: Global strategy and action plan on age ing and health. 3. Davis LL, Gilliss CL, Harper MS. (2011) Family caregiving: implications for rural practice, policy, education, and research. Rural Caregiving in the United States pp: 19–31. 1. Joseph LM. Aging in place: interprofessional approaches to Empower Infor mal caregivers. J Nurse Practitioners. 2023;19(1):104412. 1. Joseph LM. Aging in place: interprofessional approaches to Empower Infor mal caregivers. J Nurse Practitioners. 2023;19(1):104412. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 2. WHO. (2015) GSAP Age Health Draft: Global strategy and action plan on age ing and health. 3. Davis LL, Gilliss CL, Harper MS. (2011) Family caregiving: implications for rural practice, policy, education, and research. Rural Caregiving in the United States pp: 19–31.
https://openalex.org/W2619324418	http://oceanrep.geomar.de/38224/1/art_10.1038_s41598-017-02624-0.pdf	English	null	Characterization of meta-Cresol Purple for spectrophotometric pH measurements in saline and hypersaline media at sub-zero temperatures	Scientific reports	2,017	cc-by	9,728	Characterization of meta-Cresol Purple for spectrophotometric pH measurements in saline and hypersaline media at sub-zero temperatures Socratis Loucaides1,2, Victoire M. C. Rèrolle2, Stathys Papadimitriou3, Hilary Kennedy3, MatthewC Mowlem2 AndrewG Dickson4 MarthaGledhill1,5 & Eric P Achterberg1,5 Received: 2 August 2016 Accepted: 13 April 2017 Published: xx xx xxxx Received: 2 August 2016 Accepted: 13 April 2017 Published: xx xx xxxx Socratis Loucaides1,2, Victoire M. C. Rèrolle2, Stathys Papadimitriou3, Hilary Kennedy3, Matthew C. Mowlem2, Andrew G. Dickson4, Martha Gledhill1,5 & Eric P. Achterberg1,5 Accurate pH measurements in polar waters and sea ice brines require pH indicator dyes characterized at near-zero and below-zero temperatures and high salinities. We present experimentally determined physical and chemical characteristics of purified meta-Cresol Purple (mCP) pH indicator dye suitable for pH measurements in seawater and conservative seawater-derived brines at salinities (S) between 35 and 100 and temperatures (T) between their freezing point and 298.15 K (25 °C). Within this temperature and salinity range, using purified mCP and a novel thermostated spectrophotometric device, the pH on the total scale (pHT) can be calculated from direct measurements of the absorbance ratio R of the dye in natural samples as = − + − −       pH k e R e R log log 1 ( ) T T e e 2 2 1 3 2 = − + − −       pH k e R e R log log 1 ( ) T T e e 2 2 1 3 2 Based on the mCP characterization in these extended conditions, the temperature and salinity dependence of the molar absorptivity ratios and − k e log( ) T 2 2 of purified mCP is described by the following functions: e1 = −0.004363 + 3.598 × 10−5T, e3/e2 = −0.016224 + 2.42851 × 10−4T + 5.05663 × 10−5(S − 35), and − k e log( ) T 2 2 = −319.8369 + 0.688159 S −0.00018374 S2 + (10508.724 − 32.9599 S + 0.059082S2) T−1 + (55.54253 − 0.101639 S) ln T −0.08112151T. This work takes the characterisation of mCP beyond the currently available ranges of 278.15 K ≤ T ≤ 308.15 K and 20 ≤ S ≤ 40 in natural seawater, thereby allowing high quality pHT measurements in polar systems. About half of the anthropogenic carbon dioxide (CO2) released to the atmosphere since the industrial revolution has been absorbed by the oceans1. This process continues today and buffers atmospheric CO2 levels, thereby partly alleviating global warming. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 2 August 2016 Accepted: 13 April 2017 Published: xx xx xxxx www.nature.com/scientificreports/ www.nature.com/scientificreports/ saturation states of calcite and aragonite7, 8. The contemporary ocean shows the lowest buffering capacity (highest Revelle factor) in polar waters9, and it is projected that by the end of the century these regions will become under- saturated with respect to aragonite10, 11.l p g Although high latitude waters contribute disproportionally to the oceanic CO2 uptake5, 12, the flux estimates are based on data available from periods of seasonal sea ice retreat and parts of the ocean which are ice-free13. Over the last few years the role of sea ice processes in CO2 cycling has been increasingly recognised. Sea ice is a porous medium and within its pores and channels are gas pockets and residual high ionic strength liquids (brines) at thermal equilibrium with the ice14. The brine, enriched in seawater solutes rejected from the ice during freezing14, is the habitat of sympagic phototrophic and heterotrophic organisms15, 16. It has been estimated that in first- and multi-year ice packs of the Southern Ocean, primary production results in the fixation of 36 Tg C yr−1 into biomass17. It is now accepted that the sea ice pack and land fast ice are to a measurable extent CO2 permea- ble and that internal physical, chemical, and biological processes taking place during ice formation and melting may play a significant role in CO2 cycling in high latitude oceans18–20. For example, gravity drainage of CO2-rich brines during ice formation may be a significant and so far unaccounted sink of dissolved inorganic carbon (DIC) in surface waters with estimates in the order of 200–500 Tg C yr−1 for the (Arctic and Antarctic) polar oceans21. Carbonate mineral precipitation in brines during ice formation may present a potentially significant source of total alkalinity (TA) to polar surface waters following their dissolution when sea ice melts, generating an additional sink (~33–83 Tg C yr−1) of atmospheric CO2, which is equivalent to 17–42% of the air-sea CO2 flux in open high latitude ocean waters22. In addition to these mechanisms (gravity drainage, CaCO3 formation in sea ice), based on recent direct measurements of the CO2 exchange between sea ice and the atmosphere as a function of ice temperature, the Antarctic ice pack, during seasonal warming, was estimated to take up the equivalent of 58% of the atmospheric CO2 uptake of the open Southern Ocean surface waters south of 50°S23. www.nature.com/scientificreports/ The interplay between biological and physicochemical processes makes carbonate chemistry within sea ice highly complex, leading to strong gradients in pH between the ice and underlying waters with potentially significant impacts on ocean-atmosphere CO2 fluxes15, 18, 24–26. p 2l Our ability to characterize the marine carbonate system in open ocean waters has undergone major advance- ments during the last few decades, but our understanding of CO2 cycling in ice brine conditions remains limited due to theoretical and methodological constraints25. Sea ice brines exhibit a much wider range of salinity (S) and temperature (T) changes within short temporal and spatial scales than the open ocean. Specifically, brine S-T con- ditions in sea ice extend to the hypersaline region (S > 100) at temperatures much colder than the freezing tem- peratures of seawater (271.23 K at S = 35 and 0 dbar pressure)18, 20, 27. Such large ranges in T and S make the use of traditional ex situ pH and pCO2 (partial pressure of CO2) measurement techniques a challenge, because in situ temperature corrections are required post-analysis using relationships and constants that have not been validated for below- zero temperatures. The most robust method for back-calculating pH and pCO2 to in situ T relies on the solution of a thermodynamic model that describes the marine CO2 system28. This requires the knowledge of the first and second acidity constant of carbonic acid at in situ T and S. Empirical data for these constants, however, are not available to date for T < 274.15 K and S > 50 in natural seawater while non-linear extrapolation to low T and high S can potentially result in large errors in calculated pH and pCO2 values29. g p y g p p 2 Experimental determination of the carbonic acid acidity constants can be facilitated by measurements of all four variables (DIC, TA, pH, pCO2) of the marine carbonate system at the S and T of interest. Although meas- urements of TA, DIC, and pCO2 at sub-zero temperatures and hyper-saline conditions are possible using current methodologies and instrumentation28, spectrophotometric pH measurements are limited to the range of condi- tions for which indicators have been characterised. For example, the characterization of the commonly used indi- cator dye meta-Cresol Purple (mCP) is only valid for 278.15 K ≤ T ≤ 308.15 K and 20 ≤ S ≤ 4030, 31. www.nature.com/scientificreports/ Furthermore, pH measurements at low temperatures using conventional optical apparatus (spectrophotometers, glass cells, lenses etc.) are highly problematic due to the formation of condensation along the optical path.h ) g y p g p p The purpose of this work was to facilitate pH measurements in cold and hypersaline conditions, such as those encountered in the oceanic cryosphere. To this end, we extended the characterization of the pH indicator mCP (in its purified form) to below-zero temperatures down to the freezing point (267.15 K) of S = 100 brines. The salinity maximum and temperature minimum were set by the S-T range in natural sea ice brines with con- servative ionic composition and inter-ionic ratios relative to surface oceanic water. This development became possible by the recent electrochemical characterization of the pH of the Tris/HCl buffer system32 and the use of a novel, custom-made microfluidic spectrophotometric system. The lens-less design of the microfluidic chip pre- vents condensation and is thus ideal for pH measurements at a lower range of temperatures. Our work facilitates high quality in-situ measurements of pH, thereby furthering our understanding of the carbonate system in polar aquatic environments. Characterization of meta-Cresol Purple for spectrophotometric pH measurements in saline and hypersaline media at sub-zero temperatures Socratis Loucaides1,2, Victoire M. C. Rèrolle2, Stathys Papadimitriou3, Hilary Kennedy3, MatthewC Mowlem2 AndrewG Dickson4 MarthaGledhill1,5 & Eric P Achterberg1,5 The influx of CO2 into the ocean causes acidification of surface waters and leads to a decline in the saturation states of carbonate minerals (i.e. aragonite and calcite), posing a threat to marine calcifying species2–4. The capacity of ocean waters to absorb CO2 increases towards the poles because of the higher solubility of gasses at lower temperatures5. High freshwater inputs into polar waters, from ice and snow melt, reduce the seawater’s buffering capacity, as indicated by the Revelle factor6, leading to a decline in pH and 1University of Southampton, Waterfront Campus, Southampton, SO14 3ZH, UK. 2National Oceanography Centre, European Way, Southampton, SO14 3ZH, UK. 3Ocean Sciences, College of Natural Sciences, Bangor University, Menai Bridge, LL59 5AB, UK. 4Marine Physical Laboratory, Scripps Institution of Oceanography, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0244, USA. 5GEOMAR Helmholtz Centre for Ocean Research, 24148, Kiel, Germany. Correspondence and requests for materials should be addressed to S.L. (email: s.loucaides@noc.ac.uk) Scientific Reports \| 7: 2481 \| DOI:10.1038/s41598-017-02624-0 1 Methods ifi i Sulfonephthaleine pH indicator dyes are weak acids (H2I) where the acidic and basic components exhibit different colours and, therefore, absorb light at distinctly different wavelengths. For mCP, H2I is pink, HI− is yellow and I−2 is purple. The relative distribution of the indicator species is pH-dependent and can be expressed in terms of chemical equilibria with corresponding dissociation constants: + ⇔ = − + + − HI H H I K H I H HI [ ] [ ][ ] , (1) T 2 1 2 + ⇔ = − + − − + − I H HI K HI H I [ ] [ ][ ] , (2) T 2 2 2 + ⇔ = − + + − HI H H I K H I H HI [ ] [ ][ ] , (1) T 2 1 2 (1) + ⇔ = − + − − + − I H HI K HI H I [ ] [ ][ ] , (2) T 2 2 2 (2) where brackets denote concentration. At typical surface seawater pH (~8.1), mCP is present only as I−2 and HI− because pK1 T ~2 and pK2 T~ 8. At a sample pH close to the log of the indicator’s second dissociation constant (pK2 T), pH can be measured with considerable accuracy (better than 0.001) by measuring light absorption at the wavelengths of maximum absorbance of the acidic (HI−) and basic (I−) indicator species (434 and 578 nm, respectively).h p y Measurements of pH using indicator dyes require that their optical properties are carefully characterized. The characterization of mCP involves the determination under different T and S conditions of the molar absorptivity constants (εi λ) of each indicator species (i) at wavelengths (λ) of 434 and 578 nm and the second dissociation constant K2 T (equation 2). Methods ifi i Purification of meta-Cresol Purple. The mCP indicator dye was obtained as a sodium salt (Acros Organics). The indicator was purified using the preparative HPLC procedure described in Liu et al.31 using a Shimadzu HPLC system. In preparative mode, the system consisted of a system controller (SCL-10Avp), a pre- parative scale pump (LC-8A), a Rheodyne 3725i manual injector, and a diode array detector (SPD M10Avp) with a preparative flow cell. In analytical mode, the preparative pump was replaced with an analytical scale pump (LC-10ADvp) and the manual injector with an automatic injector (SIL 10AD). The HPLC column (Primesep B2) used for the purification of mCP was from SIELC Technologies. The Primesep B2 column uses a mixed- mode resin to separate analytes via ion-exchange and hydrophobic mechanisms. A preparative column (Part B2–220.250.0510, 22 × 250 mm, particle size 5 μm) was used for the purification procedure while a smaller Scientific Reports \| 7: 2481 \| DOI:10.1038/s41598-017-02624-0 2 www.nature.com/scientificreports/ analytical column (Part B2-46-250.0510, 4.6 × 250 mm, particle size 5 μm) was used for the qualitative analysis of the purified indicator.hi pi The mobile phase used for the purification was 70% acetonitrile (HPLC grade; Fisher Chemical) and 30% deionised water (Milli-Q, Millipore, MQW). A small amount (0.05%) of trifluoroacetic acid (TFA; ReagentPlus®; Sigma-Aldrich) was used as a mobile phase modifier. The un-purified mCP sodium salt was dissolved in the mobile phase at a concentration of 70 mM. The solution was sonicated in an ultrasonic bath for 15 min to ensure complete dissolution of the indicator. For each purification cycle, 7 mL of indicator solution was injected into the system. The pump flow rate was adjusted to 31 mL min−1 and the pure mCP was collected at its characteristic retention time (approximately 20 min). The pure mCP was separated from the solvent using a rotary evaporator at 40 °C under partial vacuum. Complete evaporation of the mobile phase was achieved after 2–3 h and the recovery efficiency was about 60%. The purified mCP (in acid form) was collected from the evaporation flask and its purity was tested using an analytical HPLC procedure. This was done by injecting 0.020 mL of 70 mM purified mCP (in mobile phase) through the analytical HPLC system at a flow rate of 1.5 mL min−1. The mCP purity was assessed by comparing the chromatographs of the purified and unpurified material. Characterization Procedure. Methods ifi i Solution pH can then be calculated from the absorbance (Aλ) ratio at 434 and 578 nm ( = R A A 578 434) using: = − +    − −    pH k R e e Re log( ) log (3) T T 2 1 2 3 (3) where the parameters e1, e2 and e3 are the molar absorptivity ratios defined by: ε ε ε ε ε ε = = = − − − − − − e e e ; ; (4) HI HI I HI I HI 1 578 434 2 578 434 3 434 434 2 2 (4) The derivation of equation 3 is described in detail in Zhang and Byrne33. Equation 3 can be rearranged to ref. 31: = − +    − −    pH k e R e R log( ) log 1 (5) T T e e 2 2 1 3 2 (5) which simplifies the characterization procedure since e3/e2 is determined as a single parameter in a basic solution (pH ~12) where I2− is the predominant indicator species so that: which simplifies the characterization procedure since e3/e2 is determined as a single parameter in a basic solution pH ~12) where I2− is the predominant indicator species so that: ε ε = − − e e (6) I I 3 2 434 578 2 2 ε ε = − − e e I I 3 2 434 578 2 2 (6) Applying Beer-Lambert’s law, and as long as ε − I 434 2 and ε − I 578 2 are measured in the same solutions, e3/e2 simply becomes the ratio between − A I 434 2 and − A I 578 2 eliminating the need for precise knowledge of the concentration of mCP. This, however, presents its own challenge since the absorbance of I2− at 578 nm is much higher than at 434 nm making it difficult to determine both absorbances accurately from a single measurement. To overcome this, we measured the absorbances of the same solutions in two different absorption cells: 1-cm-path length for − A I 578 2 and a 10-cm-path length for − A I 434 2 . This ensured that absorption measurements of both mCP species were within acceptable ranges and eliminated errors associated with mCP dilution preparation uncertainties. Methods ifi i However, this buffer was increasingly basic at low temperatures and high salinities (e.g., pHT = 8.09 at T = 298.15 K and S = 35; pHT = 9.19 at T = 269.15 K and S = 70)32. So, two sets of less alkaline buffers, each set with distinctly different non-equimolal Tris/HCl composition (0.06 m Tris, 0.04 m HCl; and 0.10 m Tris, 0.06 m HCl) were prepared and used for the determination of − k e log( ) T 2 2 at S = 35–100. The (0.06 m Tris, 0.04 m HCl) buffers s were characterized electro- chemically at Scripps32 and used for the mCP characterization experiments at S = 35, 45, 50, 60, 70, 85, and 100. Their pHT was calculated from the reported best-fit function, pHT = 144.4361–1.0809685 S + 0.006023772 S2 + ( 0.0618411−0.000817397 S + 4.27187 × 10−6 S2) T + (−27.233738 + 0.2329236 S–0.001281138 S2) lnT, with a standard error of 0.002 pH unit32. The (0.10 m Tris, 0.06 m HCl) buffers were used for additional mCP character- ization experiments at S = 35 and 45. The pHT of the (0.10 m Tris, 0.06 m HCl) buffers was not characterized electrochemically (except for the S = 45 buffer at 273.15 K, see below) but instead computed from the equimolal pHT (as calculated from the best-fit equation cited above) via the Henderson–Hasselbalch equation32, 34. This computation gives pHT = 8.785 at 273.15 K for the S = 45 (0.10 m Tris, 0.06 m HCl) buffer, which agrees well with the value determined electrochemically (pHT = 8.783) as described in Papadimitriou et al.32. This approach is also supported from the excellent agreement between thus computed and electrochemically determined pHT values for the (0.06 m Tris, 0.04 m HCl) buffers32. Spectrophotometric measurements. The experimental set-up used for the determination of molar absorptivity constants (εi λ) is illustrated in Fig. 1. The microfluidic flow cells used for the characterization were manufactured in tinted poly (methyl methacrylate) (PMMA). The fabrication procedure is described in detail in Ogilvie et al.35 and Floquet et al.36. Two absorption cells (1 cm and 10 cm) with cross sections of 700 µm × 700 µm were micro-milled into a single PMMA chip. A tungsten halogen light source (Ocean Optics HL-2000) was used for the absorption measurements in conjunction with a 434 nm LED used to boost light intensity at the lower end of the spectrum. Methods ifi i Absorbance measurements were made at mCP concentrations between 10 and 600 µM (n = 6–10) in NaCl solutions buffered with 0.02 M CH3COONa with ionic strength equivalent to that of seawater and brines up to S = 110. The pH of these solutions was adjusted to 4.5 by addition of small amounts of 1 M HCl. The maximum salinity used for the determination of e1 and e3/e2 (S = 110) brackets the maximum salinity at which the pHT of the Tris/HCl buffers (S = 100) has been determined32 (see below). The latter salinity sets the upper limit of the salinity range for the − k e log( ) T 2 2 determined in this study. 2 2 The molar extinction coefficients (ε − I 434 2 , ε ε − − , I HI 578 434 2 and ε − IH 578 ) were determined using the Beer-Lambert Law rearranged to ε = = λ × λ i A b C a b mCP , where a is the slope of the linear regression of absorbances versus concentrations of the mCP dilution series and b is the length of the optical cell. Although molar extinction coefficients have been traditionally determined through repeat absorption measurements of a single mCP concentration (as in a single point calibration) we have opted for a multi-point regression approach to establish the linear range of our meas- urements and to account for intercept offsets.h pf The − k e log( ) T 2 2 term in equation 5 was determined by the measurement of the absorbance ratio = R A A 578 434 in Tris/HCl buffers in synthetic seawater and synthetic seawater-derived brines (S = 35–100). The buffers were pre- pared and their pH was characterized electrochemically on the total proton scale (pHT) in the 267.15 K to 298.15 K temperature range with the Harned cell at the Marine Physical Laboratory, Scripps Institution of Oceanography, University of California San Diego32. The equimolal Tris/HCl buffer (0.08 m Tris, 0.04 m HCl) has been previously used for this purpose31, and the salinity and temperature dependence of its pHT in the current, extended S–T range has been determined [equimolal Tris/HCl: pHT = 536.08338–54.732367 S + 0.8518518 S2 + (0.1675218−1.72224095 × 10−2 S + 2.66720246 × 10−4 S2) T + (−10873.5234 + 1369.56485 S−21.34442 S2) T−1 + (−95.04342 + 9.7014355 S–0.1509014 S2) lnT (standard error: 0.001 pH unit)]32. Methods ifi i Maximum errors in the length of each absorption cell were 5 µm which translates to a maximum error of 0.045% in e1 or e3/e2 and of 0.00002 in pH. Applying Beer-Lambert’s law, and as long as ε − I 434 2 and ε − I 578 2 are measured in the same solutions, e3/e2 simply becomes the ratio between − A I 434 2 and − A I 578 2 eliminating the need for precise knowledge of the concentration of mCP. This, however, presents its own challenge since the absorbance of I2− at 578 nm is much higher than at 434 nm making it difficult to determine both absorbances accurately from a single measurement. To overcome this, we measured the absorbances of the same solutions in two different absorption cells: 1-cm-path length for − A I 578 2 and a 10-cm-path length for − A I 434 2 . This ensured that absorption measurements of both mCP species were − A I 578 2 and a 10-cm-path length for − A I 434 2 . This ensured that absorption measurements of both mCP species were within acceptable ranges and eliminated errors associated with mCP dilution preparation uncertainties. Maximum errors in the length of each absorption cell were 5 µm which translates to a maximum error of 0.045% in e1 or e3/e2 and of 0.00002 in pH. Scientific Reports \| 7: 2481 \| DOI:10.1038/s41598-017-02624-0 3 www.nature.com/scientificreports/ Absorption measurements for the determination of e3/e2 were made in mCP solutions with ionic composi- tion similar to that of seawater and pH adjusted to ~12 with 1 M NaOH. To avoid precipitation of magnesium, sulphur and carbonate salts at high pH and salinities, MgCl2 was replaced with CaCl2 and Na2SO4 and NaHCO3 with NaCl. The ionic strength of the solutions was adjusted accordingly to match that of seawater and brines up to S = 110. The e3/e2 was determined by measuring A434 and A578 in a series (n = 6–10) of mCP dilutions from 5–50 µM concentration. µ We followed the same approach as described above for the determination of e1, using the 1 cm cell to deter- mine − A HI 434 and the 10 cm cell for − A HI 578 . Methods ifi i Elution time (min) 0 10 20 30 40 50 60 Absorbance 0 2000 4000 6000 8000 10000 Un-puriﬁed Puriﬁed Elution time (min) 0 10 20 30 40 50 60 Absorbance 0 2000 4000 6000 8000 10000 Un-puriﬁed Puriﬁed Figure 2. HPLC chromatogram of un-purified (red line) and purified (blue line) mCP. Note that traces of a component eluted at approximately 36 minutes are still present in the purified indicator. Figure 2. HPLC chromatogram of un-purified (red line) and purified (blue line) mCP. Note that traces of a component eluted at approximately 36 minutes are still present in the purified indicator. mCP addition on the buffer pH was estimated by measuring pH over a wide range of mCP to buffer mixing ratios (1:25 to 1:80) and using this data to regress back to a theoretical pH where mCP concentration was zero. This range of mixing ratios was obtained from the dispersion of mCP in Tris/HCl buffer within the microfluidic chan- nels37. The measurements for the determination of − k e log T 2 2 were conducted at 273.15 K and below-zero temper- atures to near the freezing point of the synthetic buffer solutions, as well as at 298.15 K, 283.15 K, and 278.15 K for overlap and direct comparison with the existing data set for purified mCP in Liu et al.31 An estimate of the freez- ing point of the synthetic buffer solutions was computed from the empirical absolute salinity-temperature rela- tionship of thermally equilibrated sea ice brines38, SA = 1000 [1−(54.11/t)]−1 where t is the temperature in °C. mCP addition on the buffer pH was estimated by measuring pH over a wide range of mCP to buffer mixing ratios (1:25 to 1:80) and using this data to regress back to a theoretical pH where mCP concentration was zero. This range of mixing ratios was obtained from the dispersion of mCP in Tris/HCl buffer within the microfluidic chan- nels37. Methods ifi i The measurements for the determination of − k e log T 2 2 were conducted at 273.15 K and below-zero temper- atures to near the freezing point of the synthetic buffer solutions, as well as at 298.15 K, 283.15 K, and 278.15 K for overlap and direct comparison with the existing data set for purified mCP in Liu et al.31 An estimate of the freez- ing point of the synthetic buffer solutions was computed from the empirical absolute salinity-temperature rela- tionship of thermally equilibrated sea ice brines38, SA = 1000 [1−(54.11/t)]−1 where t is the temperature in °C. Methods ifi i A linear array photodiode spectrophotometer (USB4000, Ocean Optics, UK) was used as a detector. Both the light source and detector were connected to the microfluidic flow cell with 600 µm diame- ter optical fibres (Thorlabs, USA). The flow cell was submerged in a water bath (Grant TX150) filled with 15% ethylene glycol solution. The temperature was kept constant (±0.02 °C) and was monitored continuously using a precision thermometer (ASL F250 MKII). The lens-less design of the PMMA microfluidic flow cell allowed for uncompromised optical measurements of pH (no condensation issues) and superior thermostatic control at near-freezing temperatures. For the determination of the molar absorptivity constants (εi λ), experimental solutions were volumetrically premixed with mCP indicator using calibrated pipettes in 20 mL glass vials with silicone/PTFE septum tops. The vials were kept on a rack which was submerged in the water bath. Solutions were siphoned from the vials through a 0.7 mm i.d. PTFE capillary tube into the flow cell using a 1 mL disposable syringe connected to the outlet of the flow cell. The flow cell was flushed with 2 mL of the experimental solution between measurements. The absorption spectrum was recorded in replicate (n = 5) using LabVIEW® software. Reference measurements were performed in experimental solutions without added indicator. For the determination of − k e log T 2 2, the = R A A 578 434 was determined inTris/HCl buffers using the microfluidic pH sensor as described in Rérolle et al.37 but with the same spectrophotometer and light source described above. For each measurement, 4 µL of the 4 mM mCP solution was mixed with 900 µL Tris/HCl buffer. The impact of the Scientific Reports \| 7: 2481 \| DOI:10.1038/s41598-017-02624-0 4 www.nature.com/scientificreports/ Figure 1. Experimental setup used for the determination of the molar absorptivity ratios e1 and e3/e2. The microfluidic flow cells and vials with mCP solutions are submerged in a 15% ethylene glycol thermostated bath. The light is transmitted from the light source to the flow cells and to the spectrophotometer through 600 µm diameter optical fibres (Thorlabs, USA). Figure 1. Experimental setup used for the determination of the molar absorptivity ratios e1 and e3/e2. The microfluidic flow cells and vials with mCP solutions are submerged in a 15% ethylene glycol thermostated bath. The light is transmitted from the light source to the flow cells and to the spectrophotometer through 600 µm diameter optical fibres (Thorlabs, USA). = −. + . × − e 0 004363 3 598 10 T, 1 5 = −. + . × − e 0 004363 3 598 10 T, 1 5 = −. + . × − e 0 004363 3 598 10 T, 1 5 Although at pH 4.5 the dominant indicator species is HI−, small absorbance contributions at 434 and 578 nm from I2− and H2I have not been accounted for in our experiments. This may explain why, between 278.15 K and 308.15 K, the best-fit equation (7) above produces e1 values between 20% and 10%, respectively, higher than those of Liu et al.31 (Fig. 3), who found that removing this bias reduced their e1 values by a similar magnitude (14–18%). The I2− and H2I absorbance contributions are, nonetheless, relatively small, and their effect on pH measurement is minor (<0.0008 pH units) at high R values (>0.7) and slightly larger (up to 0.0034 pH units) at low R values (0.1–0.7)31. Refinement of e1 to account for the contributions of I2− and H2I is possible using an iterative proce- dure and experimental determinations of ε H I 434 2 , ε H I 578 2 , and the K1 of mCP31. This, however, requires careful and laborious experiments offering only minor gain in pH measurement performance especially at pH > 7.5. The potential error in the e1 computation from equation (7) above due to the unaccounted absorbance contributions of I2− and H2I is not necessarily propagated to the final pH determination (equation 5) but is likely “calibrated out” during the determination of − k e log( ) T 2 2 as described subsequently.if g 2 2 Changes in salinity have no significant effect on e1 between S = 35 and S = 110 (Fig. 3), consistent with the findings of Liu et al.31. Generally, e1 has a minor influence on the calculation of pH at high pH values (>8). At pH 8, it is possible to disregard the temperature dependence of e1 and use an average value with no significant impact on pH (<0.001 pH units) or disregard it altogether (e1 = 0) with only a minor effect on pH (0.002 pH units). e3/e2 as a function of temperature and salinity. The e3/e2 term in equation 5 is influenced by both the ionic strength and ionic composition31 and, for this reason, was determined in an electrolyte solution with near-seawater composition and carefully adjusted ionic strength. Results and Discussioni Purification of meta-Cresol Purple. Impurities in indicator dyes result in significant uncertainties in measured pH values31, 39. Analyses have shown that commercially available mCP indicators contain different types and quantities of light absorbing impurities, which could lead to pH offsets as large as 0.01 pH units. Therefore, characterizations of un-purified mCP are batch-specific and only valid for pH measurements using the same indicator batch. Measurements generated using uncharacterised un-purified mCP can be post-corrected as long as stocks of the un-purified indicator used are archived31. The HPLC purification procedure developed by Liu et al.31 was closely replicated here, yielding approximately 150 mg of purified mCP from each injection. Analysis of the un-purified mCP indicator following the analytical HPLC protocol of Liu et al.31 revealed a near identical chromatogram with the exception of an additional peak eluted at about 50 min (Fig. 2). Analysis of the purified material using the same protocol showed complete removal of impurities, with an exception of trace amounts (<8%) of a component eluted at 36 min. Similar residual profiles have been found after purification but have been reported to have practically no effect (<0.001 pH unit) on pH measurements in buffer solutions40. Scientific Reports \| 7: 2481 \| DOI:10.1038/s41598-017-02624-0 5 www.nature.com/scientificreports/ T (K) 265 270 275 280 285 290 295 300 e1 0.0040 0.0045 0.0050 0.0055 0.0060 0.0065 0.0070 T (oC) -5 0 5 10 15 20 25 S 35 S 60 S 85 S 110 Figure 3. Values of e1 as a function of temperature, obtained in NaCl solutions buffered with CH3COONa (pH ~4.5) with ionic strengths equivalent to salinities of 35, 60, 85 and 110. The dashed line represents the e1 relationship determined by Liu et al.31. T (K) 265 270 275 280 285 290 295 300 e1 0.0040 0.0045 0.0050 0.0055 0.0060 0.0065 0.0070 T (oC) -5 0 5 10 15 20 25 S 35 S 60 S 85 S 110 Figure 3. Values of e1 as a function of temperature, obtained in NaCl solutions buffered with CH3COONa (pH ~4.5) with ionic strengths equivalent to salinities of 35, 60, 85 and 110. The dashed line represents the e1 relationship determined by Liu et al.31. Molar absorptivity ratios as a function of temperature and salinity. e1 as a function of tempera- ture. The temperature dependence of e1 for 267.15 K ≤ T ≤ 298.15 K and 35 < S < 110 is relatively small (Fig. Results and Discussioni 3) and is described by the best-fit equation: Molar absorptivity ratios as a function of temperature and salinity. e1 as a function of tempera- ture. The temperature dependence of e1 for 267.15 K ≤ T ≤ 298.15 K and 35 < S < 110 is relatively small (Fig. 3) and is described by the best-fit equation: = −. + . × − e 0 004363 3 598 10 T, (7) 1 5 (7) = −. + . × − e 0 004363 3 598 10 T, 1 5 The pH was adjusted to ~12 with NaOH so that only the basic (I2−) form of mCP was present and interferences from HI− and H2I were negligible. The tempera- ture and salinity dependence of e3/e2 (Fig. 4) for 267.15 K < T < 298.15 K and 35 < S < 110 can be described by: = −. + . × + . × − − − e e S / 0 016224 2 42851 10 T 5 05663 10 ( 35) (8) 3 2 4 5 (8) The relationship provides e3/e2 values that are in agreement with those reported by Liu et al.31; at S = 35 and T = 298.15 K, the difference between the values obtained from equation 8 and from the relationship in Liu et al.31 is 0.0006, which corresponds to a pH discrepancy of less than 0.001 for pH values lower than 8.3. This discrep- ancy becomes even smaller at lower temperatures. At higher salinities, however, the deviation between the e3/e2 predicted by the equation of Liu et al.31 and its value computed from equation 8 above increases to about 0.005, equivalent to ΔpH = 0.010, at S = 100. The expression for e3/e2 by Liu et al.31 was optimized for S between 20 and 40, which consequently results in an enhanced discrepancy with our findings at higher salinities. Extrapolation of the Liu et al.31 e3/e2 relationship to salinities higher than S = 40 is therefore not advisable. Equation 8 was not experimentally validated at S < 35; nevertheless, it agrees well with that of Liu et al.31 at S = 20 (the low end of their experimental range), with a maximum discrepancy at 273.15 K of 0.0006 (ΔpH = 0.002).h p g p y p The pH values obtained using equation 5 are sensitive to variations in e3/e2 and, therefore, experimental deter- mination requires due care. The multi-point determination of the molar absorptivities of I2− (ε − I 434 2 , ε − I 578 2 ) showed that the intercept of the regression of absorbance versus concentration cannot always be assumed as zero. We have observed small but significant intercept offsets in the e3/e2 determination experiments that, if ignored (e.g., through single point determination), could result in pH errors of ca. 0.001 pH unit. It is not clear what the source Scientific Reports \| 7: 2481 \| DOI:10.1038/s41598-017-02624-0 6 www.nature.com/scientificreports/ Figure 4. = −. + . × − e 0 004363 3 598 10 T, 1 5 Values of e3/e2 (a) as a function of temperature, and (b) salinity at 0 °C. The measurements were obtained at pH 12 in solutions with near-seawater composition and ionic strength equivalent to salinities 35, 60, 85, and 110. The yellow square in panel (a) represents the e3/e2 value reported by Liu et al.30 for S = 35 and T = 298.15 K. Figure 4. Values of e3/e2 (a) as a function of temperature, and (b) salinity at 0 °C. The measurements were obtained at pH 12 in solutions with near-seawater composition and ionic strength equivalent to salinities 35, 60, 85, and 110. The yellow square in panel (a) represents the e3/e2 value reported by Liu et al.30 for S = 35 and T = 298.15 K. of the non-zero intercept is in our experiments, but it may be related to light instabilities of the optical system or other random errors. Benchtop dual-beam spectrophotometers are inherently more stable, allowing for higher quality optical measurements. It is therefore possible that using such instruments eliminates the need for the multi-point determination approach used in this work. This, however, remains to be tested, and it is recom- mended that, when portable spectrophotometers are used (as in this work), a multi-point determination approach is used. of the non-zero intercept is in our experiments, but it may be related to light instabilities of the optical system or other random errors. Benchtop dual-beam spectrophotometers are inherently more stable, allowing for higher quality optical measurements. It is therefore possible that using such instruments eliminates the need for the multi-point determination approach used in this work. This, however, remains to be tested, and it is recom- mended that, when portable spectrophotometers are used (as in this work), a multi-point determination approach is used. Determination of − k e log( ) T 2 2 as a function of temperature and salinity. The temperature and salinity dependence of − k e log( ) T 2 2 of purified mCP was determined by measurements of the absorbance ratio (R = A578/A434) in the Tris/HCl buffers prepared in a range of salinities (S = 35, 45, 50, 60, 70, 85, and 100) at tem- peratures ranging from their freezing point to 298.15 K. = −. + . × − e 0 004363 3 598 10 T, 1 5 Temperature (K) 270 275 280 285 290 295 Salinity 40 50 60 70 80 90 100 7.7 7.7 7.7 7.7 7.8 7.8 7.8 7.8 7.9 7.9 7.9 7.9 8.0 8.0 8.0 8.0 8.1 8.1 8.1 8.1 8.2 Salinity Figure 5. Temperature and salinity dependence of − k e log( ) T 2 2 (values on contour lines) as determined in this study from the absorbance ratio (R = A578/A434) measurements in electrochemically characterized Tris/HCl buffers in synthetic seawater and brines (S = 35, 45, 50, 60, 70, 85, and 100) between their freezing point and 298.15 K. Temperature (K) 265 270 275 280 285 290 295 Salinity 40 50 60 70 80 90 100 0.12 0.10 0.08 0.06 0.04 0.02 0.00 -0.02 -0.04 0.16 0.14 0.18 0.22 0.20 0.24 0.26 0.28 Figure 6. Differences in − k e log( ) T 2 2 calculated with the Liu et al. (2011) and current parameterizations (equation 8). The hatched rectangle represents a portion of the experimental range of Liu et al.31. Temperature (K) 265 270 275 280 285 290 295 Salinity 40 50 60 70 80 90 100 0.12 0.10 0.08 0.06 0.04 0.02 0.00 -0.02 -0.04 0.16 0.14 0.18 0.22 0.20 0.24 0.26 0.28 Figure 6. Differences in − k e log( ) T 2 2 calculated with the Liu et al. (2011) and current parameterizations (equation 8). The hatched rectangle represents a portion of the experimental range of Liu et al.31. Liu et al.31 determined the − k e log( ) T 2 2 of purified mCP for 278.15 ≤ T ≤ 308.15 and S = 20–40. Our − k e log( ) T 2 2 S-T parameterization (equation 9) and that in Liu et al.31 yield values within 0.001 at S = 35 and T = 298.15 ± 5 K and within 0.010 down to T = 283.15 K. Higher discrepancies between the two relationships at low temperatures (Fig. 6) may reflect differences between the instruments used for the − k e log( ) T 2 2 determination. The pH measur- ing system used for this work had no parts of the optical path exposed to air, thus eliminating the possibility of condensation at low temperatures. = −. + . × − e 0 004363 3 598 10 T, 1 5 The temperature and salinity dependence of − k e log( ) T 2 2 in these conditions can be described by: − = + + + k e a b c d log( ) T lnT T, (9) T 2 2 (9) The factors in the above equation were determined from our measurements using the regression routine in Excel, with a = −319.8369 + 0.688159 S−0.00018374 S2, b = 10508.724–32.9599 S + 0.059082 S2, c = 55.54253−0.101639 S, d = −0.08112151 (r2 = 0.9986, p < 0.00001, n = 47, standard error of fit: σfit = 0.007). Based on this equation, − k e log( ) T 2 2 = 8.0171 at 0 °C and S = 35, while − k e log( ) T 2 2 = 8.2475 at −6 °C and S = 100. The relatively strong temperature dependence of − k e log( ) T 2 2 (Fig. 5) highlights the importance of accurate tem- perature control (±0.05 °C) during pH measurements. Accurate knowledge of salinity is less important (±1 psu), especially within ranges associated with open ocean waters (30 < S < 40). Under these conditions, salinity varia- tions of the order of 1 psu have only a minor effect on − k e log( ) T 2 2 and pH (0.001–0.002 unit) within the uncer- tainty of the − k e log( ) T 2 2 value, based on the standard error of the best-fit S-T function above. At higher salinities (S > 50), more accurate salinity measurements (0.1 psu) are desirable to maintain the same magnitude of − k e log( ) T 2 2 and pH uncertainty (in the order of 0.001 pH unit at S = 90). Scientific Reports \| 7: 2481 \| DOI:10.1038/s41598-017-02624-0 7 www.nature.com/scientificreports/ Temperature (K) 270 275 280 285 290 295 Salinity 40 50 60 70 80 90 100 7.7 7.7 7.7 7.7 7.8 7.8 7.8 7.8 7.9 7.9 7.9 7.9 8.0 8.0 8.0 8.0 8.1 8.1 8.1 8.1 8.2 Figure 5. Temperature and salinity dependence of − k e log( ) T 2 2 (values on contour lines) as determined in this study from the absorbance ratio (R = A578/A434) measurements in electrochemically characterized Tris/HCl buffers in synthetic seawater and brines (S = 35, 45, 50, 60, 70, 85, and 100) between their freezing point and 298.15 K. = −. + . × − e 0 004363 3 598 10 T, 1 5 The condensation is more difficult to control with bench-top spectrophotom- eters as that used by Liu et al.31, although dry N2 gas was used to eliminate condensation on the optical windows at 5 °C. From this comparison, it is clear that the relationship for − k e log( ) T 2 2 by Liu et al.31 should not be extrapo- lated for pH measurements outside its range (S = 20–40, T = 278.15–303.15 K) as this can lead to large errors in pH (0.02–0.30) (Fig. 6). The relationship (equation 8) proposed here should also not be used outside its calibra- tion range (S = 35–100, T = 267.15–298.15 K). 8 Scientific Reports \| 7: 2481 \| DOI:10.1038/s41598-017-02624-0 www.nature.com/scientificreports/ ificreports/ T (K) 265 270 275 280 285 290 295 300 pHspec-pHHarned -0.015 -0.010 -0.005 0.000 0.005 0.010 0.015 0.020 S 35 S 45 S 50 S 60 S 70 S 85 S 100 Figure 7. Differences between spectrophotometrically (equations 4, 6, 7 and 8) and electrochemically (Harned) determined pHT in Tris/HCl buffer solutions in synthetic seawater and brines with conservative (seawater- derived) major ionic composition. Figure 7. Differences between spectrophotometrically (equations 4, 6, 7 and 8) and electrochemically (Harned) determined pHT in Tris/HCl buffer solutions in synthetic seawater and brines with conservative (seawater- derived) major ionic composition. Determination of pH using purified mCP at temperatures between 298.15 K and the freezing point of seawater and sea-ice brines up to salinity 100. Equations 5, 7, 8, and 9 can be used to deter- mine pH on the total proton scale by measurement of the absorption ratio R of purified mCP in seawater and seawater brines, with conservative major ionic composition, with S between 30 and 100 and T between freezing point and 298.15 K. The residuals (pHspec–pHHarned) of pH measurements in Tris/HCl buffers using purified mCP and application of eq. 4, 6, 7 and 8 indicate a relatively wide spread (Fig. 7) with an average absolute residual of 0.004 and maximum absolute residual of 0.016. = −. + . × − e 0 004363 3 598 10 T, 1 5 As the analytical precision (1 standard deviation of n = 5–10 repeat measurements of the same buffer) is significantly smaller (0.001–0.004), at least part of the observed mag- nitude of buffer residuals could be attributed to error propagation from the parameters involved in pH determi- nation (e.g., − k e log( ) T 2 2 , σfit = 0.007) and random error related to buffer preparation, bottling, and handling. Residuals are up to 3 times larger close to the freezing point than at 298.15 K possibly due to the physical/optical heterogeneity of water during the early stages of ice-crystal formation. Therefore, the proposed pH measurement protocol offers good precision (0.001–0.004) and an overall uncertainty in the order of the maximum residual values observed here (0.010–0.020 pH unit), especially at below-zero temperatures near the freezing point of concentrated brines. In comparison, extrapolation of the temperature and salinity dependence of the mCP char- acterization by Liu et al.31 to values outside their empirical range can lead to pH errors at S = 100 in the order of 0.3 pH unit. Scientific Reports \| 7: 2481 \| DOI:10.1038/s41598-017-02624-0 References pH Evolution in Sea Ice Grown at an Outdoor Experimental Facility. Mar. Chem. doi:10.1016/j marchem.2013.04.007 (2013).h marchem.2013.04.007 (2013). 26. Papadimitriou, S., Kennedy, H., Kattner, G., Dieckmann, G. S. & Thomas, D. N. Experimental evidence for carbonate precipitation marchem.2013.04.007 (2013). 26. Papadimitriou, S., Kennedy, H., Kattner, G., Dieckmann, G. S. & Thomas, D. N. 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Summary and Conclusioni y We have purified mCP and characterized it spectrophotometrically in synthetic solutions with conservative sea- water major ionic composition and salinity between 35 and 100 at temperatures ranging from the freezing point of such solutions to 298.15 K. This was made possible by the use of suitable and well characterised Tris/HCl buff- ers and a novel custom-made optical cell that was fully submerged in a water bath eliminating the possibility of condensation build-up in the optical path. This setup allowed for accurate optical measurements at temperatures down to 267.15 K. Both the experimental set-up and the S-T functions of this work will allow traceable, precise, and reliable spectrophotometric pH measurements in internal sea ice brines and other high latitude and deep waters where temperatures are often just above freezing. The current characterization of purified mCP offers major improvement of pH measurement (0.010–0.020 pH unit uncertainty) in high salinities (up to S = 100) and near-zero and below-zero temperatures to the freezing point over that obtained from the extrapolation of the previous characterization30 (0.3 pH unit uncertainty) to these S-T conditions. The important tools developed in this work provide a step forward towards the understanding of the carbonate system in the cryosphere and cold waters in general. In combination with attainable measurements of the remainder three measurable parameters of the carbonate system (DIC, TA, pCO2), the reliable pH measurements made possible in the extended salinity and temperature ranges of this investigation will facilitate the determination of several unknowns in the parame- terization of the carbonate system in these S –T conditions, including the acidity constants of carbonic acid and, following this, important geochemical indicators, such the saturation state of seawater and brines with respect to carbonate minerals in high latitude marine systems. Scientific Reports \| 7: 2481 \| DOI:10.1038/s41598-017-02624-0 9 www.nature.com/scientificreports/ Scientific Reports \| 7: 2481 \| DOI:10.1038/s41598-017-02624-0 References Pratt, K. W. Measurement of pHT values of Tris buffers in artificial seawater at varying mole ratios of Tris:Tris·HCl. Mar. Chem. 162 89–95, doi:10.1016/j.marchem.2014.03.003 (2014).l j 35. Ogilvie, I. R. G. et al. Reduction of surface roughness for optical quality microfluidic devices in PMMA and COC. Journal of Micromechanics and Microengineering 20, 065016 (2010).l g g 36. Floquet, C. F. A. et al. Nanomolar detection with high sensitivity microfluidic absorption cells manufactured in tinted PMMA for chemical analysis. Talanta 84, 235–239, doi:10.1016/j.talanta.2010.12.026 (2011).l y j 7. Rérolle, V. M. C. et al. Development of a colorimetric microfluidic pH sensor for autonomous seawater measurements. Anal. Chim Acta 786, 124–131, doi:10.1016/j.aca.2013.05.008 (2013). p Acta 786, 124–131, doi:10.1016/j.aca.2013.05.008 (2013). j 38. Assur, A. Composition of sea ice and its tensile strength in Arctic sea ice. 106–138 (National Academy of Sciences, 1958). 38. Assur, A. Composition of sea ice and its tensile strength in Arct p g ( y ) 39. Yao, W. S., Liu, X. W. & Byrne, R. H. Impurities in indicators used for spectrophotometric seawater pH measurements: Asse and remedies. Mar. Chem. 107, 167–172, doi:10.1016/j.marchem.2007.06.012 (2007).il j ( ) 40. Patsavas, M. C., Byrne, R. H. & Liu, X. W. Purification of meta-cresol purple and cresol red by flash chromatography: Procedures for ensuring accurate spectrophotometric seawater pH measurements. Mar. Chem. 150, 19–24, doi:10.1016/j.marchem.2013.01.004 (2013). Scientific Reports \| 7: 2481 \| DOI:10.1038/s41598-017-02624-0 10 www.nature.com/scientificreports/ Author Contributions S.L. conducted the experiments, analysed the data, made the figures and wrote the manuscript. V.R. developed and built the spectrophotometric pH apparatus and measured the pH of the buffers. S.P. prepared and characterised the buffers and contributed to the analysis of the data. H.K. as the project’s principal investigator managed the project. M.M. oversaw and contributed to the development of the relevant technology. A.D. lead the production and characterisation of the buffers. M.G. set up and optimised the indicator purification procedure. E.A. contributed to the data analysis and interpretation. All authors reviewed the manuscript. Scientific Reports \| 7: 2481 \| DOI:10.1038/s41598-017-02624-0 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. 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: 2481 \| DOI:10.1038/s41598-017-02624-0 11
https://openalex.org/W4389515255	https://ijpeds.iaescore.com/index.php/IJPEDS/article/download/22602/14453	English	null	Risk to pollution of aquifers associated with productive activities and environmental management of water resources: bibliometric analysis and trends	International Journal of Power Electronics and Drive Systems/International Journal of Electrical and Computer Engineering	2,024	cc-by-sa	11,757	International Journal of Power Electronics and Drive Systems (IJPEDS) Vol. 15, No. 1, March 2024, pp. 577~590 ISSN: 2088-8694, DOI: 10.11591/ijpeds.v15.i1.pp577-590 International Journal of Power Electronics and Drive Systems (IJPEDS) Vol. 15, No. 1, March 2024, pp. 577~590 ISSN: 2088-8694, DOI: 10.11591/ijpeds.v15.i1.pp577-590 577 Corresponding Author: Gloria Yaneth Florez-Yepes Grupo de Investigación en Desarrollos Tecnológicos y Ambientales (GIDTA) Facultad de Ingeniería y Arquitectura, Universidad Católica de Manizales Manizales, Cra 23 No. 60-63, Colombia Email: gyflorez@ucm.edu.co Risk to pollution of aquifers associated with productive activities and environmental management of water resources: bibliometric analysis and trends Gloria Yaneth Florez Yepes1, Angela María Alzate Álvarez1, Paola Vásquez Cardona2, Luis Alberto Vargas Marín3, Erika Juliana Aldana Arcila1, Fredy E. Hoyos4 1Grupo de Investigación en Desarrollos Tecnológicos y Ambientales (GIDTA), Facultad de Ingeniería y Arquitectura, Universidad Católica de Manizales, Manizales, Colombia 2Grupo de Investigación en Recursos Naturales y Medio Ambiente de Corpocaldas (GIRNMAC), Universidad de Manizales, Manizales, Colombia 3Centro de Investigación en Medio ambiente y desarrollo (CIMAD), Universidad de Manizales, Manizales, Colombia 4Departamento de Energía Eléctrica y Automática, Facultad de Minas, Universidad Nacional de Colombia, Medellín, Colombia ABSTRACT Article history: Received Jan 24, 2023 Revised Jun 2, 2023 Accepted Jun 25, 2023 Its analyzed the evolution and trends of the concept, and studies the models used to know the vulnerability and risk of aquifers and strategies for their management. The methodological process starts from questions guiding the knowledge of research trends, authors among others to proceed to carry out the analyses from the databases of Scopus, Web of Science and Dimensions. VOSviewer and Bibliometric (Rstudio) software were used. Among the most representative results, it was found that most of the research to determine the risk of aquifer contamination is focused on different models such as improved flux prototypes for NO2 emission from agriculture (IPNOA), pollutant origin surcharge hydraulically (POSH), intrinsic vulnerability methods ground water occurrence, overall aquifer class, depth to groundwater (GOD), depth recharge, aquifer, soil, topography, impact, hydraulic conductivity (DRASTIC), substance, infiltration, not saturated, type of coverage, topographic surface, conductivity (SINTACS) and chlorofluorocarbons (CFC) among others. Different models have been used that integrate both hydrological and hydro-geological aspects as well as social aspects including fundamental rights, other models such as the diffuse model, which has had better results in its application, the gaps in the research, are especially focused on conducting holistic research when assessing the risk of these dynamical systems. Keywords: Aquifers Bibliometric analysis Contamination Risk of pollution Water resources This is an open access article under the CC BY-SA license. Corresponding Author: Gloria Yaneth Florez-Yepes Grupo de Investigación en Desarrollos Tecnológicos y Ambientales (GIDTA) Facultad de Ingeniería y Arquitectura, Universidad Católica de Manizales Manizales, Cra 23 No. 60-63, Colombia Email: gyflorez@ucm.edu.co 1. INTRODUCTION This article takes a tour around the different subjects that from 2012 to 2021 have been carried out around the risk of aquifer pollution, as well as reviewing research trends and knowledge gaps from the creation of networks using VOSviewer and R software to identify the correlations and forms of behavior of the literature related to the topic. This analysis will allow the academic and scientific community to identify the main publications and topics associated with the risk of groundwater contamination, in addition to identifying the research gaps to be explored, main authors and research institutes that are addressing the topics, the countries that are developing this research to a higher extent, as well as the cooperation between different researchers to solve the research gaps in a global problem as important as pollution of aquifers. This document proposes some models used for the analysis of the risk of aquifer pollution and its management processes, among the most outstanding were found: the depth recharge, aquifer, soil, topography, impact, hydraulic conductivity (DRASTIC) model, which considers water depth, net recharge, aquifer lithology, soil type, topography, nature of the unsaturated zone and hydraulic conductivity. The substance, infiltration, not saturated, type of coverage, topographic surface, conductivity (SINTACS) model) where it is taken into account-water table-infiltration, self-purification effect of the unsaturated zone, typology of the cover soils, aquifer lithology, hydraulic conductivity and slope of the topographic surface, this model is a derivative of the DRASTIC, both, evaluate the vulnerability of the aquifer dividing it into cells or polygons, for which, they calculate a vulnerability index from a system by weighted ranges. The improved flux prototypes for NO2 emission from agriculture (IPNOA) model is one of the best-known methods used to assess nitrate content. The autoregressive, moving average (ARIMA) model is used to find patterns that are repeated over time to make future estimates which are based on historical data and not on independent variables. Remote sensing and GIS models, where satellite images and terrain elevation models have been used to take it to a process of mapping and spatializing the risk of contamination of aquifers. 1. INTRODUCTION Water has become one of the main sources of development of countries, while it has become one of the most important concerns for the twentieth century, given the increase in production systems and unsustainable practices which have become a serious threat to both human health and ecosystems. In the vast majority of countries of the world agriculture has been the main cause of water pollution, especially in groundwater, nitrates have been the main source of such pollution. After the green revolution, the consumption Journal homepage: http://ijpeds.iaescore.com  578 ISSN: 2088-8694 of agrochemicals has been increased both for fertilization and for the Phyto-sanitary control of crops, generating infiltration into the soil and runoff from them, turning aquifers into highly vulnerable ecosystems, the term vulnerability to aquifer pollution is used to represent the intrinsic characteristics that determine the sensitivity of the aquifer to be adversely affected by a load imposed pollutant [1], to measure this vulnerability different methods can be taken into account, including simulation models of spatial, statistical or process-based or hybrid indices. of agrochemicals has been increased both for fertilization and for the Phyto-sanitary control of crops, generating infiltration into the soil and runoff from them, turning aquifers into highly vulnerable ecosystems, the term vulnerability to aquifer pollution is used to represent the intrinsic characteristics that determine the sensitivity of the aquifer to be adversely affected by a load imposed pollutant [1], to measure this vulnerability different methods can be taken into account, including simulation models of spatial, statistical or process-based or hybrid indices. y In Latin America, researches have been limited in relation to the amount of groundwater and therefore, the degree of contamination of the same is unknown, however, these ones have served as complementary or alternative sources for the use of surface water, while they have been affected, not only by agricultural production systems as mentioned in previous lines, but also, by other systems such as the construction of tunnels, roads, mining systems, among others [2], which confirms the need to carry out research both on the supply of water resources and its state and vulnerability. 1. INTRODUCTION modular finite-difference groundwater flow (MODFLOW) model and multi-species transport in 3-dimensions (MT3D) model, is used in hydrogeology to simulate the underground flow of any aquifer and finally, one of the most used in Latin America is the pollutant origin surcharge hydraulically (POSH), which allows to identify the potential of pollutant loads to which the aquifer is exposed. 2. METHODOLOGY In Table 1, the different search equations used for the analysis of information are shown, along with the results for each equation in terms of the number of documents found in the last 10 years for each equation. We constructed five search equations which are related in Table 1. Figure 1. Methodological process Figure 1. Methodological process Table 1. Search equations Equation Search Equation Results Equation 1 (Aquifer and contamination and environmental and management) A total of 881 documents were obtained. For the last 10 years: 483 documents were obtained. Equation 2 (Underground and aquifers and contamination and environmental and management) A total of 109 documents were obtained. For the last 10 years: 53 documents were obtained. Equation 3 (Risk and of and water and contamination and environmental and management) A total of 3688 documents were obtained. For the last 10 years: 1260 documents were obtained. Equation 4 (Contamination and of and aquifers and productive and activities) In total: 18 documents were obtained. Equation 5 (Contamination and of and aquifers and agricultural and activities) A total of 522 documents were obtained. For the last 10 years: 314 documents were obtained. To achieve a better result in the information acquired, some search criteria were determined, as follows: keywords in English, different documents including articles and secondary documents, likewise, we worked with information from 2012 to 2021, the year 2022 was not included since it is the year that is in progress that would alter the statistics. For the processing of the information, the VOSviewer software was executed to establish the analysis of co-occurrence of the publications made among different countries, it was taken into account that at least five documents have been published in the subject. Additionally, bibliometric networks were generated by combining the keywords with the co- occurrence, to finally decide to analyze those declared by the author to have a higher visualization of the relationships. Subsequently, we proceeded to analyze the citations by documents considering a minimum of 10 citations, it was also taken into account the citations registered in the journals considering a minimum of five documents per journal. 2. METHODOLOGY Initially a keyword search was done to configure the search equation, for the processing of the information the following sequence was carried out. In Figure 1, the procedure carried out for the analysis of investigative processes that have been consolidated for the analysis of aquifers worldwide is shown. This procedure consists of four main phases. Firstly, the most important topics are defined, followed by a clear definition of the consolidated problem in some research questions. Then, the search equation is defined, and finally, the process and analysis of the information are conducted. Subsequently, the construction of the guiding questions was made, which are listed below: “What are the thematic areas and countries conducting the most current researches on the risk of water pollution in aquifers?”. What are the key descriptors related to the risk of water pollution in aquifers?”. “What are the relevant research trends of studies regarding the risk of water pollution in aquifers?”. “What is the relationship among countries, authors and journals on the issue of the risk of water pollution in aquifers?”. How has the process of managing the risk of aquifer contamination been? What are the main gaps in the research conducted of the risk of contamination of aquifers?”. Based on the approach of the previous questions, a quantitative analysis was carried out, using the web of science, Scopus and Dimensions databases, obtaining a better result by number of publications in the Scopus database. Initially, a keyword search had been carried out also using Google scholar database in order to identify the main descriptors considering some keywords such as pollution, pollution management, aquifers, in order to find an approximation to the search equation. It was based on the methodology carried out by [3], where a Int J Pow Elec & Dri Syst, Vol. 15, No. 1, March 2024: 577-590 579 579  579   ISSN: 2088-8694 Int J Pow Elec & Dri Syst bibliometric was made to know about the IoT in wetlands. In Table 1, the different search equations used for the analysis of information are shown, along with the results for each equation in terms of the number of documents found in the last 10 years for each equation. We constructed five search equations which are related in Table 1. bibliometric was made to know about the IoT in wetlands. 2. METHODOLOGY In the R software, other aspects were reviewed, such as the index of publication collaboration between authors, number of documents published per author, dynamics of annual publications, as well as annual citations, as well as three-phase graphic analyses were carried out, considering journals, affiliations and authors who have published against the risk of aquifer contamination and its management. 3. RESULTS It was decided to work with the search equation No 1 where 483 documents were found for the years analyzed, the criterion for its selection is because the publications found within the database had a higher relationship with the subject of study. In the bibliometric analysis it was found that there are 483 documents in the period studied, of which 83.1% corresponds to research articles, 5% to book chapters, 5% to Review, 4.8% to conference paper, 0.6% to books and the rest in other documents, likewise it is found that the area where the largest number of documents are published is in Environmental, followed by Earth Sciences and Agricultural Sciences, it is remarkable that there are also publications on the subject of study in the area of social sciences, this is because the problems of water pollution become social problems, if there are no good water health conditions, this will affect health and increase the social crisis. Figure 2 displays the knowledge domains where scientific papers related to the subject of this research are most frequently published. Figure 3 depicts the countries at the forefront of hydrological research, focusing on studies related to aquifer pollution and Risk to pollution of aquifers associated with productive activities … (Gloria Yaneth Florez Yepes)  580 ISSN: 2088-8694 management processes. The primary contributors to scientific production in this field are the United States, China, and India, as illustrated in Figure 3. management processes. The primary contributors to scientific production in this field are the United States, China, and India, as illustrated in Figure 3. Figure 2. Documents by thematic area Figure 3. Countries that have publications related to aquifer pollution and their management Figure 2. Documents by thematic area Figure 2. Documents by thematic area Figure 3. Countries that have publications related to aquifer pollution and their management According to the analysis, a total of 78 countries have contributed to publications regarding aquifer contamination and management processes. Among these countries, 32 have met the criterion of publishing at least five documents in the last 10 years. Notably, the United States leads with 98 publications, followed by China with 47 and India with 46 publications. These countries not only have the highest publication output but also exhibit substantial collaboration in terms of joint publications. Figure 4 demonstrates the collaborative relationships, with Germany and Canada displaying strong publication ties with the United States. Figure 4. nds towards the factors that generate risk to the contamination of aquifers and the Faced with the analysis of the citations, it was observed that of 484 documents, 220 fulfill the function of having been cited at least 10 times, among which we find [4], who carried out the work called “Mapping the groundwater vulnerability for pollution at the pan African scale”, where the DRASTIC method was used for the construction of a map at 1:60,000,000 scale which revealed that groundwater in Central and West Africa is highly vulnerable since the water table is very low, likewise, agricultural activities are quite influential in the contamination of the same ones, since they are shallow, in addition a sensitivity analysis was carried out that showed that the depth of the groundwater, the hydraulic conductivity and the net recharge generate significant changes in vulnerability and in the risk of contamination. Another work of great importance is related to [5] in which an assessment of the risk of nitrates in groundwater was made using intrinsic vulnerability methods: a comparative study of the environmental impact of intensive agriculture in the Mediterranean region of Sicily, Italy, where it is stated that in recent years in Italy, the consumption of agrochemicals and manure has increased which has generated a higher contribution of nitrates, which has increased the contamination of both, surface and underground sources, for the study the method of parametric models IPNOA was used in addition it was combined with the method of intrinsic vulnerability of the aquifer (SINTACS and DRASTIC) to later take them to a geographic information system (GIS) process, where it was concluded that the SINTACS method is the most appropriate to build a relevant risk map. Other relevant research is Groundwater Residence Times and Nitrate Transport in Coastal Aquifer Systems: Daweijia Area, Northeast China, where CFC method were applied to make a relationship between the distribution of the average residence time of groundwater and nitrate transport, concluding that nitrates without denitrification process can accumulate for many years in water sources increasing pollution in a considerable way [6]. Int J Pow Elec & Dri Syst The results of the bibliometric networks against the co-occurrence of the key descriptors, allow us to identify that the trends related to the contamination of aquifers and their management are intertwined with citations related to groundwater, aquifers, quality, water quality, in addition to others such as Hydrochemistry, nitrates and aquifer pollution. A total of 5865 words were identified, considering a minimum keyword co- occurrence threshold of 10. This resulted in a selection of 298 words, which are presented in Figure 5. Figure 5. Key descriptors related to the risk of aquifer contamination Figure 5. Key descriptors related to the risk of aquifer contamination 3. RESULTS Collaboration of scientific production between countries Figure 4. Collaboration of scientific production between countries Int J Pow Elec & Dri Syst, Vol. 15, No. 1, March 2024: 577-590 Int J Pow Elec & Dri Syst, Vol. 15, No. 1, March 2024: 577-590  581 581  ISSN: 2088-8694 Risk to pollution of aquifers associated with productive activities … (Gloria Yaneth Florez Yepes) nds towards the factors that generate risk to the contamination of aquifers and the In the study entitled groundwater vulnerability assessment using DRASTIC and pesticide DRASTIC models in an area of intense agriculture in the Ganges Plains, India, seven parameters were considered: depth at water level, net recharge, aquifer material, soil material, topography, vadose zone impact and hydraulic conductivity, additionally we worked with the DRASTIC for which, land use was added, from a linear regression analysis it could be established that to measure the vulnerability of groundwater the best model is the DRASTIC Pesticide, and it was found that the most vulnerable areas are those whose land use is dedicated to the intensive cultivation of vegetables with intermediate urban areas [7]. Risk to pollution of aquifers associated with productive activities … (Gloria Yaneth Florez Yepe  582 ISSN: 2088-8694 In Colombia, the Technological University of Pereira-UTP conducted research in the municipality of Pereira based on the methodological approach to process-based risk management, taking strategic prospective planning as a complement. The risk was assessed based on the interaction between the intrinsic vulnerability of the aquifer, determined by the modified DRASTIC method (DRASTIC); and the anthropic threats assessed from six key aspects: Management of Groundwater sites, solid waste, wastewater, pollution by receiving surface sources, hydrocarbons and hazardous substances and agricultural production. The results showed that the highest values of pollution risk are associated with the spill of hydrocarbons (42.9%) and the inadequate disposal of wastewaters (38.2%). The development of the strategies was based on the management of water quality, within the results of threat and risk, being these ones, the aspects that can be intervened as they are not typical of the nature of the aquifer, but of anthropic actions [8]. Through a graduate thesis from the University of La Salle in the city of Bogotá, DRASTIC was implemented in order to define the areas of vulnerability of the it Guadalupe aquifer (Tenjo, Cundinamarca) particularly to pollution, developing a useful and reliable tool for an integral management of the resource that can be the basis in future land management plans [9]. nds towards the factors that generate risk to the contamination of aquifers and the For the aquifers of Funza, in the province of Cundinamarca, the degree of intrinsic vulnerability of the aquifer systems of the study area and the potential danger represented by anthropic activities that may affect the sustainability of groundwater were established, under the GOD and DRASTIC methodologies, the latter included the assessment by pesticides [10] The University of Sucre, jointly with the regional autonomous corporation of sucre (CARSUCRE), it elaborated the maps of intrinsic and specific vulnerability to pesticides of the Morroa aquifer through the DRASTIC parametric system where the geometry of the system was defined based on the variables of the method, through the Arcgis 9.2 software. The results are part of the basis of environmental management and a starting point for research aimed at protecting the aquifer against contamination by pesticides and other substances of special environmental care [11]. In a graduate thesis from the Catholic University of Colombia, a methodology based on the DRASTIC method was proposed, in order to evaluate the potential for groundwater contamination. Relevant adjustments were made for the hydro-geological characteristics of a country such as Colombia [12]. As it has been evidenced, there are different methods to assess the risk of pollution of aquifers, in the case of Costa Rica, a pollutant load map was developed in the northern sector of the Barva aquifer from the pollutant origin surcharge hydraulically (POSH) method, to later generate the pollution risk map. This method has been widely used in Spain, Brazil, Mexico and Argentina [13]. The threat of the aquifer by contamination is known from the identification and characterization of potentially polluting sources, of which the class, concentration, volume, mode of soil disposition and time of application of the pollutant load must be known, according to [14] in [13]. The land was divided into cells of 250* 250m, which were assigned the maximum value of pollutant load potential, distributed in high, moderate, reduced and null. The pollutant load is contributed by the human activities that are generated and that are exacerbated due to an inadequate urban, industrial and agricultural planning although there are guidelines and policies for land use planning [13], which is a constant in other countries. Another concern of groundwater is the intrusion of seawater and nitrates, especially in those places where water is used for consumption. nds towards the factors that generate risk to the contamination of aquifers and the Organic compounds have become another main source of groundwater pollution, in the research entitled: “Synthetic organic compounds and their transformation products in groundwater: Occurrence, fate and mitigation”, it was made a compilation of different studies of the influence of organic compounds on groundwater, especially by pesticides, pharmaceutical products, industrial chemicals, organophosphates, likewise, the main challenges of managed groundwater, the injection and infiltration of recovered water and the filtration of the banks, in relation to the natural attenuation of organic micro-pollutants, are analyzed, and information is provided on the future chemical quality of groundwater [22]. In the Guanzhong river basin in China, an investigation was carried out to analyze the hydro- geochemical characteristics, hydro-chemical facies of groundwater and their formation mechanisms, assess the quality of groundwater for domestic use using the water quality indicator (WQI) and assess the non- carcinogenic risks of water consumption and forms of contact with the skin of men, women and children through the human health risk assessment model (HHRA) established by the united states environmental protection agency (USEPA). This study yielded essential information for the protection and management of local groundwater, which contributed to the sustainable development of drinking water in the area, generating a harmonious relationship between society and the environment. It was possible to determine the activities that were contributing pollutants to groundwater and the largest contributors of nitrogen and fluoride concentrations by the use of fertilizers [23]. In rural areas of Argentina, due to agricultural and livestock activities, nitrogen inputs to groundwater are also generated. In this case, the characteristics of groundwater quality in an unconfined aquifer in Argentina were studied, determining pollution by nitrogen concentrations, NO3 transformation processes and their relationship with different types of land uses, based on isotopic analysis and multivariate statistical analysis. This information was compared with the urban area, showing that in this one, pollution is more considerable than in the rural area, especially by the disposal of waste from sludge systems and animal pens [24]. A similar study was conducted in China, which included groundwater quality monitoring, isotope analysis, and the Markov chain monte Carlo (MCMC) model to determine the outflow amounts of riverine NO3 from four potential sources such as atmospheric deposition (AD), chemical nitrogen fertilizers (NF), soil nitrogen (SN) and manure and sludge [25]. nds towards the factors that generate risk to the contamination of aquifers and the area, where specifically, nitrogen concentrations were measured, recognized for generating a risk to the health of the population, without neglecting that in Bangladesh a large number of victims are reported, due to arsenic contamination in groundwater [18]. One of the aspects that has generated the most contamination of aquifers has been drilling for exploration and exploitation of hydrocarbons, in this sense, a study entitled “A Critical Review of the Risks to Water Resources from Unconventional Shale Gas Development and Hydraulic Fracturing in the United States” was carried out. In which, from different case studies, four potential risks are identified: contamination of shallow aquifers by gas leaks, contamination by disposal of improperly treated shale gas wastewater, accumulation of toxic gases or sediments and excessive use of water for hydraulic fracturing [19], being this, the very document with the highest number of citations among those studied in this article with a total of 966 citations. Another aspect that has generated water pollution has been Arsenic, present especially in countries with volcanic influence, such as those of Latin America, which is present in young rocks and it is generated by the oxidation of sulfated minerals that make the water have a high pH and generate imbalance in the quality of water for consumption, the study entitled: One Century of Arsenic Exposure in Latin America: A review of history reveals the risk of aquifer contamination by the presence of this mineral [20]. Another country concerned about the presence of arsenic has been India, where an investigation was made conducting geospatial interpolation of pollutants to identify poor sanitary practices in waste management, agricultural activities, industrial pollution, for such an evaluation the heavy metal contamination index was used, which was considered the best one to determine the risk to people’s health, likewise, it was identified that shallower waters had less heavy metals than more surface waters [21]. nds towards the factors that generate risk to the contamination of aquifers and the In places such as Tunisia (Africa), a study was developed to evaluate the presence of nitrates using hydro-geochemical tools, the electrical conductivity was also analyzed in order to identify the location of the main columns of intrusion of seawater and thus be able to delimit the groundwater for consumption. It is also stated that it is necessary to carry out the risk assessment of pollution in similar scenarios to carry out management processes [15]. Land use change and especially deforestation has brought with it significant impacts to aquifers, in the study conducted for the detection and prediction of the impact of land-use changes on groundwater quality, a case study in northern Kelantan, Malaysia, nitrate concentrations were used as an indicator to determine changes in land use and quality of groundwater, in addition, a geospatial modeling was carried out with which it was demonstrated that these concentrations increase significantly in agricultural wells over time, likewise, the self-regressive integrated moving average model (ARIMA) was used, in order to make the future projection of the behavior of nitrate pollution [16]. In Bangladesh, a concern is presented for the safety of drinking water for which an investigation was conducted in order to detect the quality of groundwater through physicochemical analyses and social surveys to determine the social perception against water for consumption, it resulted in an intrusion of salt water in the analyses, therefore, a potential influence of the salinization of groundwater, 100% of the samples showed that water is not suitable for drinking, complemented by the perception of the community against different diseases that have been generated by water pollution, making this study an input to generate sustainable water policies [17]. In Bangladesh, groundwater is commonly extracted through wells and is used for human consumption in arid and humid rural areas due to its availability. That is why it was necessary to develop a study that would allow to know the quality of the water from a number of considerable samples in different wells dug in the Int J Pow Elec & Dri Syst, Vol. 15, No. 1, March 2024: 577-590 583 Int J Pow Elec & Dri Syst  ISSN: 2088-8694 area, where specifically, nitrogen concentrations were measured, recognized for generating a risk to the health of the population, without neglecting that in Bangladesh a large number of victims are reported, due to arsenic contamination in groundwater [18]. nds towards the factors that generate risk to the contamination of aquifers and the In Tunisia, in the Souassi aquifer, groundwater risk was assessed using intrinsic vulnerability and threat maps and the DRASTIC indicator of the United States environmental protection agency (USEPA), which determines vulnerability through different variables such as groundwater depth (D), recharge I, saturated zone lithology (A), soil type (S), topography (T), impact of the vadose zone (I) and hydraulic conductivity (C). Additionally, the socioeconomic value of groundwater and indicators of weakness/resistance to the threat of pollution, such as age, the state of facilities and their interaction with population growth, were considered in the research. It was concluded that maps are an appropriate spatial tool for decision-making at a local scale and are the most appropriate to guide risk management and analysis [28]. In Yucatán Mexico, a study was also conducted to analyze the risk of groundwater contamination from the interaction between surface pollutant loads and the vulnerability of the aquifer due to its high permeability. For the risk analysis, an inventory of the polluting sources was carried out and with the help of geographic information systems (GIS) and the method called European cooperation in science and technology (COST) action 620 Group, the danger rates were calculated. This method yielded adequate results for the research and allowed easy and low-cost mapping and risk assessment. The vulnerability was modeled with the depth, recharge, soil, topography, Impact, land use (DRSTIL) index which is a modification of the DRASTIC index, mentioned in an earlier case. For this study, D is the depth of the water, R the recharge, S the soil, T the topography, I the impact of the vadose zone and L the land use. The vulnerability classification coincided with land use, slope, soil type, topography, and depth of the water table, as well as with urban activities, which drastically affect dangerousness [29]. In the Ecuadorian Amazon basin, a study was carried out to evaluate the risk of aquifer contamination from the overlapping of vulnerability maps using the GOD method [1] and pollutant loads applying the POSH method, to prioritize water resource protection zones. Pollutant loads are identified according to the origin of the pollutant and the increase of natural water to the aquifer [30]. nds towards the factors that generate risk to the contamination of aquifers and the In Sao Paulo – Brazil, an investigation has also been carried out on the state of aquifers, in which three indicators are proposed: (1) dependence on groundwater, (2) availability and (3) quality. The results showed that the dependence of the population on groundwater is high, specifically to the north, center and west of São Paulo since there is a great demand for the resource for different activities. As for availability, in some places there is overexploitation of the resource and in others, due to the population present, low availability for its distribution. Finally, groundwater quality is affected due to natural concentrations of chloride and fluoride and human activities in urban areas. However, quality is categorized as very good in a large portion of the territory [26]. In Argentina, specifically for intermediate cities, a Hazard Indicator (HI) was developed to identify the sources of groundwater contamination that need to be monitored and controlled frequently and to recognize the public supply wells that need to be treated. This was applied in Mar del Plata, south-east of the Province of Buenos Aires, Argentina. The proposed HI includes three variables: potential pollution discharge (PCL), effluent disposal mode (ED) and the distance between pollution sources and wastewater supply wells or well protection areas (WP) and three classes: low (10-18-green), moderate (9-30-yellow) and tall (31-50-red). As a  584 ISSN: 2088-8694 result, underground fuel tanks and mines and quarries showed a high HI, as well as 2% of industries and 90% of industries and landfills presented the lowest Risk Indicator. The recommendations based on the results were based on two strategies: mitigation or prevention. Mitigation consists of reducing HI (passing from red to yellow) and preventing the migration of HI (from yellow to red and from green to yellow) [27]. result, underground fuel tanks and mines and quarries showed a high HI, as well as 2% of industries and 90% of industries and landfills presented the lowest Risk Indicator. The recommendations based on the results were based on two strategies: mitigation or prevention. Mitigation consists of reducing HI (passing from red to yellow) and preventing the migration of HI (from yellow to red and from green to yellow) [27]. nds towards the factors that generate risk to the contamination of aquifers and the In the GODS method, G corresponds to the degree of hydraulic confinement, O is the attenuation capacity of the contaminant due to the substrate, depending on the soil parameters, D is related to the depth of the groundwater or aquifer top for the confined and S indicates the form of attenuation of the pollutant and its removal according to the amount of organic matter [1]. Vulnerability was categorized as null, negligible, low, medium, high, and extreme [30]. y g g g g As it can be seen from this international count, different methods and instruments have been used depending on the need and purpose of the study. In these cases, samplings have been used to determine groundwater quality according to its physicochemical parameters, the pollutant loads, and their potential are identified from the POSH method and the vulnerability of aquifers to pollution by the GOD method and its variations, geographic information systems, among others. All the studies seek to generate valuable information to facilitate the decision-making of the involved participants in the territories aimed at the conservation of the underground water resource, which supplies communities in urban and rural areas. Int J Pow Elec & Dri Syst, Vol. 15, No. 1, March 2024: 577-590 3.2. Research trends in the management of risk to aquifer pollution Water pollution management is limited given the scarce economic resources that are used for this purpose, therefore there are some simple alternatives to assess the threat of groundwater, in the work of [31], a tool of five different models representing common geological environments, polluting routes and transport processes is proposed, this is done in order to prioritize sites that require more immediate intervention. The tool is based on semi-analytical stationary state models that simulate different pollutant transport scenarios. In the Mediterranean, groundwater is analyzed for management and protection, especially in coastal ecosystems dependent on groundwater. A study was made from a trans-disciplinary approach among hydrology, hydrogeology, social sciences, and the fundamental right to understand the socio-economic and environmental complexity of ecosystems dependent on groundwater, it is proposed that collective management is necessary to promote the efficient use of water and reduce its conflicts [32]. Aquifer management has been approached from the perspective of sustainability, this is how the study entitled: “Towards sustainable groundwater use: setting long-term goals, back casting, and managing adaptively” , three approaches are proposed: multigenerational, back casting, and managing adaptively, the first one refers to long-term management, the second one to immediate action from public policies and the third one refers to the capacity of resilience to meet long-term objectives, This research proposes that these approaches can achieve the sustainability of aquifers while generating resilience in adjacent ecosystems [33]. Other experiences with the management of water resources have been carried out through models, such as the study for hydro-environmental management of water resources: a diffuse-based multi-objective commitment approach, where a diffuse model for the sustainability of groundwater was designed, based on Int J Pow Elec & Dri Syst, Vol. 15, No. 1, March 2024: 577-590 585  Int J Pow Elec & Dri Syst ISSN: 2088-8694 MODFLOW and MT3D models that are qualitative and quantitative simulation models and whose objective was to formulate socio-optimal and sustainable policies for hydro-environmental management, as well as the analysis of the uncertainties of conflicts around groundwater quality, finally, the best management method is the fuzzy social choice (FSC) which is used to consider a commitment of everybody for all solutions [34]. Another trend for groundwater management is in the collaborative models or models of participation of water resources used in different scenarios for decision-making [35]. 3.2. Research trends in the management of risk to aquifer pollution Biodegradation is one of the most sustainable methods used for aquifer management and decontamination, the study entitled “Biodegradation: Updating the Concepts of Control for Microbial Cleanup in Contaminated Aquifers” presents an important review of classical concepts such as redox thermodynamic zoning or the use of stationary state transport scenarios to assess rates of biodegradation, This study proposes some perspectives for biodegradation, some of these solutions are based on the control of the plume fringe and transport times [36]. Another bio-remediation work was entitled “Assessment of groundwater quality and remediation in karst aquifers: a review”, where bio- remediation models were determined combined with remote sensing tools for the analysis of different ways of approaching of aquifer pollution ,it presents an overview of hydro- geological processes and concepts related to groundwater flow, transport of pollutants in karst systems, followed by a brief discussion on the interaction of surface and groundwater to propose the aquifer pollution management process [37]. In a semi-arid basin of Brazil, a study was carried out to prioritize the areas that required actions to mitigate and monitor pollution by decision makers and involved participants, who develop public policies for the planning and management of groundwater from the generation of water quality indicator and multivariate statistical analysis to analyze parameters in groundwater; in addition, geographic information systems (GIS) were used to construct geospatial behavior maps to evaluate hydro-geochemical variables [38]. GIS is a commonly used tool to complement isotopic, statistical and groundwater quality parameter studies; as in Brazil, they were also used in the upper Blue Nile basin [39] and in the Modjo basin in Ethiopia [40]. Figure 6 displays the most cited authors and documents in the field of aquifer pollution risk. Figure 6. Authors and most cited documents at risk of aquifer contamination and management processes Figure 6. Authors and most cited documents at risk of aquifer contamination and management processes 3.3. Analysis of the most relevant authors and index of collaboration among authors With R software, the collaboration rate among authors was determined is 4.47, the number of co- authors per document is 4.77, the documents per author is 0.23, the total number of authors in the 484 documents is 2047 authors, Likewise, according to the analysis Lotka’s law, 90% of authors produce a single article, while 0.5% produce four articles. In comparison to the yearly scientific production, there has been a significant growth in recent years. Particularly, 2021 witnessed a notable increase with a total of 74 publications. From 2012 to 2014, there was an increment from 34 to 55 publications, followed by a decrease in 2016 to 34 publications once again. However, from 2018 to 2021, there has been a significant upward trend. Despite the global impact of the COVID-19 pandemic, research and scientific output continued to rise. The most prominent authors in the field of aquifer pollution risk can be found in Figure 7. The publications of the first authors have been oriented towards measuring stress or impact factors that generate stress in aquifers. The first author has participated in cooperation with others [41], for the development of research entitled “Identification of the long-term variations of groundwater and their governing factors based on hydro-chemical and isotopic data in a river basin”. In this study, the spatio-temporal variations of the hydro-chemical characteristics of groundwater were discriminated based on the results of grouping self- Risk to pollution of aquifers associated with productive activities … (Gloria Yaneth Florez Yepes)  586 ISSN: 2088-8694 organized maps (SOM) From 2001 to 2017, hydrogen and oxygen variations were also studied in a hydro- chemical facies evolution diagram (HFE-D). This study resulted in articulated self-organized maps (SOM) with stable isotope analysis, the HFE diagram, and correlation analysis could be used successfully to interpret high-dimension, non-linear multivariate systems and provide information on the mechanisms that control groundwater evolution. Figure 7. Most relevant authors on the risk of aquifer contamination Figure 7. 3.3. Analysis of the most relevant authors and index of collaboration among authors Most relevant authors on the risk of aquifer contamination Another study by this author, co-authored with others, is entitled “Effect of groundwater quality on the sustainability of water resources: a case study in the Northern China Plain” [42], in which the authors focus on approaching the implications of pollutants for sustainability, a modular reaction code for reactive transport in three-dimensional aquifers (RT3D) was developed in order to simulate the reactive process of transporting nitrogen species into the groundwater system. Subsequently, the management optimization model was made together with the reactive nitrogen transport model considering the limitations of water quality to quantify and improve the sustainability of groundwater utilization in the study area. As for the second author Bjerg highlights the publication entitled “Linking ecological health to co- occurring organic and inorganic chemical stressors in a groundwater-fed stream system” with other authors [43], where indications of the ecological consequences of the interaction of organic and inorganic chemical stress factors are presented, which are not normally evaluated together, which may provide a missing link that allows the reconnection of chemical and ecological findings, benthic meio-invertebrates were found to be promising bio-indicators of groundwater pollution. Another research of Bjerg in cooperation with other authors has been entitled “Assessing the chemical contamination dynamics in a mixed land use stream system”, where the impacts on groundwater caused by stress factors are assessed, using three methods: i) massive discharge of pollutants into the stream for source quantification, ii) toxic units, and iii) environmental standards, in this research it was observed that the biggest pollutants came from pharmaceuticals [44]. One of the most recent articles is entitled “Shallow urban aquifers under hyper-recharge equatorial conditions and strong anthropogenic constrains. Implications in terms of groundwater resources potential and integrated water resources management strategies”. By the third author who appears in the above graph in co- authorship with other researchers [45] in which the water recharge rate was calculated through water balance and water table fluctuation methods, infiltration and groundwater flow conditions were also examined by combining hydro-geological and isotopic methods. 3.4. Knowledge gaps in the face of risks of wastewater pollution g g p p It is necessary to consider a holistic approach to linking hydrogeology and eco-toxicology to positively influence the sustainable management of water resources at global level. New approaches are urgently needed to identify sources, pathways, and potential impacts relevant to the implementation of remedial measures and management of appropriate sources, further ecological and chemical analyses are needed for the study of underground sources. The publications emphasize that future research should include multiple compounds and compartments of currents and highlight the need for holistic approaches when assessing the risk of these dynamic systems. There are gaps in research regarding long-term management processes to avoid the risk of aquifer contamination. q The analysis of aquifer pollution has been more focused on the measurement of physical-chemical parameters, it is necessary to analyze different bio-indicators that allow generating a more comprehensive knowledge of the risk of water pollution, Likewise, there are gaps in knowledge regarding the behavior of pollutants over time, in few situations, it has got a retrospective of the status of aquifers in the past to make comparisons of what happens in the present. Another aspect related to gaps in scientific research regarding aquifers is the relationship of climate change with them, in the study “impacts of climate change on groundwater and dependent ecosystems” it is shown that there is uncertainty especially in management techniques and numerical modeling to understand this relationship [46]. The need for research into organic pollutants is also highlighted in the study entitled “Organic contaminants in African aquatic systems: Current knowledge, health risks, and future research directions” is mentioned the need to deepen research where it is necessary to deepen the reservoirs of critical points of CO, behavior and environmental fate, eco-toxicology, epidemiology, and interventions to minimize health risks [47]. The study “Hydrogeology and management of freshwater lenses on atoll islands: Review of current knowledge and research needs” mentions that problems of access to water and sanitation need to be addressed within the framework of the United Nations Sustainable Development Goals by 2030 [48]. More comprehensive research of advanced statistical methods associated with the analysis of geospatial technologies is required to know the status of groundwater pollution [49]. 3.3. Analysis of the most relevant authors and index of collaboration among authors On the other hand, compared to the three-fields plot analysis and considering the fields of journals, authors and affiliations, it can be observed that the journal that has the highest number of publications is “Science of the total environment”, in which authors such as Huneau F have published, Garel E, Erostate, Huggenberger P, Aquilina, Labasque T, the main affiliation of the authors is from Université de Corse Pascal Paoli, Likewise, one of the authors who has worked the most study topic has been Wu J who has two affiliations Nanjing University and Technical University of Denmark, However, the journal where this author’s publications are mainly reported is “Journal of Contaminant Hydrology”. Figure 8 displays the three-field analysis, which establishes the existing relationships between journals, authors, and universities. Thicker lines indicate stronger interconnections. It can be observed that the Int J Pow Elec & Dri Syst, Vol. 15, No. 1, March 2024: 577-590  587  ISSN: 2088-8694 Int J Pow Elec & Dri Syst University of Corsica Pascal Paoli maintains close relationships with various authors and journals, particularly with the science of the total environment. University of Corsica Pascal Paoli maintains close relationships with various authors and journals, particularly with the science of the total environment. Figure 8. Analysis Three-fields plot Figure 8. Analysis Three-fields plot 4. CONCLUSION Much of the research on groundwater pollution has been focused on those that have influence of sea water by intrusion of the same to fresh groundwater. Multidisciplinary and trans-disciplinary research between the hard sciences and the social sciences has been necessary to help generate management proposals for addressing groundwater pollution, that is, the social component from the perceptions and from the fundamental right must be incorporated in the processes of pollution management of water sources. One of the most important aspects of groundwater pollution has been drillings for hydrocarbons, nitrate concentrations due to agricultural systems and the presence of arsenic, which in some countries is high because it is mobilized from young volcanic rocks, and by the action of Sulphur-bearing minerals, Latin America is one of the most affected regions by this process. Risk to pollution of aquifers associated with productive activities … (Gloria Yaneth Florez Yepes)  588 ISSN: 2088-8694 The DRASTIC method is one of the most used for the analysis of the risk of water pollution which consists in considering for modeling different parameters such as net recharge, aquifer media, soil media, vadose zone impact and hydraulic conductivity. Some management models have been used to propose possible solutions to the risk of aquifer contamination, in which the most important thing has been the involvement of society in the process, some better-behaved used has been the fuzzy social choice. Other models for the management of aquifer pollution are the establishment of natural background levels (NBL) and threshold values (TV), which could be used as a reference for porous aquifers subject to a large amount of rain and become the basis for decision-making and planning against them. The most current trends in aquifer research are related to the development of strategies and policies that promote the conservation, recharge, and efficient use of groundwater. Advanced monitoring and modeling: Advances in sensor technology, remote sensing, and computational modeling are enhancing the ability to monitor and understand aquifers. Researchers are using real-time data to develop accurate models that can predict aquifer behavior and make informed decisions regarding their management. Surface-water groundwater interactions: there is a growing interest in understanding the interactions between surface water and groundwater, as they are closely interconnected. Studies investigate how human activities, such as irrigation or water extraction, affect the quality and quantity of water in both systems, and how to mitigate negative impacts. 4. CONCLUSION Treatment and remediation technologies: with the increasing concern over groundwater pollution, research is focused on the development of effective treatment and remediation technologies. Advanced purification methods and in-situ remediation techniques are being explored to address aquifer contamination. REFERENCES S. R. D. M. M. Foster, “Groundwater quality protection : a guide for water utilities, municipal authorities, and environme agencies,” pp. 1–114, Washington, DC: World Bank, 2002. [2] L. C. Candelo and J. S. Díaz, “El Tesoro Enterrado De Candelaria : Sus Aguas Subterráneas,” Territorio y Medio ambiente, 2017. d l d h i f hi i hi h d l d i i hi i l i [2] L. C. Candelo and J. S. Díaz, “El Tesoro Enterrado De Candelaria : Sus Aguas Subterráneas,” Territorio y Medio ambiente, 2017. [3] V. Henao-Céspedes, G. Y. Florez, and Y. A. 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BIOGRAPHIES OF AUTHORS BIOGRAPHIES OF AUTHORS Gloria Yaneth Florez Yepes is Environmental Administrator, M.Sc. Sustainable Development and the Environment, Ph.D. Sustainable Development. Professor at the Universidad Católica de Manizales Colombia, coordinator in the research group on Technological and Environmental Development. She can be contacted at email: gyflorez@ucm.edu.co. Angela María Alzate Álvarez Magister in Environmental Management from Griffith University in Brisbane, Australia; Sanitation and Environmental Development Engineer from the Catholic University of Manizales; research professor at the Catholic University of Manizales; researcher of articles published in an indexed journal; co-owner and commercial manager of Ecoingea-Engineering and Environmental Management S.A.S, PhD student in sustainable development at the University of Manizales. She can be contacted at email: aalzate@ucm.edu.co. Paola Vásquez Cardona is Geologist and specialist in Comprehensive Assessment of Environmental Impacts from the University of Caldas. She has worked as a public servant in the Regional Autonomous Corporation of Caldas-Corpocaldas-in the subject of hydrogeology for approximately 12 years. From the water resource group of which she is a part, she has led the following studies: construction of the conceptual hydrogeological model of the Santagueda-km 41, Río Grande de La Magdalena and Río Risaralda aquifers. Formulation and implementation of Management Plans for the aforementioned aquifers and operation of the groundwater monitoring network. Corpocaldas is currently part of the COL7005 project with the IAEA - International Atomic Energy Agency. She can be contacted at email: paolavasquez@corpocaldas.gov.co. Luis Alberto Vargas Marín is Economist, Master in Education - Teaching, Master in Sustainable Development and Environment, Ph.D. in Sustainable Development. Director & Research Professor of the Center for Research of the Environment and Development (CIMAD). Director of the master's program in Sustainable Development and Environment at Manizales University. Environmental Economic Consultant. Areas of research: Environmental Economics, Ecological Economics, Environmental Valuation, and Sustainability. He can be contacted at email: lvargas@umanizales.edu.co. Erika Juliana Aldana Arcila Environmental Engineer graduated from the Catholic University of Manizales. Master in Geoscience and Environment from the Paulista State University, Rio Claro-Brazil campus. She is currently a professor in the Environmental Engineering program at the Catholic University of Manizales and co-researcher in the GIDTA research group (Group for Research in Technological and Environmental Developments). She can be contacted at email: ealdana@ucm.edu.co. Fredy E. Hoyos from La Cruz, Nariño, Colombia, received his B.S. and M.S. degree from the National University of Colombia, at Manizales, Colombia, in Electrical Engineering and Industrial Automation, in 2006 and 2009, respectively, and his Ph.D. REFERENCES 619–620, pp. 1493–1514, 2018, doi: 10.1016/j.scitotenv.2017.11.121. j j y [47] W. Gwenzi and N. Chaukura, “Organic contaminants in African aquatic systems: Current knowledge, health risks, and future research directions,” Science of The Total Environment, vol. 619–620, pp. 1493–1514, 2018, doi: 10.1016/j.scitotenv.2017.11.121. [48] A. D. Werner, H. K. Sharp, S. C. Galvis, V. E. A. Post, and P. Sinclair, “Hydrogeology and management of freshwater lenses on atoll islands: Review of current knowledge and research needs ” Journal of Hydrology vol 551 pp 819–844 Aug 2017 doi: pp j [48] A. D. Werner, H. K. Sharp, S. C. Galvis, V. E. A. Post, and P. Sinclair, “Hydrogeology and management of freshwater lenses on atoll islands: Review of current knowledge and research needs,” Journal of Hydrology, vol. 551, pp. 819–844, Aug. 2017, doi: 10.1016/j.jhydrol.2017.02.047. [49] D. Machiwal, V. Cloutier, C. Güler, and N. Kazakis, “A review of GIS-integrated statistical techniques for groundw evaluation and protection,” Environmental Earth Sciences, vol. 77, no. 19, pp. 1-30, 2018, doi: 10.1007/s12665-018-78  ISSN: 2088-8694 590 BIOGRAPHIES OF AUTHORS in Engineering with an emphasis in Automation in 2012. Dr. Hoyos is currently an Associate Professor of the Mines Faculty, Department of Electrical Energy and Automation, at the National University of Colombia, at Medellin, Colombia. His research interests include nonlinear control, system modelling, nonlinear dynamics analysis, control of nonsmooth systems, and power electronics, with application within a broad area of technological process. Dr. Hoyos is a Senior Researcher in Colciencias and member of the Applied Technologies Research Group-GITA at the Universidad Nacional de Colombia. He can be contacted at email: fehoyosve@unal.edu.co. Int J Pow Elec & Dri Syst, Vol. 15, No. 1, March 2024: 577-590
https://openalex.org/W4389388821	https://wes.copernicus.org/preprints/wes-2023-135/wes-2023-135.pdf	English	null	Reply on RC2	null	2,023	cc-by	21,316	Dynamic performance of a passively self-adjusting floating wind farm layout to increase the annual energy production Mohammad Youssef Mahfouz1, Ericka Lozon2, Matthew Hall2, and Po Wen Cheng1 1Stuttgart Wind Energy at University of Stuttgart, Allmandring 5B, 70569 Stuttgart, Germany 2National Renewable Energy Laboratory (NREL), 15013 Denver W Pkwy, Golden, CO 80401, United States Correspondence: Mohammad Youssef Mahfouz (mahfouz@ifb.uni-stuttgart.de) Finally, the ultimate loads on the mooring systems of the self-adjusting FWF design 15 are lower than those on the mooring systems of the baseline design. Dynamic performance of a passively self-adjusting floating wind farm layout to increase the annual energy production Mohammad Youssef Mahfouz1, Ericka Lozon2, Matthew Hall2, and Po Wen Cheng1 1Stuttgart Wind Energy at University of Stuttgart, Allmandring 5B, 70569 Stuttgart, Germany 2National Renewable Energy Laboratory (NREL), 15013 Denver W Pkwy, Golden, CO 80401, United States Correspondence: Mohammad Youssef Mahfouz (mahfouz@ifb.uni-stuttgart.de) Abstract. One of the main differences between floating offshore wind turbines (FOWTs) and fixed-bottom turbines is the angular and translational motions of FOWTs. When it comes to planning a floating wind farm (FWF), the translational motions introduce an additional layer of complexity to the FWF layout. The ability of a FOWT to relocate its position represents an opportunity to mitigate wake losses within an FWF. By passively relocating downwind turbines out of the wake generated opportunity to mitigate wake losses within an FWF. By passively relocating downwind turbines out of the wake generated by upwind turbines, we can reduce wake-induced energy losses and enhance overall energy production. The translational 5 movements of FOWTs are governed by the mooring system attached to it. The way a FOWT relocates its position changes if the design of the mooring system attached to it changes. Additionally, the translational motion of a FOWT attached to a given mooring system is different for different wind directions. Hence, we can tailor a mooring system design for a FOWT to passively control its motions according to the wind direction. In this work, we present a new self-adjusting FWF layout by upwind turbines, we can reduce wake-induced energy losses and enhance overall energy production. The translational 5 movements of FOWTs are governed by the mooring system attached to it. The way a FOWT relocates its position changes if the design of the mooring system attached to it changes. Additionally, the translational motion of a FOWT attached to a given mooring system is different for different wind directions. Hence, we can tailor a mooring system design for a FOWT to passively control its motions according to the wind direction. In this work, we present a new self-adjusting FWF layout 5 design, and assess its performance using both static and dynamic methods. The results show that relocating the FOWTs in an 10 FWF can increase the energy production by 3% using a steady-state wake model and 1.4% using a dynamic wake model at a wind speed of 10 m/s. Moreover, we compare the fatigue and ultimate loads of the mooring systems of the self-adjusting FWF layout design to the mooring systems in a current state-of-the-art FWF baseline design. The comparison shows that with smaller mooring system diameters, the self-adjusting FWF design has similar fatigue damage compared to the baseline design with bigger mooring system diameters. https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. Dynamic performance of a passively self-adjusting floating wind farm layout to increase the annual energy production Mohammad Youssef Mahfouz1, Ericka Lozon2, Matthew Hall2, and Po Wen Cheng1 1Stuttgart Wind Energy at University of Stuttgart, Allmandring 5B, 70569 Stuttgart, Germany 2National Renewable Energy Laboratory (NREL), 15013 Denver W Pkwy, Golden, CO 80401, United States Correspondence: Mohammad Youssef Mahfouz (mahfouz@ifb.uni-stuttgart.de) 1 Introduction As the mooring system design changes, the stiffness of the FOWT’s station-keeping system changes, and the shape of the watch circle changes. The ability of a FOWT to relocate its position is a new DoF that should be considered inside a floating wind farm (FWF) for two reasons. First, the translational motions of a FOWT have an effect on the FWF’s AEP. Not considering For a FOWT, the mooring system is responsible for station-keeping, as it provides stiffness in surge, sway, and yaw degrees 30 of freedom (DoFs). The mooring system allows the FOWTs to move within a constrained region called the watch circle. As the mooring system design changes, the stiffness of the FOWT’s station-keeping system changes, and the shape of the watch circle changes. The ability of a FOWT to relocate its position is a new DoF that should be considered inside a floating wind farm (FWF) for two reasons. First, the translational motions of a FOWT have an effect on the FWF’s AEP. Not considering For a FOWT, the mooring system is responsible for station-keeping, as it provides stiffness in surge, sway, and yaw degrees 30 of freedom (DoFs). The mooring system allows the FOWTs to move within a constrained region called the watch circle. As the mooring system design changes, the stiffness of the FOWT’s station-keeping system changes, and the shape of the watch circle changes. The ability of a FOWT to relocate its position is a new DoF that should be considered inside a floating wind farm (FWF) for two reasons. First, the translational motions of a FOWT have an effect on the FWF’s AEP. Not considering these displacements increases the uncertainty in the FWF AEP calculations and hence increases the risk of investment (Bodini 35 et al., 2020). Fleming et al. (2015) show that if a downwind turbine moves in the crosswind direction with a displacement of 20% of its rotor diameter (D), the energy produced will change by 4%. The motion can either increase or decrease the FWF’s AEP depending on the direction of the motion into the wake or out of the wake. The second reason is that the ability of FOWTs to relocate their positions can be used to increase the FWF energy production. these displacements increases the uncertainty in the FWF AEP calculations and hence increases the risk of investment (Bodini 35 et al., 2020). Fleming et al. 1 Introduction To achieve our energy goals and harness more wind energy in sites with high wind resources, we cluster wind turbines into wind farms. However, this leads to energy losses inside the farm due to the generation of wakes. As the ambient wind field passes through a wind turbine, the turbine extracts energy out of the wind field, increasing its turbulence and decreasing its 0 speed. When this lower speed and higher turbulence wind field reaches a downwind turbine, the turbine produces less energy and suffers from higher fatigue loads compared to the upwind one. The differences between the energy produced by the upwind and downwind turbines inside a wind farm are known as wake losses. For fixed-bottom onshore and offshore wind farms, the current state of the art is to optimize the wind farm layout to decrease the wake losses and increase the farm’s annual energy 20 1 1 https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. production (AEP) (Baker et al., 2019). Other wind farm layout optimization techniques focus on decreasing the cable length 25 within the wind farm (Fleming et al., 2016) or using open multidisciplinary design, analysis, and optimization to decrease the wind farm’s levelized cost of energy by optimizing the different components inside the wind farm’s complex multidisciplinary system (Perez-Moreno et al., 2018). However, to the best of our knowledge, none of the current wind farm layout optimization techniques account for the ability of a floating offshore wind turbine (FOWT) to relocate its position in the horizontal plane. production (AEP) (Baker et al., 2019). Other wind farm layout optimization techniques focus on decreasing the cable length 25 within the wind farm (Fleming et al., 2016) or using open multidisciplinary design, analysis, and optimization to decrease the wind farm’s levelized cost of energy by optimizing the different components inside the wind farm’s complex multidisciplinary system (Perez-Moreno et al., 2018). However, to the best of our knowledge, none of the current wind farm layout optimization techniques account for the ability of a floating offshore wind turbine (FOWT) to relocate its position in the horizontal plane. For a FOWT, the mooring system is responsible for station-keeping, as it provides stiffness in surge, sway, and yaw degrees 30 of freedom (DoFs). The mooring system allows the FOWTs to move within a constrained region called the watch circle. 1 Introduction (2015) show that if a downwind turbine moves in the crosswind direction with a displacement of 20% of its rotor diameter (D), the energy produced will change by 4%. The motion can either increase or decrease the FWF’s AEP depending on the direction of the motion into the wake or out of the wake. The second reason is that the ability of FOWTs to relocate their positions can be used to increase the FWF energy production. Several methods have been proposed to achieve beneficial crosswind motions in an FWF. Kheirabadi and Nagamune (2019) 40 and Kheirabadi and Nagamune (2020) implemented a yaw and induction based turbine controller to reposition downwind turbines out of the wake. The idea was to use the FOWT controller to create a crosswind aerodynamic force component by yawing the FOWTs’ nacelles to move the downwind turbines out of the wake. The results showed that relocating the FOWTs will increase the FWF’s efficiency, and that the increase in efficiency is dependent on the mooring system designs because they 40 Several methods have been proposed to achieve beneficial crosswind motions in an FWF. Kheirabadi and Nagamune (2019) 40 and Kheirabadi and Nagamune (2020) implemented a yaw and induction based turbine controller to reposition downwind turbines out of the wake. The idea was to use the FOWT controller to create a crosswind aerodynamic force component by yawing the FOWTs’ nacelles to move the downwind turbines out of the wake. The results showed that relocating the FOWTs will increase the FWF’s efficiency, and that the increase in efficiency is dependent on the mooring system designs because they govern the FOWTs’ motions. Rodrigues et al. (2015) changed the positions of FOWTs inside an FWF using a pulley to change 45 the length of the mooring lines according to the wind direction. The results also showed that relocating the FOWTs can increase the FWF’s energy production. These previous studies aimed to actively control FOWT positions and showed the dependency of the FOWT motions on the mooring system design. In our previous work (Mahfouz and Cheng, 2023), we proposed a new method to passively relocate the FOWT out of the wake through mooring system design. However, we only used static tools to govern the FOWTs’ motions. Rodrigues et al. 1 Introduction (2015) changed the positions of FOWTs inside an FWF using a pulley to change 45 the length of the mooring lines according to the wind direction. The results also showed that relocating the FOWTs can increase the FWF’s energy production. These previous studies aimed to actively control FOWT positions and showed the dependency of the FOWT motions on the mooring system design. In our previous work (Mahfouz and Cheng, 2023), we proposed a new method to passively relocate the FOWT out of the wake through mooring system design. However, we only used static tools to assess the method and no dynamic verification was carried out A summary of these three methods is given in Table 1 50 assess the method, and no dynamic verification was carried out. A summary of these three methods is given in Table 1. 50 In this paper, we build on our previous work (Mahfouz and Cheng, 2023), in which we presented a methodology to passively relocate the FOWTs in an FWF to increase the farm’s AEP. The methodology customizes the mooring system design of each FOWT to increase the FWF’s AEP by allowing larger platform offsets and reducing farm wake losses. Mahfouz and Cheng (2023) showed that we can passively relocate the turbines and quantified the gain in AEP we can achieve by passively assess the method, and no dynamic verification was carried out. A summary of these three methods is given in Table 1. 50 In this paper, we build on our previous work (Mahfouz and Cheng, 2023), in which we presented a methodology to passively relocate the FOWTs in an FWF to increase the farm’s AEP. The methodology customizes the mooring system design of each FOWT to increase the FWF’s AEP by allowing larger platform offsets and reducing farm wake losses. Mahfouz and Cheng (2023) showed that we can passively relocate the turbines and quantified the gain in AEP we can achieve by passively relocating the FOWTs. However, until this point all our work was done using static tools focusing only on increasing the AEP 55 gain without assessing the performance of the FWF layout design under dynamic wind and wave loading. In this paper, we extend our work to verify the results using dynamic models. Our goal is to assess the performance of our FWF layout design in both operational and extreme loading conditions. Method We compare the energy gain between steady-state and dynamic wake models at a constant wind 65 speed of 10 m/s. We also calculate the fatigue damage on the mooring system designs and compare it to the fatigue damage of a baseline mooring system design. Finally, we assess the loads on the new mooring system designs under extreme wind and wave conditions to determine their ability to withstand ultimate loads. obtained using OpenFAST. We compare the energy gain between steady-state and dynamic wake models at a constant wind 65 speed of 10 m/s. We also calculate the fatigue damage on the mooring system designs and compare it to the fatigue damage of a baseline mooring system design. Finally, we assess the loads on the new mooring system designs under extreme wind and wave conditions to determine their ability to withstand ultimate loads. 1 Introduction We will evaluate whether the larger displacements of the FOWTs increase the fatigue and extreme loads for the novel mooring system designs compared to a traditional, state-of-the-art, baseline mooring relocating the FOWTs. However, until this point all our work was done using static tools focusing only on increasing the AEP 55 gain without assessing the performance of the FWF layout design under dynamic wind and wave loading. In this paper, we extend our work to verify the results using dynamic models. Our goal is to assess the performance of our FWF layout design in both operational and extreme loading conditions. We will evaluate whether the larger displacements of the FOWTs increase the fatigue and extreme loads for the novel mooring system designs compared to a traditional, state-of-the-art, baseline mooring 2 https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. Table 1. Different methods for relocating the FOWTs Method Description Relocates the FOWTs actively or passively? Yaw and Induction Based Turbine Repositioning (YITuR) Kheirabadi and Nagamune (2019) designed a yaw and induction based FOWT controller to relocate the FOWT in the crosswind direction. The idea is to create a thrust component in the crosswind direction that moves the FOWT. Active Movable FOWT Design Rodrigues et al. (2015) created a new FWF layout de- sign where the FOWTs change their position accord- ing to the wind direction. The FOWTs use a pulley at- tached to the floating platform to change the mooring line length and hence change their position. Active Passively relocating FOWTs using mooring system In our previous work (Mahfouz and Cheng, 2023), we created a new FWF layout design method where the mooring system design is part of the FWF layout op- timization. Each FOWT is attached to a customized mooring system that relocates it according to the wind direction. Passive Table 1. Different methods for relocating the FOWTs Table 1. Different methods for relocating the FOWTs Relocates the FOWTs actively or passively? Description Description Method Kheirabadi and Nagamune (2019) designed a yaw and induction based FOWT controller to relocate the FOWT in the crosswind direction. The idea is to create a thrust component in the crosswind direction that moves the FOWT. Yaw and Induction Based Turbine Repositioning (YITuR) Kheirabadi and Nagamune (2019) designed a yaw and induction based FOWT controller to relocate the FOWT in the crosswind direction. The idea is to create a thrust component in the crosswind direction that moves the FOWT. Rodrigues et al. (2015) created a new FWF layout de- sign where the FOWTs change their position accord- ing to the wind direction. The FOWTs use a pulley at- tached to the floating platform to change the mooring line length and hence change their position. In our previous work (Mahfouz and Cheng, 2023), we created a new FWF layout design method where the mooring system design is part of the FWF layout op- timization. Each FOWT is attached to a customized mooring system that relocates it according to the wind direction. system. Moreover, we will verify the results of the steady-state wake model, which does not consider the FOWT’s motions, 60 against the dynamic wake model, which considers the FOWT’s motions. system. Moreover, we will verify the results of the steady-state wake model, which does not consider the FOWT’s motions, 60 against the dynamic wake model, which considers the FOWT’s motions. To accomplish this, we first present the baseline layout, the passively self-adjusting FWF layout design, and the energy gain we expect to achieve using static tools. Next, we present the stiffness and natural frequency values of each mooring system design within the FWF. Then, we compare the watch circles of the mooring system designs obtained using MoorPy with those system. Moreover, we will verify the results of the steady-state wake model, which does not consider the FOWT’s motions, 60 against the dynamic wake model, which considers the FOWT’s motions. To accomplish this, we first present the baseline layout, the passively self-adjusting FWF layout design, and the energy gain we expect to achieve using static tools. Next, we present the stiffness and natural frequency values of each mooring system design within the FWF. Then, we compare the watch circles of the mooring system designs obtained using MoorPy with those obtained using OpenFAST. 2 Self-adjusting floating wind farm design In this section we present the methodology to design the passively self-adjusting layout starting from a baseline wind farm 70 layout. Additionally, we show the results of the static models for each step of the design process. The design process consists of four main steps as shown in Fig. 1. We discuss each step in the following sections. Throughout this work, we use the In- ternational Energy Agency Wind Technology Collaboration Programme (IEA Wind) 15 MW reference wind turbine (Gaertner 3 Figure 1. Workflow of designing a passively self-adjusting FWF layout https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 1. Workflow of designing a passively self-adjusting FWF layout Figure 1. Workflow of designing a passively self-adjusting FWF layout et al., 2020) coupled to the Activefloat semisubmersible floater as presented by Mahfouz et al. (2021). The wind rose intro- et al., 2020) coupled to the Activefloat semisubmersible floater as presented by Mahfouz et al. ( pll p y duced within IEA Wind Task 37 (Baker et al., 2019) presented in Fig. 2 was used in this study. Throughout this work, the wind 75 speed was assumed to be constant in all wind directions and equal to 10 m/s. We chose the value of 10 m/s to be just below the rated wind speed of 11 m/s of the FOWT reference model, as wakes have no effect on the wind farm’s energy production above the rated wind speed. duced within IEA Wind Task 37 (Baker et al., 2019) presented in Fig. 2 was used in this study. Throughout this work, the wind 75 speed was assumed to be constant in all wind directions and equal to 10 m/s. We chose the value of 10 m/s to be just below the rated wind speed of 11 m/s of the FOWT reference model, as wakes have no effect on the wind farm’s energy production above the rated wind speed. 2.1 Conventional layout optimization The first step in the novel FWF design methodology is to optimize the wind farm layout using the conventional state-of-the- 80 art methods assuming a fixed-bottom layout. This step can be skipped if the user provides a baseline layout that is already optimized. We start with this step because our goal is to show the gain in a wind farm’s AEP due to relocating the FOWTs. Starting from a baseline layout far from the optimum will overestimate the effects of relocating the FOWTs showing higher AEP gain. Instead, we compare the novel passive relocation technique to more traditional layout optimization methods with The first step in the novel FWF design methodology is to optimize the wind farm layout using the conventional state-of-the- 80 art methods assuming a fixed-bottom layout. This step can be skipped if the user provides a baseline layout that is already optimized. We start with this step because our goal is to show the gain in a wind farm’s AEP due to relocating the FOWTs. Starting from a baseline layout far from the optimum will overestimate the effects of relocating the FOWTs showing higher AEP gain. Instead, we compare the novel passive relocation technique to more traditional layout optimization methods with limited floater motions. The baseline wind farm layout we started with in this paper is shown in Fig. 3. The layout has a 85 square shape and consists of nine wind turbines. We optimized the layout using the gradient-based optimizer SNOPT (Gill 4 Figure 2. Wind rose from IEA Wind Task 37 with constant wind speed of 10 m/s https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 2. Wind rose from IEA Wind Task 37 with constant wind speed of 10 m/s et al., 2005, 2008), the objective function was to maximize the wind energy production. The optimization process had two constraints. The first was to keep all turbines within the wind farm boundaries (i.e., within a square of side length 12D). The mooring system designs are not included in this step; hence, when the mooring systems of all FOWTs in the FWF are designed, the anchors will not be within the FWF boundary. 2.1 Conventional layout optimization We will refer to this layout as the optimized wind farm layout through the rest of the text. This layout will be used as a baseline to compare the energy gain after relocating the FOWTs in the FWF. The goal is that the customized mooring system will be coupled to the optimized wind farm layout and passively relocate the FOWTs to decrease wake losses. 100 2.1 Conventional layout optimization The second constraint was that the minimum distance between 90 any two turbines in the wind farm cannot be less than 4D. 90 Figure 3. Baseline layout and optimized wind farm layout The wakes were estimated during the optimization using FLORIS v3.1 (NREL, 2023). The wake model used was the Gaussian velocity model implemented within the tool based on the works of Bastankhah and Porté-Agel (2016); Niayifar and Porté-Agel (2016), and Gauss deflection model based on the works of Bastankhah and Porté-Agel (2016) and King et al. (2021). The turbulence model used was the Crespo-Hernandez model (Crespo and Hernández, 1996). The wake deflection in 5 FLORIS only accounts for the horizontal wake deflection due to yawing the wind turbines. In this work, the yaw angle is always Figure 3. Baseline layout and optimized wind farm layout Figure 3. Baseline layout and optimized wind farm layout The wakes were estimated during the optimization using FLORIS v3.1 (NREL, 2023). The wake model used was the Gaussian velocity model implemented within the tool based on the works of Bastankhah and Porté-Agel (2016); Niayifar and Porté-Agel (2016), and Gauss deflection model based on the works of Bastankhah and Porté-Agel (2016) and King et al. (2021). The turbulence model used was the Crespo-Hernandez model (Crespo and Hernández, 1996). The wake deflection in 95 FLORIS only accounts for the horizontal wake deflection due to yawing the wind turbines. In this work, the yaw angle is always The wakes were estimated during the optimization using FLORIS v3.1 (NREL, 2023). The wake model used was the Gaussian velocity model implemented within the tool based on the works of Bastankhah and Porté-Agel (2016); Niayifar and Porté-Agel (2016), and Gauss deflection model based on the works of Bastankhah and Porté-Agel (2016) and King et al. (2021). The turbulence model used was the Crespo-Hernandez model (Crespo and Hernández, 1996). The wake deflection in 95 FLORIS only accounts for the horizontal wake deflection due to yawing the wind turbines. In this work, the yaw angle is always 95 5 https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. defined to be zero for all wind turbines within the wind farm; hence, no horizontal wake deflection occurs for all calculations done using FLORIS. The optimized wind farm layout is shown in Fig. 3. 2.2 Wind farm layout optimization per wind direction The next step in the self-adjusting FWF layout design method is to find the FWF targeted layout. The targeted layout is the optimum wind farm layout we can achieve if we allow each turbine inside the wind farm to displace in the crosswind direction for every wind direction. To achieve the targeted layout we used the optimization algorithm SNOPT and the same wake model 105 we used to get the optimized wind farm layout. We optimized the wind farm layout separately for each wind direction in The next step in the self-adjusting FWF layout design method is to find the FWF targeted layout. The targeted layout is the optimum wind farm layout we can achieve if we allow each turbine inside the wind farm to displace in the crosswind direction The next step in the self-adjusting FWF layout design method is to find the FWF targeted layout. The targeted layout is the optimum wind farm layout we can achieve if we allow each turbine inside the wind farm to displace in the crosswind direction for every wind direction. To achieve the targeted layout we used the optimization algorithm SNOPT and the same wake model 105 we used to get the optimized wind farm layout. We optimized the wind farm layout separately for each wind direction in the wind rose. The optimization objective was to increase the energy production in every wind direction. The optimization process had two constraints: First, the FOWTs were only allowed to displace in the crosswind direction. Second, the crosswind displacement could not be bigger than 0.5D, which is a distance of 120 m for the 15 MW reference wind turbine. Figure 4. Wind farm layouts with wakes modeled by FLORIS: (left) the optimized wind farm layout and (right) the targeted layout. The black dots represent the positions of the turbines from the optimized layout. Figure 4. Wind farm layouts with wakes modeled by FLORIS: (left) the optimized wind farm layout and (right) the targeted layout. The black dots represent the positions of the turbines from the optimized layout. Figure 4 shows results of the targeted layout for only one wind direction; this process was repeated for each wind direction. 110 We can see that the turbine positions are shifted in the crosswind direction to decrease wake interactions. 2.3 Mooring system database The third step in the self-adjusting FWF layout design method as show in Figure 1 is to create a mooring system design database. The goal of the database is to contain several mooring system designs with different design parameters and save the watch circle and the displacements of each mooring system design for all wind directions. The database will be used in the next step to find a customized mooring system design for each FOWT in the FWF. To create the mooring system design database, 125 we created a full factorial design matrix iterating over several design parameters while keeping other parameters fixed. The fixed parameters were the bathymetry (depth), which was set at a value of 200 m; the number of mooring lines in each mooring system design, which was set to three lines; and the line type—all lines were assumed to be studless chains made of R4S steel. step to find a customized mooring system design for each FOWT in the FWF. To create the mooring system design database, 125 we created a full factorial design matrix iterating over several design parameters while keeping other parameters fixed. The fixed parameters were the bathymetry (depth), which was set at a value of 200 m; the number of mooring lines in each mooring system design, which was set to three lines; and the line type—all lines were assumed to be studless chains made of R4S steel. The permutable design parameters used to create the full factorial mooring system design matrix w – Mooring line diameters: three nominal diameter values of 0.09 m, 0.12 m, and 0.15 m were use – Mooring line headings: 72 combinations of mooring system line headings were used covering all possible heading combinations, with an increment of 10◦, that a three-line mooring system design can have. The minimum angle between any two mooring lines was 10◦. – Mooring line headings: 72 combinations of mooring system line headings were used covering all possible heading combinations, with an increment of 10◦, that a three-line mooring system design can have. The minimum angle between any two mooring lines was 10◦. – Anchoring radius: two anchoring radius values of 2.5D and 3.5D were used. The anchoring radius is the horizontal distance from the tower’s center to the anchor. 2.2 Wind farm layout optimization per wind direction The energy gain of the targeted layout compared to the optimized layout for each wind direction is shown in Fig. 11 and was equal to 6.1% energy gain at a constant wind speed of 10 m/s. The energy gain was calculated using equation 1: Figure 4 shows results of the targeted layout for only one wind direction; this process was repeated for each wind direction. 110 We can see that the turbine positions are shifted in the crosswind direction to decrease wake interactions. The energy gain of the targeted layout compared to the optimized layout for each wind direction is shown in Fig. 11 and was equal to 6.1% energy gain at a constant wind speed of 10 m/s. The energy gain was calculated using equation 1: 110 Egain = N X i=1 Ei −E0,i E0,total , (1) Egain = N X i=1 Ei −E0,i E0,total , (1) 6 https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. where Ei is the energy of the new layout (targeted layout) at a single wind direction i, and E0,i is the energy of the optimized 115 layout at a single wind direction i, E0,total is the total energy of the optimized layout for all wind directions N, which is equal to the number of wind directions in the wind rose in Fig. 2. The targeted layout is not realistic and is impossible to achieve, as we cannot freely move the FOWTs to the displacements in all wind directions. Realistically, our goal is to get the FOWTs to displace close to the targeted layout for as many wind directions as possible. However, the FOWT displacements are governed by the mooring system design, which cannot achieve the targeted displacement for all wind directions. 120 by the mooring system design, which cannot achieve the targeted displacement for all wind directions. 120 2.3 Mooring system database In reality, the effects of the FOWT’s motions on the rotor aerodynamics are not linear, but this simplification was enough, and the results in MoorPy 5 showed good agreement with OpenFAST as presented in Section 3.3. In MoorPy we iterated through all wind directions with an increment of 5◦and found the steady state position of each https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 5. The line lengths and anchoring radii used while creating the database. design. MoorPy is a quasi-static Python-based tool for mooring system analysis and design. In MoorPy a FOWT is modeled as a point mass with 6 DoFs and its hydrostatic properties. Additionally, an external force vector was applied to the FOWT at water level. This external force represented the aerodynamic thrust force vector at a wind speed of 10 m/s. We used coordinate transformation to transform the aerodynamic force from the FOWT’s hub to the water level. This meant that the aerodynamic forces applied at the water level were functions of the roll, pitch, and yaw motions of the platform. Also, the roll, pitch, and 150 yaw motions affect the aerodynamic force vector at the hub coordinate system, as these motions change the aerodynamic angle of attack and alter the aerodynamic force vector. To account for these effects we assumed that the aerodynamic forces change linearly with the changes in roll, pitch, and yaw DoFs. We used OpenFAST (Jonkman, 2009) to quantify the changes in the aerodynamic forces per one degree change in roll, pitch, and yaw motions for the 15 MW rotor. In reality, the effects of forces applied at the water level were functions of the roll, pitch, and yaw motions of the platform. Also, the roll, pitch, and 150 yaw motions affect the aerodynamic force vector at the hub coordinate system, as these motions change the aerodynamic angle of attack and alter the aerodynamic force vector. To account for these effects we assumed that the aerodynamic forces change linearly with the changes in roll, pitch, and yaw DoFs. We used OpenFAST (Jonkman, 2009) to quantify the changes in the aerodynamic forces per one degree change in roll, pitch, and yaw motions for the 15 MW rotor. 2.3 Mooring system database – Mooring line length: three values of the mooring line length as a function of the anchoring radius were used, as shown in equation 2. In equation 2, R is the horizontal distance between the fairlead position and the anchor position, Lmax is the maximum length a mooring line can have, and Lmin is the minimum length a chain mooring line can have. The three values of β used for the mooring system design matrix were 0.5, 0.7, and 0.9. The line profiles of the three values of β at the two anchoring radii are shown in Fig. 5. 140 Lmin = p depth2 + R2 (2a) Lmax = depth + R (2b) L = Lmin + β(Lmax −Lmin) 0 ≤β ≤1 (2c) (2a) (2b) (2b) n) 0 ≤β ≤1 (2c) (2c) After defining these permutable design parameters, we ended up with a full factorial design matrix containing 419,904 possible mooring system designs. We used MoorPy (Hall et al., 2021) to calculate the watch circle of each mooring system 145 7 Figure 5. The line lengths and anchoring radii used while creating the database. https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 5. The line lengths and anchoring radii used while creating the database. design. MoorPy is a quasi-static Python-based tool for mooring system analysis and design. In MoorPy a FOWT is modeled as a point mass with 6 DoFs and its hydrostatic properties. Additionally, an external force vector was applied to the FOWT at water level. This external force represented the aerodynamic thrust force vector at a wind speed of 10 m/s. We used coordinate transformation to transform the aerodynamic force from the FOWT’s hub to the water level. This meant that the aerodynamic forces applied at the water level were functions of the roll, pitch, and yaw motions of the platform. Also, the roll, pitch, and 0 yaw motions affect the aerodynamic force vector at the hub coordinate system, as these motions change the aerodynamic angle of attack and alter the aerodynamic force vector. To account for these effects we assumed that the aerodynamic forces change linearly with the changes in roll, pitch, and yaw DoFs. We used OpenFAST (Jonkman, 2009) to quantify the changes in the aerodynamic forces per one degree change in roll, pitch, and yaw motions for the 15 MW rotor. 2.4 Customized mooring system designs The design parameters of the nine customized mooring systems attached 185 to each FOWT in the FWF can be found in Table A1 in the appendix. The positions of the final self-adjusting FWF layout relative to the optimized wind farm layout for one direction are shown in Fig. 6, which shows how the crosswind movement of the FOWTs in the layout decreases wake interactions inside the FWF as Turbines 9 and 6 move out of the wake of Turbines 5 and 3, respectively. The energy gain of final FWF layout design compared to the optimized layout at each wind direction is shown in Fig. 11. As expected, the total energy gain of the final FWF is only 190 3.1% compared to 6.1% of the targeted layout, as the mooring system designs cannot achieve all crosswind displacements like the targeted wind farm layout design. We can see in Fig. 6 that turbines are displaced in the crosswind direction, and in-wind displacements are also visible, because the FOWTs are coupled to the mooring system designs. An overall view of the floating optimized wind farm layout with the mooring system attached to each FOWT is shown in Fig. 7. to the optimized layout at each wind direction is shown in Fig. 11. As expected, the total energy gain of the final FWF is only 190 3.1% compared to 6.1% of the targeted layout, as the mooring system designs cannot achieve all crosswind displacements like the targeted wind farm layout design. We can see in Fig. 6 that turbines are displaced in the crosswind direction, and in-wind displacements are also visible, because the FOWTs are coupled to the mooring system designs. An overall view of the floating optimized wind farm layout with the mooring system attached to each FOWT is shown in Fig. 7. 2.3 Mooring system database 170 – No vertical loads on the mooring system anchors. This means that all lines have catenary shapes with a section laying on the seabed for all wind directions. – No vertical loads on the mooring system anchors. This means that all lines have catenary shapes with a section laying on the seabed for all wind directions. After applying these constraints, we ended up with 440 mooring system designs that we used to choose the customized mooring system for each FOWT in the FWF. We presented the effect each mooring system design parameter has on the shape of the watch circle and the mooring system stiffness in our work in Mahfouz et al. (2022). 175 2.4 Customized mooring system designs After creating the mooring system database, we used the targeted layout design to choose mooring system with watch circles closest to the target displacements. This process was explained in detail in our work in Mahfouz and Cheng (2023). We used brute force optimization to find the mooring system design for each FOWT to increase the overall energy production of the i FWF. Afterward, we checked that the minimum distance between any two mooring lines within the FWF was larger than 20 180 m as required in the API (2005) standard. If the distance between two mooring lines was less than 20 m, we increased the minimum acceptable distance between any two turbines while designing the optimized wind farm layout in Section 2.1. In this work, we started with the constraint of minimum distance equal to 2D, and we iterated till the required minimum distance between any two lines was larger than 20 m. Therefore, we ended up with a constraint of minimum distance equal to 4D for the FWF. Afterward, we checked that the minimum distance between any two mooring lines within the FWF was larger than 20 180 m as required in the API (2005) standard. If the distance between two mooring lines was less than 20 m, we increased the minimum acceptable distance between any two turbines while designing the optimized wind farm layout in Section 2.1. In this work, we started with the constraint of minimum distance equal to 2D, and we iterated till the required minimum distance between any two lines was larger than 20 m. Therefore, we ended up with a constraint of minimum distance equal to 4D for the optimized layout design as introduced in Section 2.1. The design parameters of the nine customized mooring systems attached 185 to each FOWT in the FWF can be found in Table A1 in the appendix. The positions of the final self-adjusting FWF layout relative to the optimized wind farm layout for one direction are shown in Fig. 6, which shows how the crosswind movement of the FOWTs in the layout decreases wake interactions inside the FWF as Turbines 9 and 6 move out of the wake of Turbines 5 and 3, respectively. The energy gain of final FWF layout design compared optimized layout design as introduced in Section 2.1. 2.3 Mooring system database In reality, the effects of the FOWT’s motions on the rotor aerodynamics are not linear, but this simplification was enough, and the results in MoorPy 155 showed good agreement with OpenFAST as presented in Section 3.3. In MoorPy, we iterated through all wind directions with an increment of 5◦and found the steady-state position of each mooring system design for each wind direction. To account for the effect of FOWT motion on the aerodynamic force vector, we updated the aerodynamic force vector whenever the FOWT displaced until the solution converged. It is necessary to include the FOWT’s motions on the rotor aerodynamics are not linear, but this simplification was enough, and the results in MoorPy 155 showed good agreement with OpenFAST as presented in Section 3.3. In MoorPy, we iterated through all wind directions with an increment of 5◦and found the steady-state position of each the effects of FOWT rotations on the aerodynamic forces and in the coordinate transformation so the watch circles produced 160 in MoorPy will better match the results from fully coupled aero-elasto-servo-hydro simulations. While creating the mooring system database the wave loads were neglected, as their effect on the steady-state displacement of the FOWTs is negligible compared to the aerodynamic loading. After calculating the mooring system watch circles and displacements for each design in the design matrix, we checked that the designs passed the following constraints: 165 – The mooring system yaw angles are between −5◦and 5◦. We chose this constraint because a higher value will lead to high energy losses in the FWF due to yaw misalignment. We are not implementing any yaw control routines on a turbine – The mooring system yaw angles are between −5◦and 5◦. We chose this constraint because a higher value will lead to high energy losses in the FWF due to yaw misalignment. We are not implementing any yaw control routines on a turbine 8 https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. level or a farm level within this paper; hence, we decided to keep the mean yaw value within these limits to avoid yaw misalignment. – The mooring system cannot displace more than 1D in the wind direction. – The mooring system cannot displace more than 1D in the wind direction. annot displace more than 1D in the wind direction. 3 Design performance and verification 195 In this section we verify the initial results that we obtained using static wind loading and a steady-state wake model against dynamic wind and wave loading and a dynamic wake model. We use the mid-fidelity, state-of-the-art aero-servo-elasto-hydro tool OpenFAST v3.4.1 to model the FOWT and assess its behavior in operational and extreme conditions. Additionally, we use 9 9 Figure 6. Wind farm layout and wakes: (left) the optimized layout and (right) the final layout. The black dots represent the positions of the turbines in the optimized layout. https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 6. Wind farm layout and wakes: (left) the optimized layout and (right) the final layout. The black dots represent the positions of the turbines in the optimized layout. Figure 6. Wind farm layout and wakes: (left) the optimized layout and (right) the final layout. The black dots represent the positions of the turbines in the optimized layout. Figure 6. Wind farm layout and wakes: (left) the optimized layout and (right) the final layout. The black dots represent the positions of the turbines in the optimized layout. FAST.Farm v3.4.1 (Jonkman and Shaler, 2020) to model the FWF and assess how much energy gain relocating the FOWTs will achieve using the dynamic wake meandering (DWM) model. 00 FAST.Farm v3.4.1 (Jonkman and Shaler, 2020) to model the FWF and assess how much energy gain relocating the FOWTs will achieve using the dynamic wake meandering (DWM) model. 0 200 3.1 Introducing baseline floating wind farm design Before starting to assess and verify the novel FWF layout design performance, we introduce a baseline mooring system design that we use as a benchmark to compare the customized mooring system designs against. A baseline is needed to reflect the difference between the current state-of-the-art mooring system designs with small translational displacements (a few meters) and the customized mooring system designs that relocate the FOWTs more than 100 m. 205 and the customized mooring system designs that relocate the FOWTs more than 100 m. 205 The baseline mooring system design process assumed a fixed anchor spacing of 600 m, a water depth of 200 m, and three mooring lines at evenly distributed heading angles. The maximum platform offset was constrained to be 12% of water depth, about 25 m. The length and diameter of the chain were varied to meet constraints on extreme tensions and fatigue damage, with safety factors of 2 and 3, respectively. The fatigue damage was checked with a simplified fatigue analysis that consisted The baseline mooring system design process assumed a fixed anchor spacing of 600 m, a water depth of 200 m, and three mooring lines at evenly distributed heading angles. The maximum platform offset was constrained to be 12% of water depth, about 25 m. The length and diameter of the chain were varied to meet constraints on extreme tensions and fatigue damage, with safety factors of 2 and 3, respectively. The fatigue damage was checked with a simplified fatigue analysis that consisted of three load cases, with wind speeds near the rated wind speed and corresponding waves. The load cases assumed aligned 210 The baseline mooring system design process assumed a fixed anchor spacing of 600 m, a water depth of 200 m, and three mooring lines at evenly distributed heading angles. The maximum platform offset was constrained to be 12% of water depth, about 25 m. The length and diameter of the chain were varied to meet constraints on extreme tensions and fatigue damage, with safety factors of 2 and 3, respectively. The fatigue damage was checked with a simplified fatigue analysis that consisted of three load cases, with wind speeds near the rated wind speed and corresponding waves. The load cases assumed aligned 210 wind and waves, and uniform distribution of wind-wave headings. The API (API, 2005) fatigue analysis process was followed, using the T-N curve for chain. 3.1 Introducing baseline floating wind farm design The video shows a top view of the wind farm layout and the mooring systems and how the FOWTs move passively as the wind direction changes. 215 3.1 Introducing baseline floating wind farm design Fatigue was the driving constraint in the design of the baseline mooring system, resulting in a chain diameter of 0 19 m The properties of the baseline mooring design are shown in Table 2 y , p y g g pi g y of three load cases, with wind speeds near the rated wind speed and corresponding waves. The load cases assumed aligned 210 wind and waves, and uniform distribution of wind-wave headings. The API (API, 2005) fatigue analysis process was followed, using the T-N curve for chain. Fatigue was the driving constraint in the design of the baseline mooring system, resulting in a chain diameter of 0.19 m. The properties of the baseline mooring design are shown in Table 2. Table 2. Parameters of baseline mooring design # of Lines Depth Anchoring radius Fairlead radius Fairlead depth Line type Line length Line diameter (m) (m) (m) (m) (m) (m) 3 200 600 42.5 15 Chain 623.3 0.1902 10 Figure 7. The optimized wind farm layout coupled to (left) the baseline mooring system and (right) the customized mooring system. https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 7. The optimized wind farm layout coupled to (left) the baseline mooring system and (right) the customized mooring system. Figure 7. The optimized wind farm layout coupled to (left) the baseline mooring system and (right) the customized mooring system. The optimized wind farm layout coupled to the baseline mooring system and the customized mooring system is shown in Fig The optimized wind farm layout coupled to the baseline mooring system and the customized m The optimized wind farm layout coupled to the baseline mooring system and the customized mooring system is shown in Fig 7 Both layouts are shown in the absence of wind and wave loading Mahfouz (2023) presents an animation video illustrating 7. Both layouts are shown in the absence of wind and wave loading. Mahfouz (2023) presents an animation video illustrating 215 the passive displacements of the FOWTs according to the wind direction. The video shows a top view of the wind farm layout and the mooring systems and how the FOWTs move passively as the wind direction changes. 7. Both layouts are shown in the absence of wind and wave loading. Mahfouz (2023) presents an animation video illustrating 215 the passive displacements of the FOWTs according to the wind direction. 3.2 Customized mooring system stiffness We compared the natural frequency of FOWTs when coupled to the customized mooring system designs versus the baseline design. We used MoorPy to calculate the stiffness of the mooring system at each wind speed and wind direction to cover the 220 FOWT operation range between cut-in and cut-out wind speeds. Then, we used the results to calculate the natural frequencies of the system over the watch circles of the mooring systems. The results for the baseline mooring system and the customized mooring system design of the first turbine (T1) in the FWF are shown in Figs. 8, and 9, respectively. The figures show the value of the natural frequencies in the x-axis and y-axis directions. Each dotted line in the plots represents the watch circle at one wind speed; the most inner line is the watch circle at a wind speed of 3 m/s, and the outermost line is the watch circle at rated 225 wind speed of 11 m/s. The color of the plots represents the value of the natural frequency over the different positions of the watch circle. In Fig. 8, the natural frequency of the baseline design does not change as the wind speed and wind direction changes. This means that for all wind excitations and for all positions inside the watch circle, the natural frequency is constant. This is because the stiffness of the baseline design is linear and almost constant for all wind speeds and wind directions. On the other hand, Fig. 230 9 shows that the natural frequencies of the customized mooring systems are rather spread; the frequencies are low at low wind speeds and increase to be within the same frequency range of the baseline mooring system design as the wind speed increases. This is because the stiffness of the customized mooring system design is nonlinear and changes with the wind speeds and the the stiffness of the baseline design is linear and almost constant for all wind speeds and wind directions. On the other hand, Fig. 230 9 shows that the natural frequencies of the customized mooring systems are rather spread; the frequencies are low at low wind speeds and increase to be within the same frequency range of the baseline mooring system design as the wind speed increases. 3.2 Customized mooring system stiffness This is because the stiffness of the customized mooring system design is nonlinear and changes with the wind speeds and the 11 Figure 8. The natural frequency in x and y directions for the FOWT when coupled to the baseline mooring system. The dotted lines represent the watch circles at wind speeds ranging from 3 m/s to 11 m/s. https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 8. The natural frequency in x and y directions for the FOWT when coupled to the baseline mooring system. The dotted lines represent the watch circles at wind speeds ranging from 3 m/s to 11 m/s. Figure 9. The natural frequency in x and y directions for the FOWT when coupled to the customized mooring system of Turbine 1. The dotted lines represent the watch circles at wind speeds ranging from 3 m/s to 11 m/s. Figure 9. The natural frequency in x and y directions for the FOWT when coupled to the customized mooring system of Turbine 1. The dotted lines represent the watch circles at wind speeds ranging from 3 m/s to 11 m/s. wind directions. In general, the frequencies of the customized mooring system are lower than the baseline design because their stiffness values are lower to allow larger mean FOWT translational displacements. 5 wind directions. In general, the frequencies of the customized mooring system are lower than the baseline design because their stiffness values are lower to allow larger mean FOWT translational displacements. 235 3.3 Steady-state behavior The reason for this difference is that OpenFAST models the full structure of the FOWT, captures the tower top deflections, and includes them while transforming the aerodynamic forces from the tower top coordinate system to the water level coordinate system. On the other hand, the MoorPy model is a simplified model and does not account for structural deflections; therefore, the forces applied at the water level are different between the two models. Moreover, we assumed in MoorPy that the aerodynamic force vector at the hub height changes linearly with the FOWT’s roll, pitch, and yaw angle, 250 which is different than OpenFAST, which recalculates the aerodynamic forces using blade element momentum theory. MoorPy that the aerodynamic force vector at the hub height changes linearly with the FOWT’s roll, pitch, and yaw angle, 250 which is different than OpenFAST, which recalculates the aerodynamic forces using blade element momentum theory. 3.3 Steady-state behavior Before applying any dynamic loads on the FOWTs, we verified that the mean steady-state displacements calculated with MoorPy were equal to the steady-state displacements in OpenFAST. This comparison was important before running FAST.Farm simulations to quantify the energy gain of displacing the FOWTs in the FWF and comparing it to the results of the static model. 12 Figure 10. (left) The watch circle of Turbine 1 in both MoorPy and OpenFAST. (right) The maximum difference between the watch circle of each turbine in the FWF in MoorPy and OpenFAST. https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 10. (left) The watch circle of Turbine 1 in both MoorPy and OpenFAST. (right) The maximum difference between the watch circle of each turbine in the FWF in MoorPy and OpenFAST. If the mean displacements of the two models are different, it will lead to a different layout for every wind direction, and we 240 cannot directly compare the energy gain of the two models. Hence, we applied a steady wind of 10 m/s at the rotor hub using OpenFAST; then, we compared the steady-state positions between the two models in Fig. 10. The figure shows the comparison of the watch circle of Turbine 1 in the FWF in both tools on the left side and the maximum difference between the watch circles produced by MoorPy and OpenFAST. The biggest difference in displacement between the two models is around 3.3 m for Turbine 6. This difference is negligible compared to the 240 m rotor diameter of the 15 MW reference 245 wind turbine. The reason for this difference is that OpenFAST models the full structure of the FOWT, captures the tower top deflections, and includes them while transforming the aerodynamic forces from the tower top coordinate system to the water level coordinate system. On the other hand, the MoorPy model is a simplified model and does not account for structural deflections; therefore, the forces applied at the water level are different between the two models. Moreover, we assumed in around 3.3 m for Turbine 6. This difference is negligible compared to the 240 m rotor diameter of the 15 MW reference 245 wind turbine. 3.4 Energy production Additionally, the simulations started with the FOWT positions set according to the values obtained from MoorPy to avoid big transient movements. The polar formulation of the DWM in FAST.Farm was used with the default settings of the wake model. The default settings were calibrated and verified by Doubrawa et al. (2018) and Jonkman et al. (2018) against large-eddy simulations for a fixed-bottom 270 time step was 0.42 s, and the OpenFAST time step was set to 0.0378 s. The significant wave height for all simulations was equal 265 to 2 m, and the wave period was equal to 6 s. The second-order wave forces were not considered in any of these simulations. The simulation time was 4200 s, and the first 600 s were excluded to avoid transient effects. Additionally, the simulations started with the FOWT positions set according to the values obtained from MoorPy to avoid big transient movements. The polar formulation of the DWM in FAST.Farm was used with the default settings of the wake model. The default settings were calibrated and verified by Doubrawa et al. (2018) and Jonkman et al. (2018) against large-eddy simulations for a fixed-bottom 270 5 MW wind turbine. FAST.Farm wake parameters are not yet calibrated for FOWTs. Figure 11. (left) The energy gain at each wind direction for the targeted wind farm layout, (center) the final wind farm layout using static wake models, and (right) the final wind farm layout using dynamic wake models. Figure 11. (left) The energy gain at each wind direction for the targeted wind farm layout, (center) the final wind farm layout using static wake models, and (right) the final wind farm layout using dynamic wake models. After omitting the first 600 s, the final 3600 s of each simulation was processed. The average power produced by each turbine in both FWF layouts (with baseline mooring system and with the customized mooring system designs) was calculated. The energy gain of the optimized layout coupled to the customized mooring system was calcula The energy gain of the optimized layout coupled to the customized mooring system was calculated relative to the optimized layout when coupled to the baseline mooring system as in equation 1. The gain from FAST.Farm was equal to 1.4%, and the 275 gain for every wind direction in the wind rose is shown on the right side in Fig. 11. 3.4 Energy production For all simulations, the resolution of the high-resolution grid in FAST.Farm in the x, y, and z directions was equal to 5 m, and the low-resolution grid’s resolution was set to 35 m. The low-resolution time step was set to 3.78 s, while the high-resolution systems, were identical for all wind directions. 260 The wind fields were generated using the Mann turbulence box (Mann, 1998). The Mann model parameters were defined following the IEC (2019) standard to achieve a turbulence intensity of 6%. The mean wind speed at the hub was equal to 10 m/s. For all simulations, the resolution of the high-resolution grid in FAST.Farm in the x, y, and z directions was equal to 5 m, and the low-resolution grid’s resolution was set to 35 m. The low-resolution time step was set to 3.78 s, while the high-resolution For all simulations, the resolution of the high-resolution grid in FAST.Farm in the x, y, and z directions was equal to 5 m, and the low-resolution grid’s resolution was set to 35 m. The low-resolution time step was set to 3.78 s, while the high-resolution time step was 0.42 s, and the OpenFAST time step was set to 0.0378 s. The significant wave height for all simulations was equal 265 to 2 m, and the wave period was equal to 6 s. The second-order wave forces were not considered in any of these simulations. The simulation time was 4200 s, and the first 600 s were excluded to avoid transient effects. Additionally, the simulations started with the FOWT positions set according to the values obtained from MoorPy to avoid big transient movements. The polar formulation of the DWM in FAST.Farm was used with the default settings of the wake model. The default settings were calibrated and verified by Doubrawa et al. (2018) and Jonkman et al. (2018) against large-eddy simulations for a fixed-bottom 270 5 MW wind turbine. FAST.Farm wake parameters are not yet calibrated for FOWTs. time step was 0.42 s, and the OpenFAST time step was set to 0.0378 s. The significant wave height for all simulations was equal 265 to 2 m, and the wave period was equal to 6 s. The second-order wave forces were not considered in any of these simulations. The simulation time was 4200 s, and the first 600 s were excluded to avoid transient effects. 3.4 Energy production In this section, our goal is to compare the FWF energy gain that we calculated using the steady-state Gaussian wake model in FLORIS with the results of the mid-fidelity DWM from FAST.Farm. FAST.Farm uses OpenFAST to account for the aero-elasto- servo-hydro dynamics of each FOWT in the FWF and the DWM model to account for wake deficits, advection, deflection, 255 In this section, our goal is to compare the FWF energy gain that we calculated using the steady-state Gaussian wake model in FLORIS with the results of the mid-fidelity DWM from FAST.Farm. FAST.Farm uses OpenFAST to account for the aero-elasto- FLORIS with the results of the mid-fidelity DWM from FAST.Farm. FAST.Farm uses OpenFAST to account for the aero-elasto- servo-hydro dynamics of each FOWT in the FWF and the DWM model to account for wake deficits, advection, deflection, 255 meandering, and merging. We simulated the optimized wind farm layout twice at every wind direction of the wind rose in Fig. 2—first with all the FOWTs in the FWF coupled to the baseline mooring system design, and second with each FOWT coupled to its customized mooring system design. Our wind rose has 16 wind directions, resulting in 32 FAST.Farm simulations. All servo-hydro dynamics of each FOWT in the FWF and the DWM model to account for wake deficits, advection, deflection, 255 meandering, and merging. We simulated the optimized wind farm layout twice at every wind direction of the wind rose in Fig. 2—first with all the FOWTs in the FWF coupled to the baseline mooring system design, and second with each FOWT coupled to its customized mooring system design. Our wind rose has 16 wind directions, resulting in 32 FAST.Farm simulations. All 13 https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. simulations settings and the wind fields for both cases, with the baseline mooring system and with the customized mooring systems, were identical for all wind directions. 260 simulations settings and the wind fields for both cases, with the baseline mooring system and with the customized mooring systems, were identical for all wind directions. 260 i systems, were identical for all wind directions. 260 The wind fields were generated using the Mann turbulence box (Mann, 1998). The Mann model parameters were defined following the IEC (2019) standard to achieve a turbulence intensity of 6%. The mean wind speed at the hub was equal to 10 m/s. 3.4 Energy production The energy gain distribution per wind direction in the FAST.Farm model follows the same trend as the gain distribution for the MoorPy-FLORIS results. However, the gain values predicted by FAST.Farm are smaller than what the steady-state models predicted. The difference between the MoorPy-FLORIS static model and FAST.Farm model can be caused by two reasons. First, the FOWTs are not being displaced by the customized mooring system designs as we predict in the design phase. However, this possibility is omitted because as 280 by the customized mooring system designs as we predict in the design phase. However, this possibility is omitted because as 280 14 https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. we presented in Section 3.3, the FOWTs relocate their positions in OpenFAST and MoorPy similarly and the difference is negligible. The second source of difference between the MoorPy-FLORIS model and the FAST.Farm model comes from the difference between the wake models implemented in FLORIS and FAST.Farm, as well as the difference in fidelity between MoorPy-FLORIS and FAST.Farm. First, FLORIS is a steady-state model that does not account for the FOWT’s mean pitch angle, which means that the rotor average wind speeds in FLORIS and OpenFAST are different. Moreover, Ramos-García et al. 285 (2022b, a), Johlas (2021), and Nanos et al. (2022) showed that the positive pitch angle of FOWTs deflects the wake upward. This deflection is captured in FAST.Farm but not in FLORIS. Therefore, we analyzed the difference between the wind speed deficit in FLORIS and FAST.Farm to understand the difference between the models. Since the main energy gain happens when the wind is blowing from west to east (wind direction = 0◦), we used this wind direction during this analysis. we presented in Section 3.3, the FOWTs relocate their positions in OpenFAST and MoorPy similarly and the difference is negligible. The second source of difference between the MoorPy-FLORIS model and the FAST.Farm model comes from the difference between the wake models implemented in FLORIS and FAST.Farm, as well as the difference in fidelity between MoorPy-FLORIS and FAST.Farm. First, FLORIS is a steady-state model that does not account for the FOWT’s mean pitch angle, which means that the rotor average wind speeds in FLORIS and OpenFAST are different. Moreover, Ramos-García et al. 285 (2022b, a), Johlas (2021), and Nanos et al. 3.4 Energy production (2022) showed that the positive pitch angle of FOWTs deflects the wake upward. This deflection is captured in FAST.Farm but not in FLORIS. Therefore, we analyzed the difference between the wind speed deficit in FLORIS and FAST.Farm to understand the difference between the models. Since the main energy gain happens when the wind is blowing from west to east (wind direction = 0◦), we used this wind direction during this analysis. 285 Figure 12. The wind flow field at the rotor hub at 4200 s. (left) The non-relocating optimized wind farm layout coupled to the baseline mooring system design compared to (right) the self-relocating optimized layout at wind direction = 0◦. Figure 12. The wind flow field at the rotor hub at 4200 s. (left) The non-relocating optimized wind farm layout coupled to the baseline mooring system design compared to (right) the self-relocating optimized layout at wind direction = 0◦. Figure 12. The wind flow field at the rotor hub at 4200 s. (left) The non-relocating optimized wind farm layout coupled to the baseline mooring system design compared to (right) the self-relocating optimized layout at wind direction = 0◦. The wind flow field at the rotor hub from FAST.Farm is presented in Fig. 12—the flow field of the optimized wind farm layout 290 with the baseline mooring system on the left and the self-relocating layout on the right. Figure 13 shows the rotor average wind speed in FLORIS and in FAST.Farm for a wind direction of 0◦. On the left, all FOWTs are coupled to the baseline mooring system with limited movement in the crosswind direction; in the middle, each FOWT is attached to its customized mooring system design, allowing crosswind movements; and on the right is the difference in rotor average wind speed of each turbine. 290 Turbines 7 and 9 are relocated out of the wake, and their rotor average wind speeds are higher, as shown in Fig. 13. From Fig. 295 13 we can conclude the following: – The rotor average wind speed of a FOWT is always slightly lower in FAST.Farm when compared to FLORIS, because FAST.Farm considers the pitch, roll, and yaw motions while FLORIS cannot capture them. 3.4 Energy production For an inflow wind field with mean wind speed of 10 m/s, the average rotor wind speed of FOWTs in FAST.Farm is 9.5 m/s, instead of 10.0 m/s in FLORIS 300 mean wind speed of 10 m/s, the average rotor wind speed of FOWTs in FAST.Farm is 9.5 m/s, instead of 10.0 m/s in FLORIS. 300 FLORIS. 300 15 https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. – In FLORIS, Turbine 4 is partially in the wake of Turbine 1 in the baseline floating optimized layout, while in FAST.Farm this is not the case. This can be caused by wake meandering, which is considered in the FAST.Farm model and not in FLORIS, so the wake is deflected and Turbine 4 is no longer in the wake of Turbine 1. It can also be caused by horizontal wake deflection, as the platform yaw angle of Turbine 1 will deflect the wake away from Turbine 4. However, this reason is omitted, as the FOWT yaw angle of Turbine 1 is almost zero. – In FLORIS, Turbine 4 is partially in the wake of Turbine 1 in the baseline floating optimized layout, while in FAST.Farm this is not the case. This can be caused by wake meandering, which is considered in the FAST.Farm model and not in FLORIS, so the wake is deflected and Turbine 4 is no longer in the wake of Turbine 1. It can also be caused by horizontal wake deflection, as the platform yaw angle of Turbine 1 will deflect the wake away from Turbine 4. However, this reason is omitted, as the FOWT yaw angle of Turbine 1 is almost zero. 305 – Looking at the change in rotor average wind speeds between the optimized layout when attached to the customized mooring system designs and the baseline mooring system design, we can see that there are small differences in the rotor average wind speeds for the turbines in the free flow wind fields (such as Turbines 1, 2, 3, and 6). These differences come from the difference in the mean pitch angle of the FOWTs affected by the mooring system design overhang. The difference in the mooring system designs has a slight effect on the floating platform’s pitch stiffness and affects the steady-state pitch angles of FOWTs. 310 – The wake losses in FAST.Farm are less than in FLORIS. 3.4 Energy production This is clear from the rotor average wind speed of Turbine 7 in both FAST.Farm and FLORIS. In Fig. 13, for the optimized layout with the baseline mooring system, the average rotor wind speed of Turbine 7 in the FLORIS model is lower than the FAST.Farm model, which indicates that the wind speed deficit is higher in FLORIS and hence there are higher losses. After the optimized layout is modelled with the customized mooring system designs and the turbines are relocated, the wind speed increases in both models, proving that relocating the layout decreases the wake losses for Turbine 7. The value of the rotor average wind speed of Turbine 7 after the FOWTs are relocated is slightly higher in the FAST.Farm model. However, the total increase in FLORIS is higher, and hence the energy gain of relocating Turbine 7 is also higher. This was seen for all the turbines in the wake for all wind directions. 320 These four differences between the FAST.Farm and the FLORIS models lead to the difference in the energy gain of relocating the wind turbines. The first three differences are expected, as they result from the lack of turbine structural modelling in FLORIS or the simplicity of the steady wake model compared to the DWM. However, the big difference in velocity deficit of the two models requires further analysis. To understand why FLORIS estimates higher wake losses than FAST.Farm and if this difference is because of the effect of FOWTs on wake recovery and wake deflection, we decided to simulate the optimized 325 wind farm layout as a fixed-bottom wind farm and compare it to the optimized layout when coupled to the baseline mooring system design. The results of the average rotor wind speed of the fixed-bottom optimized layout in FAST.Farm are shown in Fig. 14. Figure 14 shows that the FAST.Farm rotor average wind speeds of the turbines in ambient wind (Turbines 1, 2, and 3) for this difference is because of the effect of FOWTs on wake recovery and wake deflection, we decided to simulate the optimized 325 wind farm layout as a fixed-bottom wind farm and compare it to the optimized layout when coupled to the baseline mooring system design. The results of the average rotor wind speed of the fixed-bottom optimized layout in FAST.Farm are shown in Fig. 14. 3.4 Energy production In general, further work is needed to verify and calibrate the mid-fidelity DWM model in FAST.Farm with higher fidelity models to capture the effects of FOWTs on the wake with more certainty. Turbines 2 and 3. The main reason for the faster wake recovery at the hub height is the vertical deflection of the wakes. 345 Another reason is the motions of the FOWTs, which increase the turbulence of the flow field and hence increase the wake mixing and accelerate the wake recovery. In general, further work is needed to verify and calibrate the mid-fidelity DWM model in FAST.Farm with higher fidelity models to capture the effects of FOWTs on the wake with more certainty. 3.4 Energy production This proves that the difference in velocity 335 deficit is due to the motions of FOWTs and their effect on the wake rather than to the different fidelity of the steady wake model in FLORIS and the DWM in FAST.Farm. m for a FOWT. This means that the FOWT’s wake is deflected 41 m more compared to that of a fixed-bottom turbine, which 340 is equivalent to 17%D. This agrees with the results found in Ramos-García et al. (2023), which indicated that FAST.Farm results showed larger vertical wake deflection when compared to the higher fidelity wake model presented by Ramos-García et al. (2022b). Moreover, Fig. 15 shows the horizontal wind field of the wind farm at the hub height, which shows that the wake recovery is faster inside an FWF compared to a fixed-bottom wind farm. This is clear when looking at the wakes behind m for a FOWT. This means that the FOWT’s wake is deflected 41 m more compared to that of a fixed-bottom turbine, which 340 is equivalent to 17%D. This agrees with the results found in Ramos-García et al. (2023), which indicated that FAST.Farm results showed larger vertical wake deflection when compared to the higher fidelity wake model presented by Ramos-García et al. (2022b). Moreover, Fig. 15 shows the horizontal wind field of the wind farm at the hub height, which shows that the wake recovery is faster inside an FWF compared to a fixed-bottom wind farm. This is clear when looking at the wakes behind Turbines 2 and 3. The main reason for the faster wake recovery at the hub height is the vertical deflection of the wakes. 345 Another reason is the motions of the FOWTs, which increase the turbulence of the flow field and hence increase the wake mixing and accelerate the wake recovery. In general, further work is needed to verify and calibrate the mid-fidelity DWM model in FAST.Farm with higher fidelity models to capture the effects of FOWTs on the wake with more certainty. Turbines 2 and 3. The main reason for the faster wake recovery at the hub height is the vertical deflection of the wakes. 345 Another reason is the motions of the FOWTs, which increase the turbulence of the flow field and hence increase the wake mixing and accelerate the wake recovery. 3.4 Energy production This proves that the difference in velocity 335 deficit is due to the motions of FOWTs and their effect on the wake rather than to the different fidelity of the steady wake model in FLORIS and the DWM in FAST.Farm. Figure 15 shows that the wake center of Turbine 4 is deflected upward when it is floating compared to when it is fixed. After a distance of 6D, the wake center of Turbine 4 is at a height of 193 m for a fixed-bottom turbine and a height of 234 deficit of the floating optimized layout with the baseline mooring system design. This proves that the difference in velocity 335 deficit is due to the motions of FOWTs and their effect on the wake rather than to the different fidelity of the steady wake model in FLORIS and the DWM in FAST.Farm. Fi 15 h th t th k t f T bi 4 i d fl t d d h it i fl ti d t h it i fi d deficit of the floating optimized layout with the baseline mooring system design. This proves that the difference in velocity 335 deficit is due to the motions of FOWTs and their effect on the wake rather than to the different fidelity of the steady wake model in FLORIS and the DWM in FAST.Farm. Figure 15 shows that the wake center of Turbine 4 is deflected upward when it is floating compared to when it is fixed. After a distance of 6D, the wake center of Turbine 4 is at a height of 193 m for a fixed-bottom turbine and a height of 234 deficit of the floating optimized layout with the baseline mooring system design. This proves that the difference in velocity 335 deficit is due to the motions of FOWTs and their effect on the wake rather than to the different fidelity of the steady wake model in FLORIS and the DWM in FAST.Farm. Figure 15 shows that the wake center of Turbine 4 is deflected upward when it is floating compared to when it is fixed. After a distance of 6D, the wake center of Turbine 4 is at a height of 193 m for a fixed-bottom turbine and a height of 234li deficit of the floating optimized layout with the baseline mooring system design. 3.4 Energy production Fi 14 h h h FAST F i d d f h bi i bi i d (T bi 1 2 d 3) f Figure 14 shows that the FAST.Farm rotor average wind speeds of the turbines in ambient wind (Turbines 1, 2, and 3) for the fixed-bottom optimized layout are higher than the floating optimized layout but still lower compared to FLORIS. This 330 difference comes from the rotor uptilt angle of the turbines. This uptilt angle is only accounted for in FLORIS in the power coefficients look-up table, and it does not show in the rotor average wind speeds. This means that for a the fixed-bottom turbine, the mean power produced in the ambient wind field in FLORIS and FAST.Farm is equal. Additionally, Fig. 14 shows that for the fixed-bottom optimized layout, the velocity deficit for Turbine 7 in FAST.Farm is equal to FLORIS and is higher than the 16 Figure 13. The rotor average wind speed in FLORIS and FAST.Farm at wind direction = 0◦. (left) The rotor average wind speed of the optimized wind farm layout coupled to the baseline mooring system. (center) The rotor average wind speed of the optimized layout coupled to the customized mooring system design. (right) The difference between the average rotor wind speed of the two configurations. https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 13. The rotor average wind speed in FLORIS and FAST.Farm at wind direction = 0◦. (left) The rotor average wind speed of the optimized wind farm layout coupled to the baseline mooring system. (center) The rotor average wind speed of the optimized layout coupled to the customized mooring system design. (right) The difference between the average rotor wind speed of the two configurations. Figure 13. The rotor average wind speed in FLORIS and FAST.Farm at wind direction = 0◦. (left) The rotor average wind speed of the optimized wind farm layout coupled to the baseline mooring system. (center) The rotor average wind speed of the optimized layout coupled to the customized mooring system design. (right) The difference between the average rotor wind speed of the two configurations. deficit of the floating optimized layout with the baseline mooring system design. 3.5 Performance in normal operation The fatigue loads of the mooring lines were assessed at all wind directions with a step of 10◦ a turbulence intensity of 6%. The fatigue loads of the mooring lines were assessed at all wind directions with a step of 10◦ leading to 36 wind directions. The significant wave height for all simulations was set to 2 m, and the wave period was set to 355 6 s. The wind and wave headings were aligned for all wind directions. The second-order wave loads were considered for all directions, and one wave seed and wind seed were used for each wind direction. We chose to do the fatigue analysis at the rated wind speed, as from our experience, this is were the fatigue loads are the highest. In this work, we focused on estimating the mooring line fatigue and did not check the effect of relocating the turbines on the blades and tower fatigue loads. Moreover, we did not check the wake effect on the mooring system fatigue loads, as it is out of the scope of this work. 360 leading to 36 wind directions. The significant wave height for all simulations was set to 2 m, and the wave period was set to 355 6 s. The wind and wave headings were aligned for all wind directions. The second-order wave loads were considered for all directions, and one wave seed and wind seed were used for each wind direction. We chose to do the fatigue analysis at the rated wind speed, as from our experience, this is were the fatigue loads are the highest. In this work, we focused on estimating the mooring line fatigue and did not check the effect of relocating the turbines on the blades and tower fatigue loads. Moreover, Figure 16. 1 Hz DEL of the baseline mooring system design at each wind direction Figure 17. 1 Hz DEL of the customized mooring system design of Turbine 1 in the FWF at each wind direction Figure 16. 1 Hz DEL of the baseline mooring system design at each wind direction Figure 16. 1 Hz DEL of the baseline mooring system design at each wind direction Figure 17. 1 Hz DEL of the customized mooring system design of Turbine 1 in the FWF at each wind direction gure 17. 3.5 Performance in normal operation To check if relocating the FOWTs increases the fatigue loads of the customized mooring system designs compared to the 350 baseline mooring system, we calculated the damage equivalent loads (DELs) and the 25-year fatigue damage at rated wind 17 Figure 14. The rotor average wind speed of the optimized wind farm layout as fixed bottom and coupled to the baseline mooring system in FAST.Farm and FLORIS https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 14. The rotor average wind speed of the optimized wind farm layout as fixed bottom and coupled to the baseline mooring system in FAST.Farm and FLORIS Figure 14. The rotor average wind speed of the optimized wind farm layout as fixed bottom and coupled to the baseline mooring system in FAST Farm and FLORIS Figure 14. The rotor average wind speed of the optimized wind farm layout as fixed bottom and coupled to the baseline mooring system in FAST.Farm and FLORIS FAST.Farm and FLORIS Figure 15. FAST.Farm results of (top) the optimized wind farm layout as fixed bottom and (bottom) the optimized wind farm layout coupled with the baseline. The left side represents the horizontal plane at hub height; the right side is the vertical plane passing through the hub of Turbine 7. Figure 15. FAST.Farm results of (top) the optimized wind farm layout as fixed bottom and (bottom) the optimized wind farm layout coupled with the baseline. The left side represents the horizontal plane at hub height; the right side is the vertical plane passing through the hub of Turbine 7. speed of 11 m/s. OpenFAST simulations of 4200 s were done for each of the nine turbines within the FWF. The wind field was generated using the Mann turbulence generator, and its parameters were defined following the IEC (2019) standard to achieve speed of 11 m/s. OpenFAST simulations of 4200 s were done for each of the nine turbines within the FWF. The wind field was generated using the Mann turbulence generator, and its parameters were defined following the IEC (2019) standard to achieve 18 https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. a turbulence intensity of 6%. The fatigue loads of the mooring lines were assessed at all w a turbulence intensity of 6%. 3.5 Performance in normal operation 1 Hz DEL of the customized mooring system design of Turbine 1 in the FWF at each wind direction 19 DEL = ( j X i=1 Ni Tsim Sm i )1/m (3) The 1 Hz DELs were calculated using equation 3, with a Wohler exponent m = 3, and the Tsim equal to 3600 because the first 600 s from each simulation were omitted to decrease transients effects. The rainflow counting method was used to count the load cycles for the mooring line fairlead tensions. The DELs of the three mooring lines of the baseline mooring system for each wind direction are shown in Fig. 16. The maximum DEL of each of the three lines is when the wind direction is aligned 365 with the line, as this line will be carrying the highest tension. For example, when the wind direction is from east to west, Line 2 of the baseline mooring system in Fig. 16 will have the highest DEL value. Moreover, since the three lines in the baseline mooring system are identical and mooring system is symmetrical, the DELs of the lines are also symmetrical. In Fig. 17 the DELs of the lines of the customized mooring system attached to Turbine 1 can be seen. Similar to the baseline mooring system, each wind direction are shown in Fig. 16. The maximum DEL of each of the three lines is when the wind direction is aligned 365 with the line, as this line will be carrying the highest tension. For example, when the wind direction is from east to west, Line 2 of the baseline mooring system in Fig. 16 will have the highest DEL value. Moreover, since the three lines in the baseline mooring system are identical and mooring system is symmetrical, the DELs of the lines are also symmetrical. In Fig. 17 the DELs of the lines of the customized mooring system attached to Turbine 1 can be seen. Similar to the baseline mooring system, the DELs are highest when the wind is aligned with a mooring line. However, since each mooring system is asymmetric, and 370 the mooring lines are not identical, the values of the DELs of each line differ. In a wind farm design, we can benefit from the fact that the highest DELs happen when one of the lines is aligned with the wind direction to decrease fatigue loads. 3.5 Performance in normal operation The FOWTs are offset to a mean displacement under the thrust force of the wind turbine. Then, waves act on the mooring system causing oscillations in position and tension about the mean. The stiffness, or the slope of the tension-displacement response, is representative of the amplitude fatigue damage with a significantly smaller line diameter. 390 The fatigue performance of the mooring system relates to the mooring system stiffness. The stiffness at a given position is equal to the slope of the force-displacement response of the mooring system at that offset. The FOWTs are offset to a mean displacement under the thrust force of the wind turbine. Then, waves act on the mooring system causing oscillations in position and tension about the mean. The stiffness, or the slope of the tension-displacement response, is representative of the amplitudel fatigue damage with a significantly smaller line diameter. 390 The fatigue performance of the mooring system relates to the mooring system stiffness. The stiffness at a given position is equal to the slope of the force-displacement response of the mooring system at that offset. The FOWTs are offset to a mean displacement under the thrust force of the wind turbine. Then, waves act on the mooring system causing oscillations in position and tension about the mean. The stiffness, or the slope of the tension-displacement response, is representative of the amplitude of wave-frequency tension fluctuations about the mean. The customized mooring system designs have much lower stiffness 395 overall compared to the baseline, resulting in lower tension amplitudes and resulting fatigue damage. of wave-frequency tension fluctuations about the mean. The customized mooring system designs have much lower stiffness 395 overall compared to the baseline, resulting in lower tension amplitudes and resulting fatigue damage. of wave-frequency tension fluctuations about the mean. The customized mooring system designs have much lower stiffness 395 overall compared to the baseline, resulting in lower tension amplitudes and resulting fatigue damage. 3.5 Performance in normal operation This can be achieved by avoiding having any of the mooring lines aligned with the most probable wind directions. the DELs are highest when the wind is aligned with a mooring line. However, since each mooring system is asymmetric, and 370 the mooring lines are not identical, the values of the DELs of each line differ. In a wind farm design, we can benefit from the fact that the highest DELs happen when one of the lines is aligned with the wind direction to decrease fatigue loads. This can be achieved by avoiding having any of the mooring lines aligned with the most probable wind directions. Figure 18. 25-year damage for all mooring system designs Figure 18. 25-year damage for all mooring system designs N = k( T MBL)1/m (4) (4) MBL = 30.2D2(44 −80d) [MN] 375 (5) (5) 20 https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. We calculated the 25-year damage using the T-N curve for studless chain mooring lines as defined within the API (2005) standard in equation 4, with slope m = 3 and intercept k = 316. T is the fairlead tension of the mooring lines, and N represents the cycles to failures. The minimum breaking load (MBL) is a function of the mooring line diameter d as in equation 5. For the damage calculations, we assumed that for the 25-year time, the wind speed is always at 11 m/s, and no other wind speeds where considered. This is enough for the purpose of the paper comparing the customized mooring system fatigue to the baseline 380 mooring system fatigue, but it does not show whether or not the mooring system designs survive a 25-year lifetime. As each i t h diff t h di d h diff t DEL t h i d di ti d id d t th ff t f We calculated the 25-year damage using the T-N curve for studless chain mooring lines as defined within the API (2005) standard in equation 4, with slope m = 3 and intercept k = 316. T is the fairlead tension of the mooring lines, and N represents the cycles to failures. The minimum breaking load (MBL) is a function of the mooring line diameter d as in equation 5. 3.6 Performance in extreme conditions The ability of the customized mooring system designs to withstand extreme loads was also checked. We applied extreme 50- year wind and wave conditions with a wind speed of 28.35 m/s, a significant wave height of 6 m, and a wave period of 11 s. These extreme values were chosen because they were the values used to design the Activefloat platform used within this 400 work as introduced by Mahfouz et al. (2021). The extreme wind field was produced using the Mann turbulence generator with turbulence intensity of 11% as recommended by the IEC (2019) standard for extreme wind conditions. OpenFAST was used to simulate each of the customized mooring system designs and the baseline mooring system design for 36 wind directions, wind and waves were aligned for all directions, and second-order wave loads were considered. The simulation time was set to 4200 s. These extreme values were chosen because they were the values used to design the Activefloat platform used within this 400 work as introduced by Mahfouz et al. (2021). The extreme wind field was produced using the Mann turbulence generator with turbulence intensity of 11% as recommended by the IEC (2019) standard for extreme wind conditions. OpenFAST was used to simulate each of the customized mooring system designs and the baseline mooring system design for 36 wind directions, wind and waves were aligned for all directions, and second-order wave loads were considered. The simulation time was set to 4200 d th fi t 600 itt d t id t i t ff t 405 s, and the first 600 s were omitted to avoid transient effects. 405 The maximum tension of each line for all the 36 wind directions is shown in Fig. 19. The maximum tensions of the cus- tomized mooring system design are always lower than the tension of the baseline mooring system. Moreover, the maximum tensions were always lower than the maximum allowable tension for all mooring lines. The maximum allowable tension was calculated by dividing the MBL, as defined in equation 5, by a safety factor of 2. This safety factor is consistent with the safety s, and the first 600 s were omitted to avoid transient effects. 405 The maximum tension of each line for all the 36 wind directions is shown in Fig. 19. 3.5 Performance in normal operation For the damage calculations, we assumed that for the 25-year time, the wind speed is always at 11 m/s, and no other wind speeds where considered. This is enough for the purpose of the paper comparing the customized mooring system fatigue to the baseline 380 mooring system fatigue, but it does not show whether or not the mooring system designs survive a 25-year lifetime. As each mooring system has different headings and hence a different DEL at each wind direction, we decided to remove the effect of wind direction and line headings on the damage. We assumed equal wind distribution over all 36 wind directions considered. The 25-year damage results are presented in Fig. 18. the damage calculations, we assumed that for the 25 year time, the wind speed is always at 11 m/s, and no other wind speeds where considered. This is enough for the purpose of the paper comparing the customized mooring system fatigue to the baseline 380 mooring system fatigue, but it does not show whether or not the mooring system designs survive a 25-year lifetime. As each mooring system has different headings and hence a different DEL at each wind direction, we decided to remove the effect of wind direction and line headings on the damage. We assumed equal wind distribution over all 36 wind directions considered. The 25-year damage results are presented in Fig. 18. 380 The baseline mooring system fatigue damage is generally the lowest with the largest line diameter of 0.19 m. The customized 385 mooring system 25-year fatigue damage is overall comparable, except for a few lines. The mooring lines with fatigue damage greater than 1.75 have a line diameter of only 0.09 m. The remainder of lines with fatigue damage greater than 0.25 have a line diameter of 0.12 m. All of the customized mooring lines with a diameter of 0.150 m show fatigue damage of less than 0.25, which is comparable to the baseline design. This is notable because the customized mooring system designs achieve similar fatigue damage with a significantly smaller line diameter. 390 fatigue damage with a significantly smaller line diameter. 390 The fatigue performance of the mooring system relates to the mooring system stiffness. The stiffness at a given position is equal to the slope of the force-displacement response of the mooring system at that offset. 3.6 Performance in extreme conditions The maximum tensions of the cus- tomized mooring system design are always lower than the tension of the baseline mooring system. Moreover, the maximum tensions were always lower than the maximum allowable tension for all mooring lines. The maximum allowable tension was calculated by dividing the MBL, as defined in equation 5, by a safety factor of 2. This safety factor is consistent with the safety 21 Figure 19. Maximum tension of each mooring system design for all wind directions under extreme wind and wave loads https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 19. Maximum tension of each mooring system design for all wind directions under extreme wind and wave loads 9. Maximum tension of each mooring system design for all wind directions under extreme wind and wave load factor defined by the API (2005) standard. The maximum allowable tension shown in Fig. 19 stands for the value for a mooring 410 line of 0.09 m diameter, as this is the smallest diameter in the customized mooring system designs. Therefore, we can conclude from Fig. 19 that all mooring system designs can withstand the extreme loads. 4 Conclusions This paper verifies an innovative method for FWF layout design and optimization using mid-fidelity dynamic models. The novel method aims to passively relocate downwind FOWTs out of the wakes of upwind ones in an FWF to decrease wake losses. 415 The relocation of the FOWTs depends on the mooring system design to govern the FOWTs’ movements according to the wind direction. The verification process has two key objectives: First, verify the energy gain predicted by the Gaussian steady- state wake model implemented within FLORIS against the DWM model within FAST.Farm. Second, verify that the novel mooring system designs can withstand the fatigue and extreme loads in operational and extreme conditions when compared to a state-of-the-art mooring system design. 420 The findings of the paper can be summarized as follows: The findings of the paper can be summarized as follows: 1. Passively relocating the FOWTs in a FWF increases the farm’s energy production. 2. Allowing larger FOWTs excursions decreases the fatigue loads on the mooring lines. 3. Allowing larger FOWTs excursions decreases the extreme loads on the mooring lines. 4. Fatigue loads on a mooring line are highest when the mooring line is upwind and aligned with the wind direction. 4. Fatigue loads on a mooring line are highest when the mooring line is upwind and aligned with the wind direction. 425 22 https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. The paper first introduced the four steps of the FWF layout design methodology and showed the results of each step using static models. The results of the static models showed that at a constant wind speed of 10 m/s, FOWT movements can increase the energy production of the FWF by 3.1%. Afterward, we verified these results using the polar implementation of the DWM model within FAST.Farm. First, we simulated the baseline FWF with all FOWTs coupled to the baseline mooring system, which restricted the FOWTs’ movements. Then we simulated the FWF with each FOWT coupled to the customized mooring 430 system design allowing crosswind movements to mitigate the wake losses. This was repeated for all wind directions of the wind rose presented in Fig. 2. Finally, we compared the results of the energy production of the baseline FWF to the new FWF layout design. 4 Conclusions All customized mooring system 450 designs comfortably withstood extreme 50-year events for all wind directions when applying a safety factor of 2. In conclusion, this study successfully verified the potential of relocating FOWTs to increase energy production within FWFs using a mid-fidelity dynamic analysis, which is the primary goal of this research. Despite the uncertainties associated with the DWM model for wake losses, the dynamic results predicted an energy gain of 1.4% at a constant wind speed of 10 system designs were always lower than a baseline state-of-the-art mooring system design. All customized mooring system 450 designs comfortably withstood extreme 50-year events for all wind directions when applying a safety factor of 2. In conclusion, this study successfully verified the potential of relocating FOWTs to increase energy production within FWFs using a mid-fidelity dynamic analysis, which is the primary goal of this research. Despite the uncertainties associated with the DWM model for wake losses, the dynamic results predicted an energy gain of 1.4% at a constant wind speed of 10 m/s. Additionally, the FOWTs moved as predicted in the design process and the energy gain distribution in Fig. 11 was as 455 predicted by the steady-state model in the design process. Additionally, the paper showed that designing mooring systems with larger excursion limits and lower stiffness leads to lower mooring system fatigue damage and extreme loads. This means that allowing larger excursions leads to mooring lines with smaller diameters. In future work, we are planning to make our designs more realistic and consider real-life scenarios. Therefore, we are planning to include the levelized cost of energy as a design objective along with increasing the energy gain. Moreover, we are planning to consider the supply chain and ease of installation 460 m/s. Additionally, the FOWTs moved as predicted in the design process and the energy gain distribution in Fig. 11 was as 455 predicted by the steady-state model in the design process. Additionally, the paper showed that designing mooring systems with larger excursion limits and lower stiffness leads to lower mooring system fatigue damage and extreme loads. This means that allowing larger excursions leads to mooring lines with smaller diameters. In future work, we are planning to make our designs more realistic and consider real-life scenarios. Therefore, we are planning to include the levelized cost of energy as a design objective along with increasing the energy gain. 4 Conclusions Wind and wave simulations were conducted at rated wind speed of 11 m/s for all wind direction with an increment of 10◦ 440 (i.e., 36 wind directions). Then we calculated the 1 Hz DEL and the 25-year damage. The results showed that the customized mooring system designs have lower 25-year damage than a state-of-the-art mooring system design with the same mooring line diameter. This is due to the lower stiffness of the customized mooring system designs, which leads to smaller dynamic fluctua- tions in the fairlead tensions. Moreover, the results showed a direct relationship between the line headings, wind direction, and DEL values. The fatigue loads were highest when the wind was aligned with the upwind mooring line. Therefore, in any FWF 445 we should avoid aligning the mooring lines with the most probable wind direction in order to avoid having high fatigue loads concentrated at one of the mooring lines. Furthermore, we checked that the customized mooring system designs can endure 50-year extreme wind and wave events for all 36 wind directions with an increment of 10◦. The results showed that the maximum loads on the new customized mooring DEL values. The fatigue loads were highest when the wind was aligned with the upwind mooring line. Therefore, in any FWF 445 we should avoid aligning the mooring lines with the most probable wind direction in order to avoid having high fatigue loads concentrated at one of the mooring lines. Furthermore, we checked that the customized mooring system designs can endure 50-year extreme wind and wave events for all 36 wind directions with an increment of 10◦. The results showed that the maximum loads on the new customized mooring DEL values. The fatigue loads were highest when the wind was aligned with the upwind mooring line. Therefore, in any FWF 445 we should avoid aligning the mooring lines with the most probable wind direction in order to avoid having high fatigue loads concentrated at one of the mooring lines. Furthermore, we checked that the customized mooring system designs can endure 50-year extreme wind and wave events for all 36 wind directions with an increment of 10◦The results showed that the maximum loads on the new customized mooring system designs were always lower than a baseline state-of-the-art mooring system design. 4 Conclusions The energy gain calculated by FAST.Farm was 1.4% at a constant wind speed of 10 m/s; this is 45% of the energy gain predicted by the steady-state wake models. However, the results showed that FAST.Farm overpredicts the vertical 430 wake deflection for FOWTs and hence underestimates the wake losses within the baseline FWF layout. This causes the big 435 difference between the steady-state wake model and the DWM model as explained in Section 3.4. Calibration of the DWM model within FAST.Farm using higher fidelity large-eddy simulations are needed to decrease the uncertainty in the wake losses inside an FWF. The study also assessed the performance of the novel mooring system designs and their fatigue loads in operational condi- wake deflection for FOWTs and hence underestimates the wake losses within the baseline FWF layout. This causes the big 435 difference between the steady-state wake model and the DWM model as explained in Section 3.4. Calibration of the DWM model within FAST.Farm using higher fidelity large-eddy simulations are needed to decrease the uncertainty in the wake losses inside an FWF. The study also assessed the performance of the novel mooring system designs and their fatigue loads in operational condi- wake deflection for FOWTs and hence underestimates the wake losses within the baseline FWF layout. This causes the big 435 difference between the steady-state wake model and the DWM model as explained in Section 3.4. Calibration of the DWM model within FAST.Farm using higher fidelity large-eddy simulations are needed to decrease the uncertainty in the wake losses inside an FWF. The study also assessed the performance of the novel mooring system designs and their fatigue loads in operational condi- tions. Wind and wave simulations were conducted at rated wind speed of 11 m/s for all wind direction with an increment of 10◦ 440 (i.e., 36 wind directions). Then we calculated the 1 Hz DEL and the 25-year damage. The results showed that the customized mooring system designs have lower 25-year damage than a state-of-the-art mooring system design with the same mooring line diameter. This is due to the lower stiffness of the customized mooring system designs, which leads to smaller dynamic fluctua- tions in the fairlead tensions. Moreover, the results showed a direct relationship between the line headings, wind direction, and tions. 4 Conclusions Moreover, we are planning to consider the supply chain and ease of installation 460 m/s. Additionally, the FOWTs moved as predicted in the design process and the energy gain distribution in Fig. 11 was as 455 predicted by the steady-state model in the design process. Additionally, the paper showed that designing mooring systems with larger excursion limits and lower stiffness leads to lower mooring system fatigue damage and extreme loads. This means that allowing larger excursions leads to mooring lines with smaller diameters. In future work, we are planning to make our designs more realistic and consider real-life scenarios. Therefore, we are planning to include the levelized cost of energy as a design objective along with increasing the energy gain. Moreover, we are planning to consider the supply chain and ease of installation 460 23 https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. by designing a single mooring system for all FOWTs in the FWF instead of having a different mooring system design for each FOWT in the FWF. Code and data availability. The code used for FWF layout optimization, and the MoorDyn file for each mooring system design can be found here https://doi.org/10.5281/zenodo.8370977 Video supplement. An animation video of the passive displacements of the FOWTs can be found here http://doi.org/10.5446/63167. 465 Video supplement. An animation video of the passive displacements of the FOWTs can be found here http://doi.org/10.5446/63167. 5 Competing interests. The authors declare that they have no conflict of interest. Appendix A: FWF mooring system designs Table A1. Customized mooring system designs T Line 1 Line2 Line 3 Heading d Anc. r L Heading d Anc r L Heading d Anc r L [◦] [m] [m] [m] [◦] [m] [m] [m] [◦] [m] [m] [m] 1 30 0.15 840 944.91 170 0.15 840 909.85 280 0.15 840 944.91 2 210 0.15 840 909.85 0 0.15 840 909.85 100 0.15 600 707.94 3 215 0.15 840 944.91 335 0.15 840 944.91 95 0.15 840 944.91 4 80 0.15 840 909.85 210 0.09 840 909.85 350 0.12 600 674.9 5 240 0.09 840 944.91 10 0.15 840 909.85 140 0.12 840 909.85 6 270 0.12 840 944.91 30 0.15 840 944.91 160 0.15 840 909.85 7 265 0.12 840 909.85 25 0.15 840 909.85 165 0.15 600 674.9 8 70 0.15 600 674.9 200 0.12 840 909.85 340 0.15 840 909.85 9 190 0.15 840 909.85 340 0.15 840 909.85 80 0.15 600 707.94 Author contributions. MYM developed the novel method to passively relocate the FOWTs in the FWF, carried out all the static and dynamic simulations, and wrote most of the paper. EL designed the baseline mooring system, contributed to the fatigue analysis post-processing and helped in writing and reviewing the paper. MH contributed to the conceptualization of the methods and reviewed the paper. PWC supervized the work and reviewed the paper. 470 Competing interests. The authors declare that they have no conflict of interest. 24 https://doi.org/10.5194/wes-2023-135 Preprint. Discussion started: 16 October 2023 c⃝Author(s) 2023. CC BY 4.0 License. Acknowledgements. The support of Jason Jonkman, Andrew Platt and Lucas Carmo in setting up the FWF model in FAST.Farm is gratefully acknowledged. The research leading to these results has received partial funding from the European Union’s Horizon2020 research and innovation programme under grant agreement no.815083 (COREWIND). This work was authored in part by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE-AC36- 475 08GO28308. Funding provided by U.S. Department of Energy Office of Energy Efficiency and Renewable Energy Wind Energy Technologies Office. The views expressed in the article do not necessarily represent the views of the DOE or the U.S. Government. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. innovation programme under grant agreement no.815083 (COREWIND). This work was authored in part by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE-AC36- 475 08GO28308. Funding provided by U.S. Department of Energy Office of Energy Efficiency and Renewable Energy Wind Energy Technologies Office. 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https://openalex.org/W4285084453	https://www.frontiersin.org/articles/10.3389/fchem.2022.955065/pdf	English	null	Recent Progress in Doped g-C3N4 Photocatalyst for Solar Water Splitting: A Review	Frontiers in chemistry	2,022	cc-by	6,345	MINI REVIEW MINI REVIEW published: 13 July 2022 doi: 10.3389/fchem.2022.955065 Reviewed by: Huogen Yu, Reviewed by: Huogen Yu, Wuhan University of Technology, Reviewed by: Huogen Yu, Wuhan University of Technology, China Li Xiaofang, Wuhan University, China Correspondence: Yilong Yang ylyang@hncj.edu.cn Kaidong Xu 30010908@hncj.edu.cn Reviewed by: Huogen Yu, Wuhan University of Technology, China Recent Progress in Doped g-C3N4 Photocatalyst for Solar Water Splitting: A Review Yilong Yang 1, Wantong Niu 1, Liyun Dang 1, Yanli Mao 2, Junshu Wu 3 and Kaidong Xu 1* 1School of Materials and Chemical Engineering, Henan University of Urban Construction, Pingdingshan, China, 2Henan Provinc Key Laboratory of Water Pollution Control and Rehabilitation Technology, Henan University of Urban Construction, Pingdingshan China, 3Key Laboratory of Advanced Functional Materials, Ministry of Education, Faculty of Materials and Manufacturing, Beijin University of Technology, Beijing, China 1School of Materials and Chemical Engineering, Henan University of Urban Construction, Pingdingshan, China, 2Henan Province Key Laboratory of Water Pollution Control and Rehabilitation Technology, Henan University of Urban Construction, Pingdingshan, China, 3Key Laboratory of Advanced Functional Materials, Ministry of Education, Faculty of Materials and Manufacturing, Beijing University of Technology, Beijing, China Graphitic carbon nitride (g-C3N4) photocatalysis for water splitting is harvested as a fascinating way for addressing the global energy crisis. At present, numerous research subjects have been achieved to design and develop g-C3N4 photocatalysis, and the photocatalytic system still suffers from low efﬁciency that is far from practical applications. Here, there is an inspiring review on the latest progress of the doping strategies to modify g-C3N4 for enhancing the efﬁciency of photocatalytic water splitting, including non-metal doping, metal doping, and molecular doping. Finally, the review concludes a summary and highlights some perspectives on the challenges and future research of g-C3N4 photocatalysts. INTRODUCTION Wuhan University of Technology, China Wuhan University of Technology, China Li Xiaofang, Wuhan University, China The global energy demands and environmental crisis have stimulated tremendous research on the exploration of green and renewable energy due to awareness of energy conservation and environmental protection (Miao et al., 2022). Since titanium dioxide (TiO2) was discovered as the photoanode for photoelectrochemical (PEC) water splitting(Fujishima and Honda, 1972), semiconductor-based photocatalysis for solar hydrogen production has seen an upsurge in global interests (Wang and Wang, 2019; Zada et al., 2020; Qi et al., 2021; Wei et al., 2021). However, it is still very challenging to achieve high solar-to-hydrogen (STH) conversion efﬁciency toward practical applications. To make high utilization of solar energy, the exploration of visible- light-active photocatalysts is highly desirable. In 2009, Wang et al. developed the pioneering work on g-C3N4 for visible-light–driven photocatalytic water splitting(Wang et al., 2009), and g-C3N4-based photocatalysis has drawn considerable attention in the last decade (Xiao et al., 2021; Xing et al., 2021; Chen et al., 2022a) (Figures 1A–E). For uniﬁcation in this study, we will consider the two kinds of materials with triazine (C3N3) unit or tri-s-triazine (C6N7) unit (Figure 1A) to name as g-C3N4. g-C3N4 affords a lamellar structure consisting of C and N atoms which is similar to graphene and can be traced back to the original form of “melon” found by Berzelius and Liebig in 1834 (Liebig, 1834). Unlike TiO2, g-C3N4 affords a narrow bandgap of 2.7 eV (Figure 1C) with the valance band (VB) position at +1.6 eV and conduction band (CB) position at −1.1 eV vs. normal hydrogen electrode (NHE) (Wang et al., 2009) (Figure 1E). This enables g-C3N4 to drive photocatalytic reaction using visible light. Correspondence: Yilong Yang ylyang@hncj.edu.cn Kaidong Xu 30010908@hncj.edu.cn Specialty section: This article was submitted to Catalysis and Photocatalysis, a section of the journal Frontiers in Chemistry Received: 28 May 2022 Accepted: 09 June 2022 Published: 13 July 2022 Specialty section: This article was submitted to Catalysis and Photocatalysis, a section of the journal Frontiers in Chemistry Specialty section: This article was submitted to Catalysis and Photocatalysis, a section of the journal Frontiers in Chemistry Received: 28 May 2022 Accepted: 09 June 2022 Published: 13 July 2022 Edited by: Kangle Lv, South-Central University for Nationalities, China Keywords: doping, G-C3N4, photoadsorption, band structure, water splitting Citation: Yang Y, Niu W, Dang L, Mao Y, Wu J and Xu K (2022) Recent Progress in Doped g-C3N4 Photocatalyst for Solar Water Splitting: A Review. Front. Chem. 10:955065. doi: 10.3389/fchem.2022.955065 g Generally speaking, the g-C3N4 photocatalyst possesses all the following abilities: stability, non- toxicity, abundant source, visible-light-responsive absorption, and easy to control and modify. As a July 2022 \| Volume 10 \| Article 955065 1 Frontiers in Chemistry \| www.frontiersin.org Study of Doped g-C3N4 Photocatalyst Yang et al. FIGURE 1 \| (A) Schematic diagram of a perfect g-C3N4 sheet constructed from melem units, (B) experimental XRD pattern of g-C3N4, (C) ultraviolet–visible diffuse reﬂectance spectrum of the g-C3N4. Inset: Photograph of g-C3N4, (D) typical time course of H2 production from water containing 10 vol% triethanolamine as an electron donor under visible light (of wavelength longer than 420 nm) by (i) unmodiﬁed g-C3N4 and (ii) 3.0 wt% Pt-deposited g-C3N4 photocatalyst, (E) density-functional-theory band structure for polymeric melon calculated along with the chain (Γ–X direction) and perpendicular to the chain (Y–Γ direction). The position of the reduction level for H+ to H2 is indicated by the dashed blue line, and the oxidation potential of H2O to O2 is indicated by the red dashed line just above the valence band. Copyright 2009, Springer Nature; (F) illustration of the charge transfer in g-C3N4 nanosheets under visible light. Copyright 2017, Elsevier. FIGURE 1 \| (A) Schematic diagram of a perfect g-C3N4 sheet constructed from melem units, (B) experimental XRD pattern of g-C3N4, (C) ultraviolet–visible diffuse reﬂectance spectrum of the g-C3N4. Inset: Photograph of g-C3N4, (D) typical time course of H2 production from water containing 10 vol% triethanolamine as an electron donor under visible light (of wavelength longer than 420 nm) by (i) unmodiﬁed g-C3N4 and (ii) 3.0 wt% Pt-deposited g-C3N4 photocatalyst, (E) density-functional-theory band structure for polymeric melon calculated along with the chain (Γ–X direction) and perpendicular to the chain (Y–Γ direction). The position of the reduction level for H+ to H2 is indicated by the dashed blue line, and the oxidation potential of H2O to O2 is indicated by the red dashed line just above the valence band. Copyright 2009, Springer Nature; (F) illustration of the charge transfer in g-C3N4 nanosheets under visible light. Copyright 2017, Elsevier. simpliﬁed by element doping to tune the band gap, which considerably broadens the light absorption and accelerates the electron-hole pair separation (Li et al., 2020b). Citation: This work overviews the recent advances of g-C3N4 materials focusing on efﬁcient photocatalytic water splitting in doping strategies for modifying carbon nitride including non-metal doping, metal doping, and molecular doping. This review also aims to present a general summarization in boosting the g-C3N4 photocatalyst to seek new inspiration for material science. fantastic visible-light–driven photocatalyst, its remarkable property has been conducted on widely photocatalytic applications, such as H2 evolution from water (Zhao et al., 2017a; Yu et al., 2021; Zhao et al., 2022), O2 evolution (Yang et al., 2019; Zhao et al., 2019; Xu et al., 2021), overall water splitting (Chen et al., 2019; Bhagat and Dashora, 2021; Wu et al., 2021), photodegradation of pollutants (Xiong et al., 2016; Duan et al., 2019; Jing et al., 2021), CO2 reduction (Jiang et al., 2018; Yang et al., 2020a; Cheng et al., 2020), organic synthesis (Devthade et al., 2018; Camussi et al., 2019; Lima et al., 2019), and photoelectrocatalysis (Karimi-Nazarabad et al., 2019; Wu et al., 2020; Huo et al., 2021). However, the three crucial issues of light absorption efﬁciency, charge separation and transfer efﬁciency, and surface reaction efﬁciency still restrict the development of high-performance g-C3N4 photocatalysis, which is fairly difﬁcult to achieve by pristine g-C3N4. Frontiers in Chemistry \| www.frontiersin.org Basic Properties of Carbon Nitride for Solar Water Splitting g Under irradiation, the electrons of the g-C3N4 photocatalyst can be excited from the VB to CB by absorbing the photons with the energy (h]) higher than the bandgap energy, wherein holes are left in the VB (Luo et al., 2016). The large parts of the photoexcited charge carriers will combine rapidly, and only a small part of photogenerated electrons and holes can be transferred to the surface of g-C3N4 to involve the reaction. Then, the water molecules can be reduced with the photoexcited electrons for H2 evolution and oxidized by the photoexcited holes for O2 generation during the photocatalytic reaction. For H2 evolution, the CB potential of the photocatalyst should be more negative than the H2 reduction potential, while Although the g-C3N4-based photocatalysis has been fully discussed in many recent review articles(Li et al., 2020a; Li et al., 2021a; Chen et al., 2021; Xing et al., 2021; Zhu et al., 2021; Chen et al., 2022b), a relative focus review about doping strategies to modify g-C3N4 for improving the efﬁciency of water splitting is still lacking. Compared with other effective strategies for modiﬁcation of g-C3N4, like heterojunction construction, defect introduction, and nanostructure controlling, the modiﬁcation process of g-C3N4 can be comparatively July 2022 \| Volume 10 \| Article 955065 Frontiers in Chemistry \| www.frontiersin.org 2 Study of Doped g-C3N4 Photocatalyst Yang et al. the VB potential should be more positive than the water oxidation potential for the O2 evolution from water. The g-C3N4 photocatalyst possesses a CB of −1.1 eV and a VB of 1.6 eV is ﬁt for splitting water to H2 and O2. g-C3N4 was fabricated by polymerizing urea and ionic liquids ([Bmim][BF4]), which was used as the texture modiﬁer and dopant source (Lin and Wang, 2014). This research leads in a new one-pot fabrication of B and F co- doped g-C3N4. So far, visible-light photoadsorption, high chemical stability, appropriate CB and VB potentials, and strong photocatalytic activity make g-C3N4 be the most widely focused in photocatalytic water splitting. Nevertheless, the efﬁciency of water splitting by g-C3N4 is still low, mainly due to limited photoadsorption in the visible-light region, limited ability of electron transport along or across the g-C3N4 sheets (Figure 1F), and recombination of photoexcited electron-hole pairs. In addition to F, other halogen elements we cannot ignore are chloride (Cl), bromine (Br), and iodine (I) in the doping area. Basic Properties of Carbon Nitride for Solar Water Splitting Br-doped g-C3N4 was also successfully synthesized by using ionic liquid as the Br source and soft-template for photoredox water splitting (Zhao et al., 2017b). The Br- doping tuned light absorption and band structure without destroying the major construction of the g-C3N4 polymer. Similarly, I doped g-C3N4 materials also lead to positive effects that enlarged the speciﬁc surface area, enhanced optical absorption, narrowed the bandgap, and accelerated the photoinduced charge carrier transfer rate, leading to an increasing H2 evolution rate (Iqbal et al., 2020). In a word, due to dramatic development of g-C3N4 photocatalysts in the STH conversion ﬁeld, a review focusing on the photocatalytic water splitting is still necessary to provide researchers a state-of-art progress in this dynamic research ﬁeld. This review presents a brief discussion of the current doping research, accompanied with the challenges and future direction of g-C3N4 photocatalysts for photocatalytic applications. g 2 ( q ) In addition, introducing other non-metal elements are also effective strategies in promoting the photocatalytic property (Deng et al., 2019; Li et al., 2021b; Li et al., 2021c; Liu et al., 2021; Yan et al., 2022). For instance, carbon (C) self-doped g-C3N4 was prepared via a combined method of melamine- cyanuric acid complex supramolecular pre-assembly and solvothermal pre-treatment (Li et al., 2021b). The H2 evolution rate for optimized g-C3N4 was 18 times higher than that of bulk g-C3N4, and the enhanced performance derives from the extended optical absorption, accelerated photoactivated charge carrier separation, and transfer efﬁciency. Unique oxygen-doped g-C3N4 materials were synthesized, which realized the synergetic control of the electronic structure and morphology and possessed the advantages of enlarged surface area, increased exposed active edges, and improved separation efﬁciency (Yan et al., 2022). Some other high-performance non- metal doped materials including phosphorus-doped g-C3N4(Yang et al., 2018; Lin et al., 2020; Zhao et al., 2020) and sulfur-doped g-C3N4 (Li et al., 2019a; Yang et al., 2020b; Long et al., 2020) have also been developed. For instance, Yang et al. reported a ﬂower-like P-doped g-C3N4 which was prepared using phosphoric acid and cyanuric acid-melamine complex, which served as the P source and the precursor of g-C3N4, respectively, (Yang et al., 2018). The as-prepared P-doped g-C3N4 showed a high visible-light photocatalytic H2 evolution rate of 256.4 μmol h−1 50 mg−1 and almost 24-folds higher than those of the pristine g-C3N4. Basic Properties of Carbon Nitride for Solar Water Splitting A sulfur (S) doped-g-C3N4 nanosheet with terminal- methylate was presented with a tunable bandgap (Li et al., 2019a). The VB near the Fermi level was split due to S atoms into methylated melon units, which generated a new empty mid- gap electronic state and improved the light-responsive property up to 700 nm. Furthermore, the photocatalytic activity restricted by intralayered hydrogen bonds should also be considered. Yang et al. reported an S-doped g-C3N4 through poly-condensation and the mixture of dicyandiamide and thioacetamide, resulting in greatly enhanced visible-light-response ability and n →π electron transition. The substituting of sp2-hybridized N with S atoms contributed to break intralayered hydrogen bonds, which resulted in photocatalytic H2 production (Yang et al., 2020b) (Figures 2A,B). Doping Strategies for Modifying Carbon Nitride To develop advanced g-C3N4 photocatalysts, a doping strategy is considered an appealing way to modulate physicochemical properties such as band structure tailoring and light adsorption improving, which, therefore, enhance the performance of photocatalyst (Patnaik et al., 2021). Based on the arrangements of doping elements, the classiﬁcation of doping can be divided into non-metal doping, metal doping, and molecular doping. Frontiers in Chemistry \| www.frontiersin.org Non-Metal Doping Copyright 2018, Elsevier; (E) Chemical production of polymeric carbon nitride semiconductors from nucleobases and urea, (F) Effect of cytosine amount on the HER, (G) wavelength dependence of the HER with CNC30 loaded with 3 wt% Pt. Inset: time-dependence of the HER with CNC30 at different irradiation wavelengths. Copyright 2017 John Wiley and Sons, Inc. FIGURE 2 \| (A) Schematic illustration for the formation of multi-layered cake-like porous g-C3N4 with broken hydrogen bonds by S-doping, (B) UV–visible diffuse reﬂectance spectra of samples. Copyright 2020, Elsevier; (C) Schematic illustration of the KOH-assisted hydrothermal-reformed melamine strategy for achieving simultaneous K-doping and exfoliation of g-C3N4, (D) band structure alignments for the CN, HCN, and CNK sample. Copyright 2018, Elsevier; (E) Chemical production of polymeric carbon nitride semiconductors from nucleobases and urea, (F) Effect of cytosine amount on the HER, (G) wavelength dependence of the HER with CNC30 loaded with 3 wt% Pt. Inset: time-dependence of the HER with CNC30 at different irradiation wavelengths. Copyright 2017 John Wiley and Sons, Inc. Non-Metal Doping p g The fabrication of non-metal doped carbon nitride is effective in modulating the electronic structure of g-C3N4 by distorting the π-conjugated orbital. Boron-doped g-C3N4 was prepared by microwave heating for hydrogen evolution, and boric acid was used as a doping source combined with melamine and urea for thermal condensation (Chen et al., 2018). g-C3N4 nanosheets can be in situ modiﬁed by boron atoms to improve the photoadsorption, hinder the annihilation of charge carriers, and prolong the lifetime of photogenerated electrons. Combining g-C3N4 with strongly electronegative dopants such as ﬂuorine to form an F-doped material not only raised the valence band but also affected the thermodynamic driving force for H2 reduction (Zhu et al., 2017). Fluorinated (F) carbon nitride solids were also reported with excellent visible-light photocatalytic activity (Shevlin and Guo, 2016). Fluorination not only provided a modiﬁed texture but also enabled effective adjustment of the electronic band structure, which was demonstrated by improved activities. To further explore the structural distortion-dependent photoreactivity, it is more desirable to exploit co-doping that may be a good attempt to further improve the photocatalytic activity of g-C3N4 through the synergistic effects of the two dopants. For instance, B/F co-doped July 2022 \| Volume 10 \| Article 955065 Frontiers in Chemistry \| www.frontiersin.org 3 Study of Doped g-C3N4 Photocatalyst Yang et al. FIGURE 2 \| (A) Schematic illustration for the formation of multi-layered cake-like porous g-C3N4 with broken hydrogen bonds by S-doping, (B) UV–visible diffuse reﬂectance spectra of samples. Copyright 2020, Elsevier; (C) Schematic illustration of the KOH-assisted hydrothermal-reformed melamine strategy for achieving simultaneous K-doping and exfoliation of g-C3N4, (D) band structure alignments for the CN, HCN, and CNK sample. Copyright 2018, Elsevier; (E) Chemical production of polymeric carbon nitride semiconductors from nucleobases and urea, (F) Effect of cytosine amount on the HER, (G) wavelength dependence of the HER with CNC30 loaded with 3 wt% Pt. Inset: time-dependence of the HER with CNC30 at different irradiation wavelengths. Copyright 2017 John Wiley and Sons, Inc. Yang et al. Study of Doped g-C3N4 Photocatalyst FIGURE 2 \| (A) Schematic illustration for the formation of multi-layered cake-like porous g-C3N4 with broken hydrogen bonds by S-doping, (B) UV–visible diffuse reﬂectance spectra of samples. Copyright 2020, Elsevier; (C) Schematic illustration of the KOH-assisted hydrothermal-reformed melamine strategy for achieving simultaneous K-doping and exfoliation of g-C3N4, (D) band structure alignments for the CN, HCN, and CNK sample. Metal Doping b d modiﬁed g-C3N4 (K-g-C3N4) nanosheets were synthesized (Sun et al., 2019) (Figures 2C,D). Photocatalytic H2 production tests under visible light irradiation showed high photocatalytic activities of K-g-C3N4 nanosheets (up to about 13-folds higher than that of original g-C3N4) as well as an apparent quantum efﬁciency (AQE) of 6.98% at 420 nm. In addition, lanthanum (La) and Co co-doped g-C3N4 was prepared by the wet impregnation method (Tasleem and Due to abundant orbital electrons, the electron structure and optical property of g-C3N4 can also be affected by metal element doping, leading to focusing on the metal doping method. The cobalt (Co)-doped g-C3N4 nanosheet was investigated to provide more separation centers by forming Co–N bond, which can celebrate charge transfer and enhance photocatalytic performance (Yang et al., 2021). Moreover, potassium- July 2022 \| Volume 10 \| Article 955065 Frontiers in Chemistry \| www.frontiersin.org 4 Study of Doped g-C3N4 Photocatalyst Yang et al. Tahir, 2021). The H2 evolution by La/Co co-doped-g-C3N4 showed the highest H2 production of 250 μmol g−1h−1 among the samples, which was 2.5, 1.35, and 1.25 times higher than that of original g-C3N4, La-g-C3N4, and Co-g-C3N4, respectively. The enhanced activity can be contributed to the celebrated charge separation, which was originally the electron trapping capability of La and Co. the photocatalytic performance of g-C3N4. In short, doping is a feasible and effective strategy to regulate photo-absorbance, redox potentials, and transfer of photo-induced charge carriers and one of the attractive strategies to tune the physicochemical properties of g-C3N4. g To date, doping provides an innovative approach to promote the efﬁciency of g-C3N4 photocatalyst. However, some issues like nonuniform doping, formation of surface trapping center, or low oxidizing and reducing capability resulting from narrowing the bandgap still existed, while the mechanisms in this ﬁeld are at the primary stage and further systematic investigations are still needed because of the relatively low visible-light photocatalytic efﬁciency, which is far from the requirements of practical applications. Some issues that must be resolved for doped g-C3N4 photocatalyst involve the fact that 1) the doping mechanisms of enhanced photocatalytic property is not clear. For example, many explanations of doping technology still stay at the stage of “the enhanced photocatalytic activity is contributed to the doping method” with no discussion about mechanism and essential meaning of element doping. CONCLUSION AND OUTLOOK This review presents a promising visible-light–driven photocatalyst, g-C3N4, beneﬁting from its unique microstructure, resistance against acids, and bases and fantastic band structure. Nevertheless, the pristine g-C3N4 suffers from some shortcomings, including limited photoadsorption capability and fast recombination of photoexcited electron-hole pairs, which largely restricts practical applications. The present review depicts a focus review on the doping strategies to design efﬁcient g-C3N4 in the use of photocatalytic water splitting. In summary, doping can introduce the impurity levels in the band gap region to create a new band edge potential, which can extend the spectral response region with decreased band gap. In addition, the hetero dopants get settled either in the lattice or insert in the interlayers of g-C3N4, inducing the formation of hybridized orbitals. The hybridization between g-C3N4 and dopant orbital remarkably affects the charge transportation, life time of charge carriers, and Metal Doping b d 2) It is still challenging to bring forth new ideas on doping methods, and ﬁnding the right balance of lower redox ability and higher photocatalytic activity is highly desired. To overcome the challenges, lots of attempts are still needed. The heteroatom-doping assisted with theoretical simulation calculation can be a feasible method to analyze the doping effect. Especially, it is signiﬁcant to develop a crystalline g-C3N4 (CCN) doped by metals or non-metals, which improves the charge separation, increases the reactive facet exposing, and shows dramatic photocatalytic water splitting performance. Furthermore, a broad range of heterostructures, including quantum dots/g-C3N4 junction, polymer/g-C3N4 junction, semiconductor/g-C3N4 junction, cocatalyst modiﬁcation of single atoms and defects engineering, as well as nanostructure and crystalline control, should also be considered for improved photocatalysis to increase the photoabsorption, accelerating the charge separation and transfer, elongating the charge carrier lifetime, and boosting the photocatalytic water splitting. Focusing on the perspective of green and renew energy, it is no doubt that g-C3N4-based photocatalyst will draw more attention on the research of water splitting in the future. Molecular Doping p g Heteroatom doping as discussed above is often used to modulate the atomic and band structure of g-C3N4 to promote light harvesting and celebrate electron-hole separation and transfer. Especially, integrating another structure-matching aromatic structure with g-C3N4 is a unique method to tune the intrinsic features (Yang et al., 2017; Li et al., 2018; Li and Zhang, 2018; Li et al., 2019b; Li et al., 2019c; Liu et al., 2019; Li et al., 2020c; Jiang et al., 2021; Zhou et al., 2021). The thermal co-polymerization of the aromatic comonomers and precursors of g-C3N4 can narrow the band gaps of g-C3N4, which extends the visible light absorption edge to enhance the utilization of sunlight. For instance, Liu et al. designed in-plane benzene-ring doped g-C3N4 nanosheets by copolymerizing urea and 4, 4′- sulfonyldiphenol. It exhibited dramatic H2 generation efﬁciency with a PHE rate of 12.3 mmol h−1 g−1, which was almost 12-folds higher than that of intrinsic g-C3N4 and the AQE of 17.7% at 420 nm (Jiang et al., 2021). 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Parent-child relationships and offspring’s positive mental wellbeing from adolescence to early older age Citation for published version: Stafford, M, Kuh, D, Gale, C, Mishra, G & Richards, M 2016, 'Parent-child relationships and offspring’s positive mental wellbeing from adolescence to early older age', The Journal of Positive Psychology, vol. 11, no. 3, pp. 326-337. https://doi.org/10.1080/17439760.2015.1081971 Citation for published version: Stafford, M, Kuh, D, Gale, C, Mishra, G & Richards, M 2016, 'Parent-child relationships and offspring’s positive mental wellbeing from adolescence to early older age', The Journal of Positive Psychology, vol. 11, no. 3, pp. 326-337. https://doi.org/10.1080/17439760.2015.1081971 Digital Object Identifier (DOI): 10.1080/17439760.2015.1081971 Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record Published In: The Journal of Positive Psychology Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record 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 Date: 07 December 2016, At: 02:04 The Journal of Positive Psychology The Journal of Positive Psychology ISSN: 1743-9760 (Print) 1743-9779 (Online) Journal homepage: http://www.tandfonline.com/loi/rpos20 Parent–child relationships and offspring’s positive mental wellbeing from adolescence to early older age Behavioural control, on the other hand, has been linked to lower risk of psychological symptoms (Wang, Pomerantz, & Chen, 2007). Despite the large literature relating parental care and control to psychologi- cal disorder, studies investigating their links with positive mental well-being are far fewer in number and these are summarised below. Positive mental well-being is now recognised as being multidimensional, having hedonic and eudaimonic aspects, and being more than the absence of psychological disorder. Positive mental well- being and psychological disorder do not necessarily have identical risk factors (Huppert & Whittington, 2003; Ryan & Deci, 2001; Ryff & Singer, 1998). There is therefore a need to know whether parent–child relation- ships inﬂuence positive well-being indicators, such as happiness, life satisfaction and positive psychological functioning, in adolescence and beyond. © 2015 The Author(s). Published by Taylor & Francis. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unre- stricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Corresponding author. Email: m.stafford@ucl.ac.uk Parent–child relationships and offspring’s positive mental wellbeing from adolescence to early older age Mai Stafford, Diana L. Kuh, Catharine R. Gale, Gita Mishra & Marcus Richards To cite this article: Mai Stafford, Diana L. Kuh, Catharine R. Gale, Gita Mishra & Marcus Richards (2016) Parent–child relationships and offspring’s positive mental wellbeing from adolescence to early older age, The Journal of Positive Psychology, 11:3, 326-337, DOI: 10.1080/17439760.2015.1081971 To cite this article: Mai Stafford, Diana L. Kuh, Catharine R. Gale, Gita Mishra & Marcus Richards (2016) Parent–child relationships and offspring’s positive mental wellbeing from adolescence to early older age, The Journal of Positive Psychology, 11:3, 326-337, DOI: 10.1080/17439760.2015.1081971 p g © 2015 The Author(s). Published by Taylor & Francis Published online: 15 Sep 2015. Submit your article to this journal Article views: 4598 View related articles View Crossmark data Citing articles: 1 View citing articles © 2015 The Author(s). Published by Taylor & Francis Published online: 15 Sep 2015. Submit your article to this journal Article views: 4598 View related articles View Crossmark data Citing articles: 1 View citing articles Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=rpos20 Download by: [The University of Edinburgh] The Journal of Positive Psychology, 2016 Vol. 11, No. 3, 326–337, http://dx.doi.org/10.1080/17439760.2015.1081971 The Journal of Positive Psychology, 2016 Parent–child relationships and offspring’s positive mental wellbeing from adolescence to early older age Mai Stafforda, Diana L. Kuha, Catharine R. Galeb,c, Gita Mishrad and Marcus Richardsa aMRC Unit for Lifelong Health and Ageing at UCL, 33 Bedford Place, London WC1B 5JU, UK; bMRC Lifecourse Epidemiology Unit, University of Southampton, Southampton, UK; cDepartment of Psychology, Centre for Cognitive Ageing and Cognitive Epidemiology, University of Edinburgh, Edinburgh, UK; dSchool of Population Health, University of Queensland, Brisbane, Australia (Received 16 December 2014; accepted 5 July 2015) We examined parent-child relationship quality and positive mental well-being using Medical Research Council National Survey of Health and Development data. Well-being was measured at ages 13–15 (teacher-rated happiness), 36 (life satisfaction), 43 (satisfaction with home and family life) and 60–64 years (Diener Satisfaction With Life scale and Warwick Edinburgh Mental Well-being scale). The Parental Bonding Instrument captured perceived care and control from the father and mother to age 16, recalled by study members at age 43. Greater well-being was seen for offspring with higher combined parental care and lower combined parental psychological control (p < 0.05 at all ages). Controlling for maternal care and paternal and maternal behavioural and psychological control, childhood social class, parental separation, mother’s neuroticism and study member’s personality, higher well-being was consistently related to paternal care. This suggests that both mother–child and father–child relationships may have short and long-term consequences for positive mental well-being. Keywords: well-being; longitudinal; birth cohort study; life satisfaction Bogels, 2008; McLeod, Wood, & Weisz, 2007; Radziszewska, Richardson, Dent, & Flay, 1996; Rodgers, 1996a; Shaw, Krause, Chatters, Connell, & Ingersoll- Dayton, 2004). Behavioural control, on the other hand, has been linked to lower risk of psychological symptoms (Wang, Pomerantz, & Chen, 2007). Despite the large literature relating parental care and control to psychologi- cal disorder, studies investigating their links with positive mental well-being are far fewer in number and these are summarised below. Positive mental well-being is now recognised as being multidimensional, having hedonic and eudaimonic aspects, and being more than the absence of psychological disorder. Positive mental well- being and psychological disorder do not necessarily have identical risk factors (Huppert & Whittington, 2003; Ryan & Deci, 2001; Ryff & Singer, 1998). There is therefore a need to know whether parent–child relation- ships inﬂuence positive well-being indicators, such as happiness, life satisfaction and positive psychological functioning, in adolescence and beyond. Bogels, 2008; McLeod, Wood, & Weisz, 2007; Radziszewska, Richardson, Dent, & Flay, 1996; Rodgers, 1996a; Shaw, Krause, Chatters, Connell, & Ingersoll- Dayton, 2004). © 2015 The Author(s). Published by Taylor & Francis. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unre- stricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2015 The Author(s). Published by Taylor & Francis. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licens stricted use, distribution, and reproduction in any medium, provided the original work is properly cited. y y istributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unre- roduction in any medium, provided the original work is properly cited. Introduction Paternal care and psychological control were more consistently associated with well-being than maternal care and control. Men were not included in that study. relationships with their parents are more securely attached to those parents; are more likely to be securely attached in later relationships; and are at lower risk of psychological disorder (Bowlby, 1988). Rutter (1995) highlighted the particular contribution of bonding with the father ﬁgure. High parental care is hypothesised to promote social- and neuro-cognitive development for positive interpersonal relations and behaviour (Richards & Hatch, 2011) as well as positive adult relationships (Rodgers, 1996b). Interpersonal relations, including social support and strong social networks, are important determinants of positive mental well-being (Charles & Carstensen, 2010). Another pathway is through positive psychological functioning facilitated by the parent. Psychological con- trol can reduce a child’s autonomy and impair the development of self-regulation (Grolnick & Ryan, 1989), whilst behavioural control can increase a child’s compe- tence (Grolnick & Pomerantz, 2009). A combination of intrusive parenting and low parental care is associated with greater psychological inﬂexibility among the off- spring in adolescence (Williams, Ciarrochi, & Heaven, 2012), greater emotion suppression in late childhood (Jaffe, Gullone, & Hughes, 2010) and poorer emotion regulation in early adulthood (Manzeske & Stright, 2009), which can result in fewer positive thoughts and emotions (Gross & John, 2003). The development of personality traits may also be implicated, since parental care and control have been associated prospectively with adult offspring’s optimism, empathy and expressiveness (Huppert, Abbott, Ploubidis, Richards, & Kuh, 2010; Johnson, Liu, & Cohen, 2011; Korkeila et al., 2004), though it is recognised that the relationship between par- enting practices and child’s personality is likely to be bidirectional (van Zeijl et al., 2007). Optimism and neu- roticism are highly correlated with positive mental well-being (Gale, Booth, Mõttus, Kuh, & Deary, 2013; Huppert et al., 2010). y It remains unclear whether mother–child or father– child relationships are equally important for mental well- being. Conceptually, the attachment of the child to the primary caregiver (typically the mother) is the key tem- plate for future relationships (Bowlby, 1988). Two small, non-random samples of young people in education showed that maternal care was a stronger correlate of happiness when mother– and father–child relationships were considered together (Cheng & Furnham, 2004; Furnham & Cheng, 2000), and that only maternal care and control were related to offspring’s self-esteem, neu- roticism and extraversion (Furnham & Cheng, 2000). Introduction Parent–child relationships are central to psychological development and several studies have shown that sub- optimal parenting is an important risk factor for psycho- logical disorder in clinical and representative community samples (Blatt & Homann, 1992; Enns, Cox, & Clara, 2002). Many studies have captured parent–child relation- ships along two principal dimensions. The parental care dimension reﬂects a continuum from affectionate, warm, responsive parenting to cold and unresponsive parenting. The demandingness or control dimension reﬂects the extent to which the parent demands and monitors stan- dards for their child’s conduct (Baumrind, 1991). The concept of parental control has been further distinguished as behavioural and psychological forms of control (Barber, 1996). Behavioural control can provide a struc- tured and predictable environment for the child and encourages their socially acceptable behaviour. But there is a need to balance individual autonomy with confor- mity to social norms and the association between beha- vioural control and outcomes may be curvilinear (Barber, 1996). Psychological control refers to parenting that is intrusive and manipulates the child’s emotional develop- ment (Barber, 1996). A high level of psychological con- trol has been consistently associated with an elevated risk of psychological disorder among the offspring in adolescence and adulthood (van der Bruggen, Stams, & Drawing on evidence from literature on the develop- ment of psychological disorder as well as positive mental wellbeing, we suggest that socio-relational, psychological and socio-economic pathways may link parental care and control to mental well-being in adulthood. Attachment theory details one such pathway. This suggests that infants who experience caring and responsive The Journal of Positive Psychology 327 well-being scales was positively associated with recalled or perceived parental care summed across mothers and fathers, with effect sizes being larger in men (Rothrauff, Cooney, & An, 2009). In one of the few studies to relate prospectively assessed measures of parental care and con- trol to later well-being indicators, daughters (but not sons) of non-controlling mothers were found to have higher life satisfaction at age 30 (Flouri, 2004). Father–child relation- ships were not assessed in that study. Data from Britain’s oldest birth cohort study, the Medical Research Council (MRC) National Survey of Health and Development (NSHD), indicated that perceived parental care was posi- tively associated and parental psychological control nega- tively associated with several aspects of positive mental wellbeing among midlife women, including Ryff’s pur- pose in life, self-acceptance and positive relations scales (Huppert et al., 2010). Introduction Based on the literature on psychological disorders, there is a suggestion that the effect of maternal care and con- trol may be somewhat larger, though this is not a univer- sal ﬁnding. In the US National Comorbidity Survey, lower levels of maternal and paternal care were related to adult psychopathology but with larger effect sizes for maternal care (Enns et al., 2002). In contrast, in the NSHD, Rodgers (1996a) examined paternal and maternal care and control scales as correlates of psychiatric symp- toms at age 43 and found that maternal care did not con- tribute independently of the other three scales. However, sizeable intercorrelations between these four scales were noted. Finally, socio-economic pathways may also be impli- cated. High parental care and parenting characterised by a combination of high care and high behavioural control is associated with higher educational attainment, occupa- tional position and income of the offspring in adulthood, independently of family socio-economic circumstances (Blondal & Adalbjarnardottir, 2009; Dornbusch, Ritter, Leiderman, Roberts, & Fraleigh, 1987; Singh-Manoux, Fonagy, & Marmot, 2006). Education and adult socio- economic position are strongly related to elements of positive mental well-being in adulthood (Diener, Ng, Harter, & Arora, 2010). The aim of the current study was to describe associa- tions of well-being indicators in adolescence, mid-adult- hood and early old age with perceived parental care, behavioural control and psychological control and to assess the contributions of mother–child and father–child relationships to well-being. We hypothesised that well- being would be positively associated with parental care and negatively associated with parental psychological control at each life stage. Based on previous analysis of parental care in this cohort in relation to affective Only a handful of studies have examined associations between these aspects of parenting and multiple dimen- sions of positive mental well-being. In a nationally repre- sentative study of men and women aged 25–74 in the US, well-being captured by summing Ryff’s psychological M. Stafford et al. 328 symptoms in men and women at age 43 (Rodgers, 1996a) and mental well-being in women at age 52 (Huppert et al., 2010), we expected to ﬁnd that paternal care would be more strongly related to well-being than maternal care in mutually adjusted models. two weeks. Statements include ‘I’ve been feeling good about myself’, ‘I’ve been feeling close to other people’, ‘I’ve been interested in new things’ and ‘I’ve been feeling optimistic about the future’. Positive mental well-being Positive mental well-being in childhood was based on teacher ratings when study members were aged 13 and 15 years, using a forerunner of the Rutter A Scale (Rutter, 1967). Four of these items were positively worded: ‘very popular with other children’ (age 13 years only), ‘unusually happy and contented’, ‘makes friends extremely easily’ and ‘extremely energetic, never tired’. Each of these items was scored as 1 representing the relevant positive rating or 0 representing an average or negative rating. These ratings were summed and grouped into none, one and two or more positive ratings, which yielded one large group with no positive ratings, and two modest and approximately equal-sized groups, respectively (Richards & Huppert, 2011). Introduction Validation work indi- cates good construct validity for a single factor structure as well as good criterion validity and test–retest reliabil- ity and supports its use in general population samples (Tennant et al., 2007). Where three or fewer items were missing, the individual’s mean for complete items was imputed. Items loaded on a single component were summed using equal weights to create scores which theoretically ranged from 14 to 70, with 70 indicating highest well-being. The Diener Satisfaction with Life Scale was also completed at age 60–64 (Diener, Emmons, Larsen, & Grifﬁn, 1985). The scale captures cognitive-judgemental aspects of well-being using ﬁve items, such as ‘In most ways my life is close to my ideal’, ‘I am satisﬁed with my life’ and ‘So far I have got the important things I want in life’. Study population The NSHD (Kuh et al., 2011) was originally established to investigate the cost of childbirth and the quality of associated care in the immediate post-war years. A sam- ple of 5362, comprising all single births within marriage to families of non-manual and agricultural occupation and a random one in four sample of single births within marriage to families of manual occupation, was recruited from all births registered in 1 week of March 1946 in England, Wales and Scotland. Data have been collected on 23 occasions from birth with the latest data collection in 2006–2011. The well-being measures up to age 43 are non-stan- dard and pre-date the development and more widespread use of the validated positive well-being instruments now available. It was not possible, using these data, to examine changes in well-being. Results Table 1 summarises the ﬁve well-being indicators from ages 13–15 through to age 60–64. Women expressed greater satisfaction with their home and family at age 43 but gender differences in well-being were not apparent at other ages. Women recalled higher levels of paternal care and higher behavioural and psychological control from both parents compared with men. The correlation between perceived maternal and paternal care was moderate (ρ = 0.47) and did not differ between male and female study members. Similarly, the coefﬁcient for the correlation between perceived maternal and paternal behavioural control was 0.60 and there were no gender differences. The correlation between perceived maternal and paternal psychological control was slightly larger for men than for women (ρ = 0.56 for men and 0.48 for women). Parental separation by age 16 was experienced by 5.6% of study members. In childhood, 45% of study members had a father in a non-manual occupation. Covariates We selected potential confounding variables which have previously been shown to be related to the quality of the child–parent relationships and to either positive mental well-being or psychological distress (Gale et al., 2013; Kiernan & Huerta, 2008; Rodgers, 1996a, 1996b). Paren- tal separation before the study member was aged 16 was derived from parental marital status collected prospec- tively throughout the childhood years. Childhood social class was based on father’s occupation at age 4 (or age 11 or 15 if this was missing (n = 54)) classiﬁed accord- ing to the Registrar General’s scheme and included in analysis as a continuous variable from 1 to 6 (with 6 indicating the lowest social class). Maternal mental health was captured by the neuroticism scale of the Maudsley Personality Inventory (Shaw & Hare, 1965) when study members were aged 15. Conceptually, off- spring’s personality may mediate the association between parent–child relationships and mental well-being, or it may select children into different levels of parental care and control. Study members’ neuroticism and extraver- sion were measured by the Maudsley Personality Inventory at age 26. Of the 5362 original cohort members, 250 had died by age 15, 323 by age 36, 361 by age 43 and 718 by age 60–64. Well-being data were complete for 3699 cohort members at ages 13–15, 3322 at age 36, 3262 at age 43 and 1984 (Diener SWLS) or 1976 (WEMWBS) at age 60–64. Those with missing well-being at age 36 recalled lower paternal care and higher paternal psycho- logical control than those with complete well-being data at age 36. Those with missing well-being at age 60–64 recalled higher paternal psychological control compared with those who had complete well-being data at age 60–64. The magnitude of these differences was small (the largest being 0.15 standard deviations of the parental bonding scale) and no other differences in PBI scales were seen for those with and without each well-being indicator. Parent–child relationships Items are worded positively and respondents are asked to indicate how frequently, on a ﬁve-point scale, they have experienced each statement over the last The Journal of Positive Psychology 329 control provided an improved ﬁt to the data. For this rea- son, we elected to retain separate scales for perceived care and the two forms of control, rather than dichoto- mise and combine them. adjusted for covariates of interest: parental separation before age 16, childhood social class, maternal mental health and study member’s neuroticism and extraversion. Regression models were based on 20 multiply imputed data-sets combined using Rubin’s rules, with additional variables included for the imputation model (household crowding at ages 0, 2, 4, 6, 8, 11; father’s and mother’s educational attainment). Outcomes were included in the imputation model but not used for the estimation part of the model. Analyses were conducted in Stata v12SE. Parent–child relationships The quality of relationships with the mother and father in childhood were identiﬁed from the PBI which was reported retrospectively on a self-completion question- naire by the study member during a home visit when they were aged 43. Study members were asked how they remembered the attitudes and behaviours of their mother and father in their ﬁrst 16 years with responses to 24 items for each parent reported on a four-point Likert scale. Ratings from this instrument are stable over long follow-up periods (Wilhelm, Niven, Parker, & Hadzi- Pavlovic, 2005). Previous factor analysis of the items demonstrated three dimensions of parenting which we refer to here as perceived parental care (12 items e.g. ‘Was affectionate to me’ and ‘Praised me’, internal con- sistency Cronbach’s α = 0.93 for father and 0.91 for mother), perceived parental behavioural control (5 items e.g. ‘Let me go out as often as I wanted’ (reverse coded) and ‘Let me do those things I liked doing’ (reverse coded), α = 0.75 for father and 0.77 for mother), and perceived parental psychological control (7 items e.g. ‘Invaded my privacy’ and ‘Tried to make me dependent on her/him’, α = 0.83 for father and 0.80 for mother) (Huppert et al., 2010). Although some researchers clas- sify participants into combinations of high/low care and control based on cut-points of these continuous scales, analysis in this cohort and others (Huppert et al., 2010; Rodgers, 1996a; Rothrauff et al., 2009; Schoﬁeld et al., 2012) did not indicate that the interaction of care and Life satisfaction at age 36 was captured with a single item with binary response ‘Would you say that, on the whole, life has been good to you?’ At age 43, concur- rently with the Parental Bonding Instrument (PBI), satis- faction with life in the home and family domain was captured with a single item with seven possible response options ‘Looking back, how satisﬁed are you with what you have accomplished in your home and family life?’ Study members self-completed the 14-item Warwick- Edinburgh Mental Well-being Scale (WEMWBS) at age 60–64. The scale was designed to capture a broad con- cept of wellbeing, including both eudaimonic and hedo- nic aspects, and includes items on positive affect, satisfying interpersonal relationships and positive mental functioning. Statistical analysis The six PBI scale scores were standardised. Ordered logistic regression (for the outcomes happy/sociable child at age 13–15 and satisfaction with the home and family at age 43) and logistic regression (for life satis- faction at age 36) were used to estimates associations with perceived parental care and control. WEMWBS and Diener SWLS scores were approximately normally dis- tributed and their associations with perceived parental care and control were estimated using linear regression. We ﬁrst examined the associations between well-being indicators and care and control for mothers and fathers combined (that is, with each scale score summed across both parents). We then examined care and control from each parent separately (model 1). We examined whether associations between well-being indicators and the PBI scales differed by gender. Where gender interactions were not statistically signiﬁcant (at the 5% level), we present gender-adjusted analysis. We then included pater- nal and maternal care and control together (i.e. the six PBI scales were mutually adjusted, model 2). We com- pared the magnitudes of the estimates for paternal vs. maternal care (and paternal vs. maternal behavioural and psychological control) using Wald tests. Finally, we Regression estimates based on multiply imputed data were similar in magnitude and direction to those based on complete cases and the latter will not be described further. Associations between well-being at ages 13–15, 36, 43 and SWLS scores at 60–64 and the parental bonding scales are summarised in Table 2. There was no evidence of effect modiﬁcation by gender so gender-ad- justed analyses are presented. Higher levels of combined 330 M. Stafford et al. Table 1. Well-being indicators across the life course, parental care and control in men and women from the MRC National Survey of Health and Development. Statistical analysis Men % (n) Women % (n) P for gender difference Teacher-rated happiness/sociability at age 13–15 0 56.3 (1083) 59.3 (1053) 0.18 1 positive rating 23.7 (455) 21.6 (384) 2+ positive ratings 20.0 (385) 19.1 (340) Life been good at age 36 0.14 No 4.7 (78) 5.9 (97) Yes 95.3 (1568) 94.1 (1552) Satisfaction with home and family life at age 43 0.03 Very dissatisﬁed 0.9 (15) 0.5 (8) Dissatisﬁed 1.9 (31) 1.1 (17) Somewhat dissatisﬁed 3.3 (54) 2.9 (47) Neither satisﬁed nor dissatisﬁed 3.3 (53) 2.3 (37) Fairly satisﬁed 18.2 (294) 18.6 (300) Satisﬁed 29.0 (468) 26.9 (434) Very satisﬁed 43.4 (701) 47.8 (771) Mean (SD; n) Mean (SD; n) Diener Satisfaction with Life Scale at age 60–64 26.9 (5.8; 930) 26.5 (6.2; 1054) 0.1 Warwick Edinburgh Mental Wellbeing scale at age 60–64 51.6 (7.9; 930) 51.6 (8.3; 1046) 0.9 Paternal care (scale 0–36) 21.1 (7.4; 1507) 23.2 (8.1; 1479) <0.001 Paternal behavioural control (0–15) 13.7 (4.2; 1516) 12.6 (4.5; 1468) <0.001 Paternal psychological control (0–21) 3.3 (2.9;1514) 3.9 (3.3;1493) <0.001 Maternal care (0–36) 24.7 (6.2; 1543) 25.0 (7.2; 1522) 0.3 Maternal behavioural control 13.9 (3.9; 1553) 13.2 (4.1; 1544) <0.001 Maternal psychological control 4.4 (3.4;1557) 4.8 (3.5;1554) 0.008 Note: SD, standard deviation. 60–64 years. The magnitude of the maternal psychologi- cal control estimate was statistically signiﬁcantly greater than that of the paternal psychological control estimate at age 43. parental care were associated with higher well-being at ages 36, 43 and 60–64. Higher levels of combined par- ental psychological control were associated with poorer well-being at all ages (see also Table 3 for results for the WEMWBS scale). Associations between parental beha- vioural control and well-being were inconsistent in magnitude and direction across the different well-being indicators. In further analysis, we found no evidence of a non-linear association between well-being and beha- vioural control (data available on request). Gender modiﬁed the association between paternal care and WEMWBS scores at age 60–64, such that the positive association was larger in men than in women (Table 3, model 1). Paternal psychological control was negatively associated with WEMWBS scores. These associations remained on adjustment for maternal care and control (Table 3, model 2). In the model which con- sidered only the mother–child relationship, maternal care was positively and maternal psychological control nega- tively associated with WEMWBS score. Statistical analysis However, the association between maternal care and WEMWBS score was fully attenuated on adjustment for paternal care and control. Wald tests conﬁrmed a larger magnitude of association between paternal care (compared with mater- nal care) and well-being in men only (p = 0.001). When the relationship with each parent was consid- ered separately (Table 2, model 1), there was a consistent pattern of higher paternal care and higher maternal care (the latter with the exception of age 13–15) being associ- ated with greater well-being. There was also a general trend of higher paternal and maternal psychological control being associated with poorer well-being when parents were considered separately, although this did not achieve formal statistical signiﬁcance at all ages. Esti- mates from model 2 (which included care and control from both parents) were broadly similar, although adjust- ment for paternal care and control considerably attenu- ated the associations between maternal care and well- being at ages 36, 43, and 60–64 years, and between maternal psychological control and well-being at age 36. Post-estimation Wald tests revealed that the magnitude of the paternal care estimate was larger than that of the maternal care estimate at ages 13–15, 43 and Positive associations between paternal care and well- being at ages 13–15, 43 and 60–64 years remained on adjustment for childhood social class, parental separa- tion, mother’s mental health, extraversion and neuroti- cism at age 26, and the other ﬁve PBI scales (Table 4). Inverse associations between paternal psychological con- trol and two well-being indicators (at age 36 and WEMWBS score at age 60–64) also remained on adjust- ment for these covariates. Inverse associations between 331 The Journal of Positive Psychology Table 2. Associations between the PBI and four well-being indicators. M. Stafford et al. Model 1: includes care and control from one parent. Model 2: includes gender, care and control from both parents (mutually adjusted). psychological control with lower well-being at all ages. The same patterns were seen for paternal and maternal care and psychological control when each parent–child relationship was considered separately. In models which included the relationship with both parents, associations were attenuated, especially for maternal care and control. In interpreting these mutually adjusted estimates, it is important to note that our models estimated the statisti- cally independent associations of the six parental bond- ing scales but in reality, these are not independent. There is moderate correlation between mother’s and father’s care and control in this study and others’ (Belsky & Fearon, 2004; Martin, Ryan, & Brooks-Gunn, 2007), and other work indicates concordance in mother’s and father’s parenting is itself associated with children’s cognitive development (Van Bakel & Riksen-Walraven, 2002). maternal behavioural control and well-being at age 43 and between maternal psychological control and well-be- ing (at age 43 and WEMWBS score at age 60–64) also remained. More disadvantaged childhood social class was associated with lower likelihood of positive happi- ness/sociability at age 13–15 and of endorsing that life had been good up to age 36. It was not associated with the well-being indicators at later ages. As expected, greater extraversion at age 26 generally showed positive associations with mental well-being and greater neuroti- cism was associated with less positive mental well-being. However, personality did not substantially attenuate the associations of interest. Statistical analysis Happiness/sociability at age 13–15 (n = 3699 imputed) Life been good at age 36 (n = 3295 imputed) Satisfaction with home and family at age 43 (n = 3230 imputed) Diener Satisfaction With Life Scale at age 60–64 (n = 1984 imputed) Values are odds ratios (95% conﬁdence interval) Values are odds ratios (95% conﬁdence interval) Values are odds ratios (95% conﬁdence interval) Values are regression estimates (standard errors) Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 Model 1 Mode 2 Combined care 1.02 (0.97,1.07) 1.22 (1.10,1.37) 1.14 (1.08,1.19) 0.62 (0.10)** Combined behavioural control 1.00 (0.95,1.05) 1.01 (0.90,1.13) 0.97 (0.93,1.02) −0.21 (0.10)* Combined psychological control 0.95 (0.90,0.99)* 0.83 (0.76,0.92)** 0.95 (0.91,0.99)* −0.09 (0.09) Paternal care 1.08 (0.99,1.19) 1.13 (1.02,1.26)* 1.36 (1.12,1.66)* 1.27 (1.00,1.61)* 1.26 (1.16,1.36) 1.26 (1.15,1.38) 1.18 (0.17) 1.16 (0.20) Paternal behavioural control 1.03 (0.92,1.14) 1.07 (0.94,1.23) 0.97 (0.79,1.19) 0.95 (0.73,1.23) 1.00 (0.92,1.09) 1.11 (1.00,1.24) −0.39 (0.17)* −0.28 (0.23) Paternal psychological control 0.95 (0.87,1.04) 0.98 (0.88,1.10) 0.73 (0.62,0.87)** 0.77 (0.63,0.94)* 0.93 (0.87,1.00) 0.97 (0.90,1.06) −0.10 (0.16)** 0.02 (0.20) Maternal care 0.97 (0.88,1.05) 0.92 (0.83,1.03) 1.26 (1.03,1.53)* 1.16 (0.93,1.44) 1.13 (1.05,1.23)* 1.03 (0.94,1.12) 0.58 (0.17)** 0.07 (0.19) Maternal behavioural control 0.96 (0.87,1.06) 0.93 (0.81,1.05) 1.01 (0.82,1.23) 1.07 (0.83,1.38) 0.90 (0.83,0.97)* 0.85 (0.77,0.95)* −0.41 (0.18)* −0.15 (0.22) Maternal psychological control 0.92 (0.84,1.01) 0.93 (0.83,1.04) 0.81 (0.67,0.96)* 0.90 (0.73,1.10) 0.91 (0.85,0.98)* 0.92 (0.85,0.99)* −0.16 (0.15) −0.22 (0.19) Wald test for difference in coefﬁcients for paternal and maternal care p = 0.03 p = 0.6 p = 0.006 p = <0.001 Wald test for difference in coefﬁcients for paternal and maternal behavioural control p = 0.2 p = 0.6 p = 0.007 p = 0.9 Wald test for difference in coefﬁcients for paternal and maternal psychological control p = 0.6 p = 0.3 p = 0.4 p = 0.4 Notes: *p < 0.001; p < 0.05. Model 1: includes care and control from one parent. Model 2: includes gender, care and control from both parents (mutually adjusted). Notes: *p < 0.001; p < 0.05. Model 1: includes care and control from one parent. Model 2: includes gender, care and control from both parents (mutually adjusted). M. Stafford et al. M. Stafford et al. 332 ociations between the PBI and Warwick Edinburgh Mental Well-being scale at age 60–64. Table 3. Associations between the PBI and Warwick Edinburgh Mental Well-being scale at age 60–64. Table 3. Associations between the PBI and Warwick Edinburgh Mental Well-being scale at age 60–64. Warwick Edinburgh Mental Well-being at age 60–64 (n = 1976 imputed) Values are regression estimates (standard errors) Model 1 Model 2 Paternal care 1.15 (0.28) 1.03 (0.31) Paternal care × female −0.85 (0.39)* −0.85 (0.39)* Paternal behavioural control −0.00 (0.23) −0.13 (0.30) Paternal psychological control −1.00 (0.20)** −0.72 (0.25)* Maternal care 0.84 (0.23) 0.26 (0.26) Maternal behavioural control 0.04 (0.24) 0.25 (0.30) Maternal psychological control −0.86 (0.21) −0.51 (0.25)* Combined care 0.83 (0.13) Combined behavioural control 0.07 (0.13) Combined psychological control −0.61 (0.12) Wald test for difference in coefﬁcients for paternal and maternal care: men p = 0.001 Women p = 0.1 Wald test for difference in coefﬁcients for paternal and maternal behavioural control p = 0.4 Wald test for difference in coefﬁcients for paternal and maternal psychological control p = 0.8 Notes: *p < 0.001; p < 0.05; SE standard error. Model 1: includes care and control from one parent. Model 2: includes gender, care and control from both parents (mutually adjusted). Warwick Edinburgh Mental Well-being at age 60–64 (n = 1976 imputed) Model 1 Model 2 Paternal care 1.15 (0.28) 1.03 (0.31) Paternal care × female −0.85 (0.39)* −0.85 (0.39)* Paternal behavioural control −0.00 (0.23) −0.13 (0.30) Paternal psychological control −1.00 (0.20)** −0.72 (0.25)* Maternal care 0.84 (0.23) 0.26 (0.26) Maternal behavioural control 0.04 (0.24) 0.25 (0.30) Maternal psychological control −0.86 (0.21) −0.51 (0.25)* Combined care 0.83 (0.13) Combined behavioural control 0.07 (0.13) Combined psychological control −0.61 (0.12) Wald test for difference in coefﬁcients for paternal and maternal care: men p = 0.001 Women p = 0.1 Wald test for difference in coefﬁcients for paternal and maternal behavioural control p = 0.4 Wald test for difference in coefﬁcients for paternal and maternal psychological control p = 0.8 Notes: *p < 0.001; p < 0.05; SE standard error. Model 1: includes care and control from one parent. Model 2: includes gender, care and control from both parents (mutually adjusted). Wald test for difference in coefﬁcients for paternal and maternal behavioural control Wald test for difference in coefﬁcients for paternal and maternal psychological control Notes: *p < 0.001; p < 0.05; SE standard error. Discussion We have shown that recalled parental care and psycho- logical control are associated with positive mental well- being at several points in the life course. Associations between well-being and parental behavioural control were generally not found, though we note that this con- struct was captured by only ﬁve items and had accept- able though somewhat lower internal consistency than the other two parental bonding scales considered here. The PBI has sometimes been used to categorise par- ticipants into groups described as having an authoritative parenting style (that is, high care and high control), indulgent style (that is, high care and low control) or authoritarian style (that is, low care and high control). Although a combination of high care and high beha- vioural control is theoretically optimal, several studies ﬁnd, as we did, that high care is beneﬁcial for well-be- ing, self-esteem and social competence irrespective of the level of control (García & Gracia, 2009; Rothrauff et al., 2009; Schoﬁeld et al., 2012). It has been proposed that caring parenting, even in the absence of parental behavioural control, may encourage children to model We hypothesised that well-being would be positively associated with parental care and negatively associated with parental psychological control. This hypothesis was supported. Combined levels of parental care from both parents were associated with higher well-being through- out adulthood and combined levels of parental 333 The Journal of Positive Psychology Table 4. Fully adjusted associations between the PBI and well-being indicators from age 13–15 to age 60–64. Discussion Happiness/sociability at age 13–15 (n = 3699 imputed) Life been good at age 36 (n = 3295 imputed) Satisfaction with home and family at age 43 (n = 3230 imputed) Diener Satisfaction With Life Scale at age 60–64 (n = 2214 imputed) Warwick Edinburgh Mental Wellbeing at age 60–64 (n = 1976 imputed) Odds ratio (95% CI) Odds ratio (95% CI) Odds ratio (95% CI) Regression coefﬁcient (SE) Regression coefﬁcient (SE) Paternal care 1.11 (1.00,1.23)* 1.20 (0.95,1.52) 1.24 (1.13,1.36) 1.08 (0.20) 0.94 (0.30)** Paternal care × female −0.67 (0.38) Paternal behavioural control 1.05 (0.92,1.20) 0.94 (0.72,1.21) 1.10 (0.99,1.23) −0.32 (0.23) −0.22 (0.30) Paternal psychological control 0.99 (0.88,1.10) 0.78 (0.61,0.95)* 0.98 (0.90,1.07) 0.07 (0.19) −0.67 (0.24)* Maternal care 0.91 (0.82,1.02) 1.14 (0.92,1.41) 1.02 (0.93,1.12) 0.02 (0.19) 0.15 (0.25) Maternal behavioural control 0.94 (0.82,1.06) 1.03 (0.80,1.33) 0.86 (0.78,0.96)* −0.13 (0.22) 0.28 (0.30) Maternal psychological control 0.94 (0.84,1.05) 0.93 (0.76,1.14) 0.92 (0.85,1.00)* −0.20 (0.18) −0.48 (0.24)* Female gender 0.94 (0.82,1.08) 0.95 (0.67,1.34) 1.26 (1.09,1.45)* −0.12 (0.28) 0.68 (0.38) Childhood social classa 0.94 (0.89,0.99)* 0.81 (0.71,0.91)* 1.04 (0.99,1.10) 0.08 (0.10) −0.07 (0.14) Parental separation 1.07 (0.81,1.41) 0.83 (0.46,1.48) 1.09 (0.82,1.45) 0.48 (0.58) 0.88 (0.78) Mother’s neuroticismb 1.00 (0.96,1.04) 0.90 (0.81,0.99)* 1.00 (0.96,1.05) −0.06 (0.10) −0.33 (0.12)* Extraversion at age 26 1.09 (1.07,1.12) 1.02 (0.96,1.07) 1.05 (1.03,1.08) 0.12 (0.04)* 0.29 (0.06) Neuroticism at age 26 1.00 (0.98,1.02) 0.91 (0.86,0.95) 0.97 (0.95,0.98) −0.20 (0.04) −0.28 (0.05) Notes: p < 0.001; *p < 0.05; CI conﬁdence interval; SE standard error. aBased on father’s occupation with higher values indicating more disadvantaged class. bBased on Maudsley Personality Inventory with higher values indicating greater neuroticism. M. Stafford et al. 334 their parents’ supportive, prosocial behaviour, and that the school and other social environments beyond the family may provide discipline and control (Rothrauff et al., 2009). maternal role, and because fathers (more so than mothers) may respond in different ways to sons and daughters (Parke, 2002). We found some evidence that paternal care was more strongly associated with well-being than maternal care. The relative importance of the father–child and mother– child relationships for well-being in adulthood has previ- ously been assessed in only three studies. The largest of these highlighted the importance of paternal care for Ryff’s well-being scales and used data from a subsample of women from the same cohort as the current study (Huppert et al., 2010). Limitations of the current study Parental care and control were reported retrospectively by the study members themselves when they were aged 43, at which time associations with well-being were strongest and may plausibly be due to memories of parental care and control being inﬂuenced by current well-being. Nevertheless, we have considered a range of well-being indicators and not all were completed concur- rently with the PBI, suggesting that the associations seen at age 43 are not simply due to reporting or recall bias. Recalled parental bonding on this instrument shows a high level of agreement between twins and other siblings in other studies (Mackinnon, Henderson, & Andrews, 1991; McCrae & Costa, 1988) and correlates in the expected direction with prospectively assessed childhood variables in this cohort (Rodgers, 1996a), thus providing some validation for its retrospective administration. However, relationships with parents and the perceptions of those relationships may change with age and we are unable to account for this. We do not have information about parental care and control in speciﬁc phases of the ﬁrst 16 years of life. In particular, the effect on psycho- logical distress of parental control may be stronger in critical childhood periods (Rutter, 1995) and appears to vary between childhood and adolescence in some studies (Verhoeven, Bogels, & van der Bruggen, 2012). Father’s care has been theorised to be more closely related to positive psychological development in adolescence, whereas mother’s care may be more important in child- hood (Bögels & Phares, 2008), though evidence is inconsistent (McLeod et al., 2007). The well-being indicators included in earlier years of this study were not validated and reﬂect a lesser emphasis on positive mental outcomes prevalent at the time. We were unable to look at changes in well-being and, as noted, the indicator used and the age at which the outcome was assessed were confounded. However, our aim was to explore consis- tency or otherwise of associations between parenting and a range of well-being indicators. Despite the differences in measurement, our ﬁndings are consistent in highlight- ing the potential importance of perceived paternal care. The extent to which these ﬁndings can be generalised is unclear. Unmarried mothers were not part of the original sampling frame for this cohort and lone parents now make up a larger proportion of all parents than they did in earlier decades (Haskey, 1998). Discussion Our results showed that associa- tions between paternal care and well-being at ages 13–15, 43 and 60–64 years were larger in magnitude than associations between maternal care and well-being at these ages and are in accordance with Huppert and colleagues. However, at age 43 (but not at other ages), we found that maternal psychological control was more strongly related to well-being than paternal psychological control. Two smaller, non-representative studies found greater importance of maternal compared with paternal care for happiness in young adulthood (Cheng & Furnham, 2004; Furnham & Cheng, 2000). One reason for the differing ﬁndings may be that studies used differ- ent outcomes. The importance of maternal vs. paternal care may depend on the outcome of interest. Others have noted the equal importance of father’s and mother’s care for infant and childhood outcomes (Boyce et al., 2006; Flouri & Buchanan, 2003), though through different mechanisms (Grossman et al., 2002). Another explana- tion may be that the perceived relationship with one’s mother is more important at particular life stages, possi- bly related to one’s own experience as a parent. This is speculative, however, and the current study is limited in not being able to separate the outcome assessment from the measurement age. Conclusion Our study provides evidence from a large, general pop- ulation study that the quality of the parent–child relation- ship, along the dimensions of perceived care and psychological control, may have short and long-term consequences for positive mental well-being. These results indicate that relationships with fathers and moth- ers which are supportive, affectionate and allow the child appropriate autonomy may promote good psychological functioning across life up to and including the seventh decade. Our ﬁndings support the view that the founda- tions for lifelong well-being begin in the early years, or before (HM Government, 2011), and have identiﬁed par- ent–child relationships as a key part of that foundation. This study shows that there are potential long-term gains to taking better emotional care of children, not only by reducing burden of physical (Stewart-Brown, Fletcher, & Wadsworth, 2005) and mental illness (Rodgers, 1996a), but also by improving population mental well-being. These ﬁndings reiterate the need to develop parenting programmes and other initiatives which support the development and maintenance of relationships charac- terised by high levels of care and low levels of psycho- logical control between children and their fathers as well as mothers. Bowlby, J. (1988). A secure base: Clinical applications of attachment theory. London: Routledge. Boyce, W. T., Essex, M. J., Alkon, A., Goldsmith, H. H., Kraemer, H. C., & Kupfer, D. J. (2006). Early father involvement moderates biobehavioral susceptibility to men- tal health problems in middle childhood. Journal of the American Academy of Child & Adolescent Psychiatry, 45, 1510–1520. Charles, S., & Carstensen, L. L. (2010). Social and emotional aging. Annual Review of Psychology, 61, 383–409. Cheng, H., & Furnham, A. (2004). Perceived parental rearing style, self-esteem and self-criticism as predictors of happiness. Journal of Happiness Studies, 5, 1–21. pp f pp Diener, E., Emmons, R. A., Larsen, R. J., & Grifﬁn, S. (1985). The satisfaction with life scale. Journal of Personality Assessment, 49, 71–75. Diener, E., Ng, W., Harter, J., & Arora, R. (2010). Wealth and happiness across the world: Material prosperity predicts life evaluation, whereas psychosocial prosperity predicts posi- tive feeling. Journal of Personality and Social Psychology, 99, 52–61. Dornbusch, S. M., Ritter, P. L., Leiderman, P. H., Roberts, D. F., & Fraleigh, M. J. (1987). The relation of parenting style to adolescent school performance. Child Development, 58, 1244–1257. Enns, M. W., Cox, B. J., & Clara, I. (2002). Disclosure statement No potential conﬂict of interest was reported by the authors. Flouri, E., & Buchanan, A. (2003). The role of father involve- ment in children’s later mental health. Journal of Adolescence, 26, 63–78. Conclusion Parental bonding and adult psychopathology: Results from the US National Comorbidity Survey. Psychological Medicine, 32, 997–1008. Flouri, E. (2004). Psychological outcomes in midadulthood associated with mother’s child-rearing attitudes in early childhood. Evidence from the 1970 British birth cohort. European Child & Adolescent Psychiatry, 13, 35–41. Limitations of the current study However, it is impor- tant to note that recalled parental care and control were not patterned by childhood socio-economic circum- stances in this cohort, thus eliminating an important confounding structure that may affect later cohorts For four of the ﬁve well-being indicators, we found no evidence of effect modiﬁcation by gender. For mental well-being measured by the Warwick–Edinburgh Mental Well-being scale at age 60–64, paternal care was posi- tively associated in both genders, but especially in men. Scholars have suggested that identiﬁcation with the same-gender parent would be greater and that this would encourage sons (daughters) to internalise the behaviours, values and cognitions of their fathers (mothers) (Basuil & Casper, 2012; Hoeve et al., 2009). In this way, boys may be more likely than girls to internalise their fathers caring, supporting and warm attitudes in ways which improve their own interpersonal functioning and affect, as captured by the Warwick–Edinburgh Mental Well-be- ing scale. It has been suggested that child gender may be particularly important to consider when examining father–child relationships because there may be greater ﬂexibility in the paternal role compared with the The Journal of Positive Psychology 335 Belsky, J., & Fearon, R. M. P. (2004). Exploring marriage-parent- ing typologies and their contextual antecedents and developmental sequelae. Development and Psychopathology, 16, 501–523. (Kiernan & Huerta, 2008). As with all longitudinal stud- ies, those lost to follow-up tend to be more socio-eco- nomically disadvantaged and in poorer health than those remaining in the study to early older age, yet the cohort continues to be broadly representative on several key demographics, including occupational social class and employment status (Stafford et al., 2013). (Kiernan & Huerta, 2008). As with all longitudinal stud- ies, those lost to follow-up tend to be more socio-eco- nomically disadvantaged and in poorer health than those remaining in the study to early older age, yet the cohort continues to be broadly representative on several key demographics, including occupational social class and employment status (Stafford et al., 2013). Blatt, S. J., & Homann, E. (1992). 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Stafford, M., Black, S., Shah, I., Hardy, R., Pierce, M., Richards, M., … Kuh, D. (2013). Using a birth cohort to study ageing: Representativeness and response rates in the The Journal of Positive Psychology 337 National Survey of Health and Development. European Journal of Ageing, 10, 145–157. Wang, Q., Pomerantz, E. M., & Chen, H. (2007). The role of parents’ control in early adolescents’ psychological func- tioning: A longitudinal investigation in the United States and China. Child Development, 78, 1592–1610. f g g Stewart-Brown, S. L., Fletcher, L., & Wadsworth, M. E. (2005). Parent–child relationships and health problems in adulthood in three UK national birth cohort studies. The European Journal of Public Health, 15, 640–646. Wilhelm, K., Niven, H., Parker, G., & Hadzi-Pavlovic, D. (2005). The stability of the Parental Bonding Instrument over a 20-year period. References Psychological Medicine, 35, 387–393. Tennant, R., Hiller, L., Fishwick, R., Platt, S., Joseph, S., Weich, S., … Stewart-Brown, S. (2007). The Warwick-Edinburgh Mental Well-being Scale (WEMWBS): Development and UK validation. Health and Quality of Life Outcomes, 5, 63. Williams, K. E., Ciarrochi, J., & Heaven, P. C. L. (2012). Inﬂexible parents, inﬂexible kids: A 6-year longitudinal study of parenting style and the development of psycho- logical ﬂexibility in adolescents. Journal of Youth and Adolescence, 41, 1053–1066. Van Bakel, H. J. A., & Riksen-Walraven, J. M. (2002). Parent- ing and development of one-year olds: Links with parental, contextual, and child characteristics. Child Development, 73, 256–273. van Zeijl, J., Mesman, J., Stolk, M. N., Alink, L. R., van IJzendoorn, M. H., Bakermans-Kranenburg, M. J., … Koot, H. M. (2007). Differential susceptibility to discipline: The moderating effect of child temperament on the association between maternal discipline and early childhood externaliz- ing problems. 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https://openalex.org/W4283210710	https://zenodo.org/record/6676410/files/russo_2022_oi_220519_1654705551.26492.pdf	English	null	Efficacy and Safety of Varenicline for Smoking Cessation in Patients With Type 2 Diabetes	JAMA network open	2,022	cc-by	10,246	Original Investigation \| Diabetes and Endocrinology Efficacy and Safety of Varenicline for Smoking Cessation in Patients With Type 2 Diabetes A Randomized Clinical Trial Cristina Russo, MD; Magdalena Walicka, MD, PhD; Pasquale Caponnetto, PhD; Fabio Cibella, MD; Marilena Maglia, MD; Angela Alamo, MD; Davide Campagna, MD; Lucia Frittitta, MD, PhD; Maurizio Di Mauro, MD, PhD; Grazia Caci, MD; Arkadiusz Krysinski, MD, PhD; Edward Franek, MD, PhD; Riccardo Polosa, MD, PhD OBJECTIVE To evaluate the efficacy and safety of varenicline in patients with type 2 diabetes with an intention to quit smoking. OBJECTIVE To evaluate the efficacy and safety of varenicline in patients with type 2 diabetes with an intention to quit smoking. DESIGN, SETTING, AND PARTICIPANTS This multicenter, double-blind, placebo-controlled randomized clinical trial recruited patients from 6 outpatient clinics in 5 hospitals in Catania, Italy. Patients with type 2 diabetes, who were smoking at least 10 cigarettes a day, and who intended to quit smoking were screened for eligibility. Eligible patients were randomized to either varenicline or placebo treatment. The trial consisted of a 12-week treatment phase followed by a 40-week follow-up, nontreatment phase. Intention-to-treat data analysis was performed from December 2020 to April 2021. INTERVENTIONS Varenicline, 1 mg, twice daily or matched placebo administered for 12 weeks. Patients in both treatment groups also received smoking cessation counseling. Meaning Findings of this study suggest that varenicline should be included in diabetes education programs to help patients with type 2 diabetes quit smoking. MAIN OUTCOMES AND MEASURES The primary efficacy end point of the study was the continuous abstinence rate (CAR) at weeks 9 to 24. Secondary efficacy end points were the CAR at weeks 9 to 12 and weeks 9 to 52 as well as 7-day point prevalence of abstinence at weeks 12, 24, and 52. RESULTS A total of 300 patients (mean [SD] age, 57.4 [0.8] years; 117 men [78.0%] in varenicline group and 119 men [79.3%] in placebo group) were randomized to receive varenicline (n = 150) or placebo (n = 150). The CAR at weeks 9 to 24 was significantly higher for the varenicline than placebo group (24.0% vs 6.0%; odds ratio [OR], 4.95; 95% CI, 2.29-10.70; P < .001). The CARs at weeks 9 to 12 (31.3% vs 7.3%; OR, 5.77; 95% CI, 2.85-11.66; P < .001) and weeks 9 to 52 (18.7% vs 5.3%; OR, 4.07; 95% CI, 1.79-9.27; P < .001) as well as the 7-day point prevalence of abstinence at weeks 12, 24, and 52 were also significantly higher for the varenicline vs placebo group. The most frequent adverse events occurring in the varenicline group compared with the placebo group were nausea (41 [27.3%] vs 17 [11.4%]), insomnia (29 [19.4%] vs 19 [12.7%]), abnormal dreams (19 [12.7%] vs 5 [3.4%]), anxiety (17 [11.4%] vs 11 [7.3%]), and irritability (14 [9.4%] vs 8 [5.4%]). Serious adverse events were infrequent in both groups and not treatment-related. (continued) Open Access. OBJECTIVE To evaluate the efficacy and safety of varenicline in patients with type 2 diabetes with an intention to quit smoking. This is an open access article distributed under the terms of the CC-BY-NC-ND License. Open Access. This is an open access article distributed under the terms of the CC-BY-NC-ND License. June 21, 2022 1/14 JAMA Network Open. 2022;5(6):e2217709. doi:10.1001/jamanetworkopen.2022.17709 + Visual Abstract + Multimedia + Supplemental content Author affiliations and article information are listed at the end of this article. Key Points Question Is varenicline efficacious and safe in achieving long-term abstinence in patients with type 2 diabetes who are willing to quit smoking? Question Is varenicline efficacious and safe in achieving long-term abstinence in patients with type 2 diabetes who are willing to quit smoking? IMPORTANCE Evidence of effective smoking cessation interventions in patients with diabetes is limited. The unique behavioral and metabolic characteristics of smokers with type 2 diabetes warrants a randomized clinical trial of the smoking cessation drug varenicline. Findings In this randomized clinical trial involving 300 patients with type 2 diabetes who smoked, varenicline was efficacious at weeks 12, 24, and 52 of the trial compared with placebo. Nausea, insomnia, abnormal dreams, anxiety, and irritability were reported at higher frequency among patients in the varenicline vs placebo group. No treatment-related serious adverse events were noted. Original Investigation \| Diabetes and Endocrinology Original Investigation \| Diabetes and Endocrinology Original Investigation \| Diabetes and Endocrinology Efficacy and Safety of Varenicline for Smoking Cessation in Patients With Type 2 Diabetes A Randomized Clinical Trial Introduction Cigarette smoking is a major risk factor for cardiovascular disease1 and is highly prevalent among patients with type 2 diabetes.2 It is not surprising that smoking exacerbates the combined harmful effects of hyperglycemia and other risk factors, accelerating vascular damage in patients with diabetes.3 Increased risk of total mortality, coronary heart disease, stroke, and peripheral arterial disease has been observed in smokers with diabetes compared with nonsmokers with diabetes.3,4 Consistent with these observations is that stopping smoking has been associated with reduced mortality risk in patients with type 2 diabetes.5,6 Moreover, patients with type 2 diabetes who participated in smoking cessation programs were found to have better glycemic control and lower cardiometabolic risk factors.7 Therefore, counseling on quitting smoking and treatment of tobacco addiction should be components of routine diabetes care.8,9 Behavioral counseling and smoking cessation medications are generally combined in efforts to help people quit smoking. Consideration of smoking cessation methods should include the individual circumstances and underlying diseases of the smoker. High-quality evidence supports the benefits of individually delivered smoking cessation counseling,10 and combining smoking cessation medications with counseling can substantially reduce smoking rates.11-13 Nonetheless, the evidence for efficacious smoking cessation interventions in patients with diabetes is limited.9,14 Randomized clinical trials (RCTs) have indicated that varenicline is more efficacious for smoking cessation than placebo, bupropion,15,16 or nicotine replacement therapy.17 Varenicline has been shown to be efficacious also in smokers with cardiovascular disease,18 chronic obstructive pulmonary disease,19 or depressive disorders.20 The efficacy and safety of varenicline in smokers with diabetes have not been extensively studied, however. A retrospective pooled analysis of 15 RCTs including 323 smokers with type 2 diabetes found that varenicline was efficacious for these patients and had a safety profile that was similar to that for smokers without diabetes.21 Smokers with diabetes may differ from other smokers regarding not only risks but also reasons for smoking and motivations to quit. Evidence suggests that they are less motivated to quit than other smokers, possibly because they fear weight gain.22,23 When assisting patients with diabetes in quitting smoking, health care professionals should closely monitor metabolic parameters and not assume that these parameters will improve.9 In particular, prevention of weight gain after smoking cessation must be considered a top priority because of its detrimental implications for several metabolic parameters and diabetic control. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT01387425 JAMA Network Open. 2022;5(6):e2217709. doi:10.1001/jamanetworkopen.2022.17709 Introduction For example, Lycett et al24 found among 3131 adult smokers with type 2 diabetes who quit smoking, an increase in hemoglobin A1c (HbA1c) level within the first year after quitting. However, to our knowledge, there has been no randomized, prospective study conducted of varenicline use in patients with type 2 diabetes. Rigorous research is required to guide the decisions of health authorities and clinicians. The aim of this multicenter, double-blind, placebo-controlled RCT was to evaluate the efficacy and safety of varenicline in patients with type 2 diabetes with an intention to quit smoking. JAMA Network Open. 2022;5(6):e2217709. doi:10.1001/jamanetworkopen.2022.17709 June 21, 2022 2/14 Abstract (continued) Abstract (continued) CONCLUSIONS AND RELEVANCE Results of this trial showed that inclusion of varenicline in a smoking cessation program is efficacious in achieving long-term abstinence without serious adverse events. Varenicline should be routinely used in diabetes education programs to help patients with type 2 diabetes stop smoking. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT01387425 Downloaded From: https://jamanetwork.com/ on 06/21/2022 JAMA Network Open \| Diabetes and Endocrinology Efficacy and Safety of Varenicline in Patients With Type 2 Diabetes June 21, 2022 2/14 Participants Patients with type 2 diabetes who were receiving regular treatment in an outpatient diabetic clinic, smoking at least 10 cigarettes a day, and intending to quit were screened for eligibility. The first 300 consecutive patients who were eligible were recruited from 6 outpatient clinics located in 5 hospitals in Catania, Italy (Ambulatorio di Diabetologia, Unità Operativa Complessa [UOC]; Medicina Interna E D'Urgenza, Policlinico Universitario; Centro Diabetologico-UOC Andrologia Ed Endocrinologia, Policlinico Universitario; Centro per il Diabete e l'Obesità-UOC Endocrinologia, Ospedale Garibaldi Nesima; and Centro per il Diabete, Ospedale Cannizzaro). Inclusion criteria were as follows: (1) aged 75 years or younger; (2) met the American Diabetes Association diagnostic criteria for type 2 diabetes25; (3) type 2 diabetes diagnosis for more than 12 months; (4) HbA1c level between 7.0% and 12.0% (to convert to proportion of total hemoglobin, multiply by 0.01); (5) smoked 10 or more cigarettes per day for the past year; and (6) willingness to quit smoking as confirmed by a yes answer to both of these questions, Do you plan to quit smoking within the next 30 days? and Do you wish to participate in a smoking cessation program? Exclusion criteria were as follows: (1) mental illness, including major depression, panic disorder, psychosis, or bipolar disorder that was diagnosed and treated by psychiatrists or clinical psychologists; (2) history of alcoholism or substance use disorder within 12 months before screening; (3) use of medications that could interfere with the efficacy of the study drug (eg, nicotine replacement therapy); (4) body mass index (BMI; calculated as weight in kilograms divided by height in meters squared) of less than 18.5 or greater than 34.9; (5) known clinically significant medical condition, including any recent (within the past 4 weeks) acute decompensation of type 2 diabetes that required hospital treatment and any history of neurological, gastrointestinal, kidney, hepatic, cardiovascular, psychiatric, respiratory, metabolic, endocrine, hematologic, or other major disorder that, in our opinion, would compromise the safety or participation of the patient; and (6) current pregnancy or breast feeding or intention to become pregnant during the trial. Eligible patients were randomized to receive the active drug or placebo. The flow diagram of patients is shown in Figure 1. Recruitment started in June 2011 and concluded in December 2014. Most screening failures were associated with patients’ unwillingness to quit and the presence of clinically significant comorbidities. Last follow-up visit was completed in December 2015. Downloaded From: https://jamanetwork.com/ on 06/21/2022 JAMA Network Open \| Diabetes and Endocrinology JAMA Network Open \| Diabetes and Endocrinology Efficacy and Safety of Varenicline in Patients With Type 2 Diabetes Methods The ethical review board of the leading site, Azienda Ospedaliero Universitaria Policlinico-Vittorio Emanuele, Università di Catania, reviewed and approved this RCT (Supplement 1). All enrolled patients provided written informed consent before participation in the trial. We followed the Consolidated Standards of Reporting Trials (CONSORT) reporting guideline. Trial Design and Study Visits This RCT compared the efficacy and safety of varenicline, 1 mg, administered twice daily for 12 weeks vs placebo (1 mg, administered twice daily, for 12 weeks) for smoking cessation in patients with type 2 diabetes. The total duration of the study was 52 weeks. The 12-week treatment phase was followed by a 40-week nontreatment phase (Figure 2). Details of the trial design and protocol were described in a previous publication.26 After screening, eligible patients were invited to attend the baseline visit (visit 1), during which the eligibility criteria were reviewed and patients were randomized 1:1 to either varenicline or placebo. The list for treatment randomization was generated using SAS software (SAS Institute). The size of the blocks was a variable of 3, and the sequence of blocks was randomized and blinded. Sociodemographic characteristics, medical history, drug prescriptions, smoking history, and motivation for quitting were self-reported at baseline. Race and ethnicity were investigator observed and included White individuals. JAMA Network Open. 2022;5(6):e2217709. doi:10.1001/jamanetworkopen.2022.17709 June 21, 2022 3/14 ork Open. 2022;5(6):e2217709. doi:10.1001/jamanetworkopen.2022.1 Downloaded From: https://jamanetwork.com/ on 06/21/2022 JAMA Network Open \| Diabetes and Endocrinology Efficacy and Safety of Varenicline in Patients With Type 2 Diabetes The following baseline measures were recorded: number of cigarettes smoked per day, exhaled carbon monoxide (eCO) levels, blood pressure, heart rate, BMI, waist circumference, scores on various questionnaires, and adverse events (AEs). Blood and urine samples were collected for laboratory tests (chemistry profile, including fasting blood glucose, HbA1c level, fasting insulin level, and lipid profile; microalbuminuria; and proteinuria). Patients received smoking cessation counseling and were instructed to set a target quit date within the next 5 to 8 days. Before checkout, patients were given a full week’s supply of the study drug, either varenicline or placebo, depending on the treatment group to which they were randomized. Study drugs were dispensed in accordance with the plan (eTable 1 in Supplement 2) After visit 1, patients were invited to return to their respective outpatient clinic on a weekly basis for the next 12 weeks (visits 2-13), except for visits 6, 8, 10, and 12, which were conducted by telephone contact. Patients attending visit 2 for their target quit date must have abstained from smoking or substantially reduced cigarette consumption. Regardless of their level of cigarette consumption, all patients were followed up. At each visit, patients were reminded of the importance for the next 12 weeks (visits 2 13), except for visits 6, 8, 10, and 12, which were conducted by telephone contact. Patients attending visit 2 for their target quit date must have abstained from smoking or substantially reduced cigarette consumption. Regardless of their level of cigarette consumption, all patients were followed up. At each visit, patients were reminded of the importance Figure 1. Downloaded From: https://jamanetwork.com/ on 06/21/2022 Study Flow Diagram of Study Participants 514 Patients assessed for eligibility 214 Excluded 124 Were unwilling to quit smoking 53 Had clinically significant comorbidities 19 Had current or past major depressive disorders 10 Had hemoglobin A1c level greater than 12% 8 Had other reasons 24-wk Nontreatment phase 10 Lost to follow-up 100 Completed follow-up 150 Included in analysis 24-wk Nontreatment phase 4 Lost to follow-up 101 Completed follow-up 150 Included in analysis 52-wk Nontreatment phase 6 Lost to follow-up 94 Completed follow-up 150 Included in analysis 52-wk Nontreatment phase 9 Lost to follow-up 92 Completed follow-up 150 Included in analysis 12-wk Treatment phase 150 Randomized to varenicline 150 Received intervention 40 Discontinued treatment 110 Completed treatment phase 12-wk Treatment phase 150 Randomized to placebo 150 Received intervention 45 Discontinued treatment 105 Completed treatment phase 300 Randomized To convert hemoglobin A1C to proportion of total hemoglobin, multiply by 0.01. Figure 1. Study Flow Diagram of Study Participants 514 Patients assessed for eligibility 214 Excluded 124 Were unwilling to quit smoking 53 Had clinically significant comorbidities 19 Had current or past major depressive disorders 10 Had hemoglobin A1c level greater than 12% 8 Had other reasons 24-wk Nontreatment phase 10 Lost to follow-up 100 Completed follow-up 150 Included in analysis 24-wk Nontreatment phase 4 Lost to follow-up 101 Completed follow-up 150 Included in analysis 52-wk Nontreatment phase 6 Lost to follow-up 94 Completed follow-up 150 Included in analysis 52-wk Nontreatment phase 9 Lost to follow-up 92 Completed follow-up 150 Included in analysis 12-wk Treatment phase 150 Randomized to varenicline 150 Received intervention 40 Discontinued treatment 110 Completed treatment phase 12-wk Treatment phase 150 Randomized to placebo 150 Received intervention 45 Discontinued treatment 105 Completed treatment phase 300 Randomized To convert hemoglobin A1C to proportion of total hemoglobin, multiply by 0.01. Figure 1. Study Flow Diagram of Study Participants To convert hemoglobin A1C to proportion of total hemoglobin, multiply by 0.01. Figure 2. Schematic Diagram of the Study Design Baseline Quit date Week Treatment phase Nontreatment phase 1 0 2 3 4 5 6 7 8 9 10 11 12 13 24 36 44 52 Screening Randomization Placebo N = 300 Varenicline 1 mg twice a day Smokers with type 2 diabetes who intended to quit in the next 30 days were randomized to receive either varenicline, 1 mg, twice daily for 12 weeks or matched placebo for 12 weeks. Downloaded From: https://jamanetwork.com/ on 06/21/2022 Study Flow Diagram of Study Participants 514 Patients assessed for eligibility 214 Excluded 124 Were unwilling to quit smoking 53 Had clinically significant comorbidities 19 Had current or past major depressive disorders 10 Had hemoglobin A1c level greater than 12% 8 Had other reasons 24-wk Nontreatment phase 10 Lost to follow-up 100 Completed follow-up 150 Included in analysis 24-wk Nontreatment phase 4 Lost to follow-up 101 Completed follow-up 150 Included in analysis 52-wk Nontreatment phase 6 Lost to follow-up 94 Completed follow-up 150 Included in analysis 52-wk Nontreatment phase 9 Lost to follow-up 92 Completed follow-up 150 Included in analysis 12-wk Treatment phase 150 Randomized to varenicline 150 Received intervention 40 Discontinued treatment 110 Completed treatment phase 12-wk Treatment phase 150 Randomized to placebo 150 Received intervention 45 Discontinued treatment 105 Completed treatment phase 300 Randomized To convert hemoglobin A1C to proportion of total hemoglobin, multiply by 0.01. Figure 2. Schematic Diagram of the Study Design Baseline Quit date Week Treatment phase Nontreatment phase 1 0 2 3 4 5 6 7 8 9 10 11 12 13 24 36 44 52 Screening Randomization Placebo N = 300 Varenicline 1 mg twice a day Smokers with type 2 diabetes who intended to quit in the next 30 days were randomized to receive either varenicline, 1 mg, twice daily for 12 weeks or matched placebo for 12 weeks. Participants were prospectively reviewed for up to 52 weeks during which smoking habits, exhaled carbon monoxide levels, questionnaire answers, adverse events, vital signs, waist circumference, body mass index, and laboratory test results were assessed at each visit. Dashed lines indicate follow-up phase; telephone symbol, telephone contact. JAMA Network Open. 2022;5(6):e2217709. doi:10.1001/jamanetworkopen.2022.17709 (Reprinted) June 21, 2022 4/14 Figure 1. Downloaded From: https://jamanetwork.com/ on 06/21/2022 Participants were prospectively reviewed for up to 52 weeks during which smoking habits, exhaled carbon monoxide levels, questionnaire answers, adverse events, vital signs, waist circumference, body mass index, and laboratory test results were assessed at each visit. Dashed lines indicate follow-up phase; telephone symbol, telephone contact. Week June 21, 2022 4/14 Downloaded From: https://jamanetwork.com/ on 06/21/2022 Downloaded From: https://jamanetwork.com/ on 06/21/2022 JAMA Network Open \| Diabetes and Endocrinology JAMA Network Open \| Diabetes and Endocrinology Efficacy and Safety of Varenicline in Patients With Type 2 Diabetes of quitting smoking and were offered professional counseling to help with relapse prevention. Number of cigarettes smoked per day, eCO levels, blood pressure, heart rate, and any AEs were recorded in the case report form at each study visit; BMI and waist circumference were recorded only at the week-12 visit. At week 2 (visit 3) and week 12 (visit 13), blood and urine samples were collected for laboratory tests. Study drugs were dispensed before checkout in accordance with the plan (eTable 1 in Supplement 2) Completion of the treatment phase (visits 2-13) was followed by the nontreatment phase (visits 14-18). Only patients who completed their treatment schedule were invited to the nontreatment follow-up phase. Except for visits 16 and 17 (telephone contact), patients were invited to return to the clinic for week 13 (visit 14), week 24 (visit 15), and week 52 (visit 18). Number of cigarettes smoked per day, eCO levels, blood pressure, and heart rate were recorded in the case report form at each study visit; BMI and waist circumference were recorded only at the week-24 and week-52 visits. At week 24 (visit 15) and week 52 (visit 18), blood and urine samples were collected for laboratory tests. All randomized patients received study treatments and were included in the efficacy and safety analyses. We attempted to continue to follow-up participants who stopped treatment during the 12-week treatment phase, and all of those who stopped treatment were considered to be smokers in the analysis. Outcome Measures In keeping with previous varenicline RCTs14,15,27 and recommendations by the Society for Research on Nicotine and Tobacco,28 we established the primary efficacy end point as the continuous abstinence rate (CAR) at weeks 9 to 24. Abstinence from smoking was defined as eCO level–verified (<10 ppm) self-reported abstinence from cigarette smoking. Secondary efficacy end points were the CAR at weeks 9 to 12 and weeks 9 to 52 as well as 7-day point prevalence of abstinence at weeks 12, 24, and 52. Safety reporting details were described in a previous study.23 In brief, safety end points included information on the number of AEs and serious AEs occurring between treatment randomization (visit 1) and 1 week after treatment discontinuation (visit 14). Secondary analyses of metabolic and cardiovascular parameters by smoking phenotype classification (continuous quitters vs reducers vs failures) are planned. Interventions Varenicline, 0.5 mg, tablets and matched placebo tablets were supplied by Pfizer and packaged by Depo Pack. Secondary packaging of these tablets was sent to the hospital pharmacy for double- blinding preparation. Labels from the containers of the secondary packaging were removed, and the unlabeled study drugs were repackaged in coded containers. Blinding was ensured by the identical appearance of the tablets and containers. Patients who were randomized to varenicline were titrated to full dose by the time of their target quit date (0.5 mg daily for 2-3 days, 0.5 mg twice daily for 4 to 5 days, and then 1 mg twice daily for 11 weeks). All patients in both treatment groups received the same smoking cessation counseling throughout the duration of the trial. One-on-one counseling was provided at each visit for a total of 10 minutes by psychologists who were trained in motivational interviewing. Assessments Assessments were carried out during the study visits and included the following laboratory tests: complete blood count, chem-20 panel, urinalysis, lipid profile, fasting blood glucose, HbA1c level, and insulin level. Other measurements taken and questionnaires completed included (1) resting heart rate; (2) systolic and diastolic blood pressure; (3) BMI; (4) waist circumference; (5) eCO levels; (6) Fagerström Test for Cigarette Dependence, a 6-item questionnaire for classifying cigarette dependence into 3 levels with a score range of 0 to 3 points indicating mild, 4 to 6 points indicating moderate, and 7 to 10 points indicating severe dependence29; (7) Beck Depression Inventory II, a June 21, 2022 5/14 JAMA Network Open. 2022;5(6):e2217709. doi:10.1001/jamanetworkopen.2022.17709 June 21, 2022 5/14 Statistical Analysis The sample size calculation for this RCT was based on quit rates from previous smoking cessation studies.17,33,34 We computed that 174 patients (87 per study group) were required to have 90% power with a 2-sided α error rate of .05 to detect a difference of at least 18.7% between treatment groups in CAR at weeks 9 to 24. Allowing for a conservative attrition rate of approximately 40%, the target number of participants was increased to a total of 300 (150 per each study group). Baseline and demographic data were listed for all treatment groups. Summary statistics were provided for each treatment group. At baseline, differences in primary end point (CAR at weeks 9-24) between varenicline and placebo groups were evaluated by means of 1-way analysis of variance for normally distributed continuous variables and by Mann-Whitney test for not normally distributed continuous variables. The χ2 test was used for categorical variables, and differences in primary end point were evaluated by means of χ2 test. Secondary end points were analyzed using procedures similar to those for the primary end point. Intention-to-treat analysis was adopted for efficacy evaluation, on the assumption that patients who were lost to follow-up continued smoking. Safety data were summarized for both treatment groups, and summary statistics were provided. Any events that were documented from the point of treatment initiation until 1 week after discontinuation of randomized treatment (visit 14) were considered to be relevant to the safety analysis. Additional analyses of interest included baseline clinical and demographic variables that could influence smoking cessation rate at weeks 12, 24, and 52. Aiming to identify these variables, we built a multiple logistic regression model in which the CAR at weeks 9 to 24 (but also the CAR at weeks 9-12 and weeks 9-52 in separate models) was the outcome variable and the possible independent variables were introduced along with all the available confounders or effect modifiers. Independent variables were selected by an a priori evaluation of the variables that were able to act as determinants, effect modifiers, or confounders of success in quitting sessions. Despite the analysis of clinical and demographic features that could influence the outcome, this is not the main point of the study. We believe such analysis provides information on when varenicline can be routinely included in diabetes education programs to help patients quit smoking. Downloaded From: https://jamanetwork.com/ on 06/21/2022 Downloaded From: https://jamanetwork.com/ on 06/21/2022 JAMA Network Open \| Diabetes and Endocrinology JAMA Network Open \| Diabetes and Endocrinology Efficacy and Safety of Varenicline in Patients With Type 2 Diabetes 21-item self-rating of characteristics, attitudes, and symptoms of depression30 with a score range of 0 to 9 points indicating no or minimal, 10 to 18 points indicating mild to moderate, and 19 to 29 points indicating moderate to severe depression; (8) Beck Anxiety Inventory, a 21-item self-rating of physiological and cognitive symptoms of anxiety31 with a score range of 0 to 7 points indicating minimal, 8 to 15 points indicating mild, 16 to 25 points indicating moderate, and 26 to 63 points indicating severe level of anxiety; (9) Glover-Nilsson Smoking Behavioral Questionnaire (GN-SBQ), a self-rating of behaviors or thoughts about smoking32 with a score less than 12 points indicating mild, 12 to 22 points indicating moderate, 23 to 33 points indicating strong, and higher than 33 points indicating very strong dependence; and (10) visual analog scale (VAS) for self-efficacy and motivation, with a score range of 0 points indicating low to 10 points indicating high self-efficacy or motivation. eTable 1 in Supplement 2 has a full list of the assessments performed and the instruments used. Statistical Analysis We performed an a priori selection of variables that were able to act as determinants, effect modifiers, or confounders of success in quitting sessions. Number of cigarettes smoked per day was excluded from the multiple logistic analysis because of its strong association with pack-years. Intention-to-treat data analyses were carried out from December 2020 to April 2021, using Statistical Package for Social Sciences for Windows, version 20 (SPSS Inc). All tests were 2-sided, and P < .05 was considered to be significant. June 21, 2022 6/14 JAMA Network Open. 2022;5(6):e2217709. doi:10.1001/jamanetworkopen.2022.17709 Trial Participants Three hundred patients were randomized to either varenicline (n = 150) or placebo (n = 150) treatment. These patients had a mean (SD) age of 57.4 (0.8) years and included 117 men (78.0%) and 33 women (22.0%) in the varenicline group and 119 men (79.3%) and 31 women (20.7%) in the placebo group). A total of 215 participants completed the treatment phase, of whom 73.3% (n = 110 of 150) were in the varenicline group and 70.0% (n = 105 of 150) were in the placebo group. For the nontreatment phase, 201 participants completed the 24-week study visits, of whom 66.6% (n = 100 of 150) were in the varenicline group and 67.3% (n = 101 of 150) were in the placebo group; 195 patients completed the 52-week study visits, of whom 66.6% (n = 100 of 150) were in the varenicline group and 63.3% (n = 95 of 150) were in the placebo group. Baseline characteristics of the patients are comparable between groups (Table 1). Despite our best efforts to keep and support all randomized participants, treatment discontinuation was frequent (approximately 30%). In general, most participants were White, middle-aged (approximately 57 years), and male (approximately 78%) individuals who had smoked about 25 cigarettes daily for at least 40 years. They had a Fagerström Test for Cigarette Dependence score of 6, indicating moderate to marked nicotine dependence; a VAS motivation score of 9, indicating high motivation to quit; and a VAS self-efficacy score of 5, indicating a low perception of their ability to quit smoking. A similar proportion of patients in both treatment groups (56% [n = 84] for the Table 1. Baseline Characteristics of Patients by Treatment Group Characteristic No. (%) Varenicline group (n = 150) Placebo group (n = 150) Sex Female 33 (22.0) 31 (20.7) Male 117 (78.0) 119 (79.3) Age, mean (SE), y 57.3 (0.8) 57.4 (0.8) Educational level Primary 21 (14.0) 26 (17.3) Secondary, first degree 59 (39.3) 63 (42.0) Secondary, second degree 54 (36.0) 51 (34.0) Doctoral degree 16 (10.7) 10 (6.7) No. of cigarettes smoked per d, median (IQR) 25 (20-35) 25 (20-40) Duration of smoking, median (IQR), y 42 (35-48) 41 (35-47) No. Downloaded From: https://jamanetwork.com/ on 06/21/2022 Downloaded From: https://jamanetwork.com/ on 06/21/2022 JAMA Network Open \| Diabetes and Endocrinology Efficacy and Safety of Varenicline in Patients With Type 2 Diabetes Trial Participants of packs smoked per y, median (IQR) 50.8 (36.4-70.8) 53.4 (35.0-70.0) Proportion with quitting attempts 84 (56.0) 75 (50.0) Self-efficacy score by VAS 5 (5-7) 5 (4-8) Motivation to quit score by VAS 9 (6-10) 9 (7-10) FTCD score, median (IQR) 6 (4-8) 6 (5-8) BAI score, median (IQR) 6 (3-12) 8 (2-16) BDI-II score, median (IQR) 7 (3-13) 8 (3-14) GN-SBQ score, median (IQR) 18 (11-23) 19 (12-22) Hemoglobin A1c level, mean (SE), % 7.68 (0.12) 7.63 (0.13) Type 2 diabetes medications Metformin 124 (82.7) 129 (86.0) Insulin or insulin analogs 74 (49.3) 77 (51.3) Statins 65 (43.3) 62 (41.3) GLP-1 receptor agonist 44 (29.3) 45 (30.0) Sulphonylureas (includes repaglinide) 35 (23.3) 38 (25.3) DPP-4 inhibitors 28 (18.7) 23 (15.3) α-Glucosidase inhibitors (acarbose) 18 (12.0) 14 (9.3) Glitazones 3 (2.0) 5 (3.4) Table 1. Baseline Characteristics of Patients by Treatment Group Characteristic No. (%) Varenicline group (n = 150) Placebo group (n = 150) Sex Female 33 (22.0) 31 (20.7) Male 117 (78.0) 119 (79.3) Age, mean (SE), y 57.3 (0.8) 57.4 (0.8) Educational level Primary 21 (14.0) 26 (17.3) Secondary, first degree 59 (39.3) 63 (42.0) Secondary, second degree 54 (36.0) 51 (34.0) Doctoral degree 16 (10.7) 10 (6.7) No. of cigarettes smoked per d, median (IQR) 25 (20-35) 25 (20-40) Duration of smoking, median (IQR), y 42 (35-48) 41 (35-47) No. of packs smoked per y, median (IQR) 50.8 (36.4-70.8) 53.4 (35.0-70.0) Proportion with quitting attempts 84 (56.0) 75 (50.0) Self-efficacy score by VAS 5 (5-7) 5 (4-8) Motivation to quit score by VAS 9 (6-10) 9 (7-10) FTCD score, median (IQR) 6 (4-8) 6 (5-8) BAI score, median (IQR) 6 (3-12) 8 (2-16) BDI-II score, median (IQR) 7 (3-13) 8 (3-14) GN-SBQ score, median (IQR) 18 (11-23) 19 (12-22) Hemoglobin A1c level, mean (SE), % 7.68 (0.12) 7.63 (0.13) Type 2 diabetes medications Metformin 124 (82.7) 129 (86.0) Insulin or insulin analogs 74 (49.3) 77 (51.3) Statins 65 (43.3) 62 (41.3) GLP-1 receptor agonist 44 (29.3) 45 (30.0) Sulphonylureas (includes repaglinide) 35 (23.3) 38 (25.3) DPP-4 inhibitors 28 (18.7) 23 (15.3) α-Glucosidase inhibitors (acarbose) 18 (12.0) 14 (9.3) Glitazones 3 (2.0) 5 (3.4) Abbreviations: BAI, Beck Anxiety Inventory; BDI-II, Beck Depression Inventory II; DPP-4, dipeptidyl peptidase-4; FTCD, Fagerström Test for Cigarette Dependence; GLP-1, glucagon-like peptide-1; GN-SBQ, Glover-Nilsson Smoking Behavioral Questionnaire; VAS, visual analog scale. SI conversion: To convert to proportion of total hemoglobin, multiply by 0.01. JAMA Network Open. 2022;5(6):e2217709. doi:10.1001/jamanetworkopen.2022.17709 Trial Participants June 21, 2022 7/14 Smoking Abstinence Rates (%) OR (95% CI) P value CAR at weeks 9-12 47 (31.3) 11 (7.3) 5.77 (2.85-11.66) <.001 9-24 36 (24.0) 9 (6.0) 4.95 (2.29-10.70) <.001 9-52 28 (18.7) 9 (5.3) 4.07 (1.79-9.27) <.001 7-d point prevalence, wk 12 60 (40.0) 16 (11.0) 7.67 (3.91-15.05) <.001 24 43 (29.0) 13 (8.3) 4.44 (2.17-9.07) <.001 52 35 (23.7) 14 (9.5) 3.27 (1.57-6.78) <.001 Abbreviations: CAR, continuous abstinence rate; OR, odds ratio. JAMA Network Open \| Diabetes and Endocrinology Efficacy and Safety of Varenicline in Patients With Type 2 Diabetes varenicline group and 50% [n = 75] for the placebo group) had made at least 1 serious quit attempt (defined as complete abstinence for at least 24 hours) in the past. The mean (SE) HbA1c level was 7.68% (0.12%) for the varenicline group and 7.63% (0.13%) for the placebo group, indicating a suboptimal level of control. Smoking Abstinence Rates g The eCO level–verified (<10 ppm) CARs at weeks 9 to 12, 9 to 24, and 9 to 52 are shown in Figure 3 and Table 2. We found that the CARs were significantly higher for the varenicline group vs the placebo group at each interval: 31.3% vs 7.3% (odds ratio [OR], 5.77; 95% CI, 2.85-11.66; P < .001) at weeks 9 to 12; 24.0% vs 6.0% (OR, 4.95; 95% CI, 2.29-10.70; P < .001) at weeks 9 to 24; and 18.7% vs 5.3% (OR, 4.07; 95% CI, 1.79-9.27; P < .001) at weeks 9 to 52. The 7-day point prevalence of smoking abstinence was also significantly higher for the varenicline group than the placebo group at each time point: 40.0% vs 11.0% (OR, 7.67; 95% CI, 3.91-15.05; P < .001) at week 12; 29.0% vs 8.3% (OR, 4.44; 95% CI, 2.17-9.07; P < .001) at week 24; 23.7% vs 9.5% (OR, 3.27; 95% CI, 1.57-6.78; P < .001) at week 52. P < .001) at week 52. Clinical and Demographic Features Influencing Smoking Abstinence In a multiple logistic regression model, including baseline characteristics (sex, age, educational level, cohabiting smoker, previous quitting attempt, VAS self-efficacy, VAS motivation, Beck Anxiety Inventory score, Beck Depression Inventory II score, GN-SBQ score, number of cigarette pack-years, and study group) as independent variables, we found that the varenicline group vs the placebo group had an OR of 4.80 (95% CI, 2.06-11.16; P < .001) for the CAR at weeks 9 to 24. Those in the varenicline group with strong behavioral cigarette dependence (GN-SBQ score >22 points) had reduced odds for CAR at weeks 9 to 24 (OR, 0.27; 95% CI, 0.11-0.65; P < .01), indicating that strong behavioral cigarette dependence impairs the success of continuous abstinence with varenicline. A Figure 3. Continuous Abstinence Rates for Weeks 9 to 12, 9 to 24, and 9 to 52 35 30 25 20 15 10 5 0 Continuous abstinence rate, % P <.001 Weeks 9-12 P <.001 Weeks 9-24 P <.001 Weeks 9-52 Placebo group Varenicline group Proportion of participants who reported abstinence from smoking was defined by exhaled carbon monoxide level–verified (<10 ppm) self-reported abstinence. Primary efficacy end point was the continuous abstinence rate at weeks 9 to 24. Table 2. Primary and Secondary Efficacy Outcomes Outcome Varenicline group, No. (%) Placebo group, No. Clinical and Demographic Features Influencing Smoking Abstinence In a multiple logistic regression model, including baseline characteristics (sex, age, educational level, cohabiting smoker, previous quitting attempt, VAS self-efficacy, VAS motivation, Beck Anxiety Inventory score, Beck Depression Inventory II score, GN-SBQ score, number of cigarette pack-years, and study group) as independent variables, we found that the varenicline group vs the placebo group had an OR of 4.80 (95% CI, 2.06-11.16; P < .001) for the CAR at weeks 9 to 24. Those in the varenicline group with strong behavioral cigarette dependence (GN-SBQ score >22 points) had reduced odds for CAR at weeks 9 to 24 (OR, 0.27; 95% CI, 0.11-0.65; P < .01), indicating that strong behavioral cigarette dependence impairs the success of continuous abstinence with varenicline. A Figure 3. Continuous Abstinence Rates for Weeks 9 to 12, 9 to 24, and 9 to 52 35 30 25 20 15 10 5 0 Continuous abstinence rate, % P <.001 Weeks 9-12 P <.001 Weeks 9-24 P <.001 Weeks 9-52 Placebo group Varenicline group Proportion of participants who reported abstinence from smoking was defined by exhaled carbon monoxide level–verified (<10 ppm) self-reported abstinence. Primary efficacy end point was the continuous abstinence rate at weeks 9 to 24. Table 2. Primary and Secondary Efficacy Outcomes Outcome Varenicline group, No. (%) Placebo group, No. (%) OR (95% CI) P value CAR at weeks 9-12 47 (31.3) 11 (7.3) 5.77 (2.85-11.66) <.001 9-24 36 (24.0) 9 (6.0) 4.95 (2.29-10.70) <.001 9-52 28 (18.7) 9 (5.3) 4.07 (1.79-9.27) <.001 7-d point prevalence, wk 12 60 (40.0) 16 (11.0) 7.67 (3.91-15.05) <.001 24 43 (29.0) 13 (8.3) 4.44 (2.17-9.07) <.001 52 35 (23.7) 14 (9.5) 3.27 (1.57-6.78) <.001 Abbreviations: CAR, continuous abstinence rate; OR, odds ratio. Figure 3. Continuous Abstinence Rates for Weeks 9 to 12, 9 to 24, and 9 to 52 35 30 25 20 15 10 5 0 Continuous abstinence rate, % P <.001 Weeks 9-12 P <.001 Weeks 9-24 P <.001 Weeks 9-52 Placebo group Varenicline group Proportion of participants who repo from smoking was defined by exhal monoxide level–verified (<10 ppm) s abstinence. Primary efficacy end po continuous abstinence rate at week Figure 3. Proportion of participants who reported abstinence from smoking was defined by exhaled carbon monoxide level–verified (<10 ppm) self-reported abstinence. Primary efficacy end point was the continuous abstinence rate at weeks 9 to 24. Downloaded From: https://jamanetwork.com/ on 06/21/2022 Downloaded From: https://jamanetwork.com/ on 06/21/2022 JAMA Network Open \| Diabetes and Endocrinology JAMA Network Open \| Diabetes and Endocrinology Adverse Events A total of 105 patients (70.0%) who received varenicline and 96 patients (64.0%) who received placebo reported AEs. Most AEs were rated as mild or moderate and rarely led to treatment discontinuations in either group (6 [4.0%] in the varenicline group; 5 [3.3%] in the placebo group). The most common AEs that resulted in treatment discontinuation were anxiety and depression. The AEs that occurred more frequently in the varenicline group than in the placebo group were nausea (41 [27.3%] vs 17 [11.4%]), insomnia (29 [19.4%] vs 19 [12.7%]), abnormal dreams (19 [12.7%] vs 5 [3.4%]), anxiety (17 [11.4%] vs 11 [7.3%]), and irritability (14 [9.4%] vs 8 [5.4%]) (eTable 3 in Supplement 2). There were few AEs reported (n = 380), and only a few withdrawals from the study occurred as a result (n = 11). Patients did not report suicidal ideation, worsening of depressive symptoms, or serious AEs (with the exception of 1 intervertebral disc protrusion in the varenicline arm at visit 11 as well as 1 severe hypoglycemia and 1 hypertensive crisis in the placebo group at visit 6 and visit 8). No significant changes in mean (SE) resting heart rate and systolic and diastolic blood pressure during treatment were observed between and within treatment groups (eTable 6 in Supplement 2). Similarly, no significant differences in BMI, waist circumference, fasting blood glucose, HbA1c level, insulin level, and creatinine level were observed between and within treatment groups (eTable 6 in Supplement 2). Clinical and Demographic Features Influencing Smoking Abstinence Continuous Abstinence Rates for Weeks 9 to 12, 9 to 24, and 9 to 52 35 30 25 20 15 10 5 0 Continuous abstinence rate, % P <.001 Weeks 9-12 P <.001 Weeks 9-24 P <.001 Weeks 9-52 Placebo group Varenicline group Proportion of participants who reported abstinence from smoking was defined by exhaled carbon monoxide level–verified (<10 ppm) self-reported abstinence. Primary efficacy end point was the continuous abstinence rate at weeks 9 to 24. Table 2. Primary and Secondary Efficacy Outcomes Outcome Varenicline group, No. (%) Placebo group, No. (%) OR (95% CI) P value CAR at weeks 9-12 47 (31.3) 11 (7.3) 5.77 (2.85-11.66) <.001 9-24 36 (24.0) 9 (6.0) 4.95 (2.29-10.70) <.001 9-52 28 (18.7) 9 (5.3) 4.07 (1.79-9.27) <.001 7-d point prevalence, wk 12 60 (40.0) 16 (11.0) 7.67 (3.91-15.05) <.001 24 43 (29.0) 13 (8.3) 4.44 (2.17-9.07) <.001 52 35 (23.7) 14 (9.5) 3.27 (1.57-6.78) <.001 Abbreviations: CAR, continuous abstinence rate; OR, odds ratio. JAMA Network Open. 2022;5(6):e2217709. doi:10.1001/jamanetworkopen.2022.17709 (Reprinted) June 21, 2022 8/14 Table 2. Primary and Secondary Efficacy Outcomes JAMA Network Open. 2022;5(6):e2217709. doi:10.1001/jamanetworkopen.2022.17709 June 21, 2022 8/14 Downloaded From: https://jamanetwork.com/ on 06/21/2022 higher number of pack-years reduced the odds of the CAR at weeks 9 to 24 (OR, 0.21; 95% CI, 0.09- 0.50; P < .001) (eTable 2 in Supplement 2). Similar findings were observed for the CAR at weeks 9 to 12 (OR, 0.28; 95% CI, 0.13-0.60; P < .001) and weeks 9 to 52 (OR, 0.38; 95% CI, 0.16-0.92; P = .03) (eTable 4 and eTable 5 in Supplement 2). JAMA Network Open \| Diabetes and Endocrinology Efficacy and Safet JAMA Network Open \| Diabetes and Endocrinology JAMA Network Open \| Diabetes and Endocrinology Efficacy and Safety of Varenicline in Patients With Type 2 Diabetes higher number of pack-years reduced the odds of the CAR at weeks 9 to 24 (OR, 0.21; 95% CI, 0.09- 0.50; P < .001) (eTable 2 in Supplement 2). Similar findings were observed for the CAR at weeks 9 to 12 (OR, 0.28; 95% CI, 0.13-0.60; P < .001) and weeks 9 to 52 (OR, 0.38; 95% CI, 0.16-0.92; P = .03) (eTable 4 and eTable 5 in Supplement 2). Discussion To our knowledge, this RCT was the first to investigate the efficacy and safety of varenicline, 1 mg, twice daily for smoking cessation among patients with type 2 diabetes. The findings suggest that substantial changes in smoking habits may occur among patients with type 2 diabetes who intend to stop smoking, leading to prolonged abstinence from smoking. These findings were not associated with differences in AEs between treatment groups, suggesting a good safety profile for varenicline in smokers with type 2 diabetes. Moreover, the findings confirmed the results of a previous meta- analysis of Pfizer-sponsored varenicline trials.22 Throughout the trial, patients in the varenicline group were at least 3 times more likely to quit smoking compared with patients in the placebo group; eCO level–verified CAR in the varenicline group was 31.3% at weeks 9 to 12, 24.0% at weeks 9 to 24, and 18.7% at weeks 9 to 52. The ORs for the varenicline group in the present trial were higher than the ORs in previous RCTs,17-19 which resulted from lower abstinence rates in the placebo group. These findings support the notion that patients with type 2 diabetes who smoke have greater difficulty quitting when provided with only cessation counseling. When investigating clinical and demographic features that influenced the CARs, we found that lifetime cumulative smoking history and stronger cigarette dependence, as assessed by GN-SBQ, resulted in less probability of smoking abstinence over time. Smokers with high cigarette dependence were found to have a difficult time quitting,35 and lifetime cumulative smoking history was shown to have a significant effect on varenicline efficacy.24 A smoking cessation program alone was significantly less likely to be successful in patients with or affected by smoking rituals (ie, high GN-SBQ scores >23 points). This finding was not surprising given that smoking cessation medications may only alleviate nicotine withdrawal symptoms and reduce reinforcement from smoking but cannot replace the need for smoking-related rituals. A study that examined the effect of adding a nicotine-free cigarette-like plastic stick to a smoking cessation program found that participants who were assigned to use the plastic stick had quit rates that were more than 3-fold higher if their GN-SBQ June 21, 2022 9/14 JAMA Network Open. 2022;5(6):e2217709. doi:10.1001/jamanetworkopen.2022.17709 Limitations This study has several limitations. First, findings in an ambulatory population of asymptomatic patients with type 2 diabetes cannot be extended to those with untreated or uncontrolled diabetes or with clinically significant comorbidities. Therefore, the potential efficacy and safety of varenicline in these subpopulations could not be assessed. Second, we excluded patients who were being treated for major depression, panic disorder, psychosis, bipolar disorder, or other conditions frequently associated with major depressive disorders, thus limiting the generalizability of the results. Third, treatment discontinuation was frequent (approximately 30%) and occurred across both treatment groups, but it was unrelated to the occurrence of AEs. Discontinuation could have affected safety outcomes by reducing the statistical power to detect small changes. Fourth, findings were restricted to a selected population of patients who were highly motivated and ready to quit in the next 30 days, further limiting the generalizability of the results. Downloaded From: https://jamanetwork.com/ on 06/21/2022 Downloaded From: https://jamanetwork.com/ on 06/21/2022 JAMA Network Open \| Diabetes and Endocrinology JAMA Network Open \| Diabetes and Endocrinology Efficacy and Safety of Varenicline in Patients With Type 2 Diabetes scores were high, whereas those with low GN-SBQ scores showed no advantage from using the plastic stick.36 Although it is highly unlikely that varenicline would be less effective in smokers with diabetes, there may be potential concern about the safety of varenicline in patients with type 2 diabetes. Given that varenicline is exclusively excreted in the urine through glomerular filtration and active tubular secretion,37 patients with type 2 diabetes may have some concerns about oral antidiabetic drugs (such as metformin, glucagon-like peptide-1 agonists, and most dipeptidyl peptidase-4 inhibitors),38 which are also eliminated via the kidneys by glomerular filtration and tubular secretion and can accelerate kidney failure. The safety profile of varenicline in these patients was similar to that observed in previous varenicline trials involving smokers in the general population,16,17 which reported nausea, insomnia, abnormal dreams, anxiety, and irritability as the most common AEs. There were no differences in metabolic and cardiovascular parameters between or within treatment groups. No weight gain was observed, which was somewhat surprising given that smoking cessation is typically associated with increase in body weight.39,40 Similarly, lack of post–smoking cessation weight gain was reported in a retrospective pooled analysis of 15 RCTs of varenicline use among smokers with type 2 diabetes.22 Most patients in the present trial were receiving metformin, which might have contributed to preventing weight gain after smoking cessation. Conclusions Smoking cessation in diabetes is largely neglected, and the efficacy and safety of the antismoking medication varenicline have not been investigated in patients with type 2 diabetes. The findings of the present RCT indicate that inclusion of varenicline in a smoking cessation program for patients with type 2 diabetes with the intention to quit may result in prolonged abstinence without serious AEs. This evidence supports the use of varenicline in diabetes education programs to help patients with type 2 diabetes stop smoking. Accepted for Publication: May 2, 2022. Published: June 21, 2022. doi:10.1001/jamanetworkopen.2022.17709 Open Access: This is an open access article distributed under the terms of the CC-BY-NC-ND License. © 2022 Russo C et al. JAMA Network Open. Corresponding Author: Riccardo Polosa, MD, PhD, Centro per la Prevenzione e Cura del Tabagismo (CPCT), Azienda Ospedaliero Universitaria Policlinico-V. Emanuele dell’Università di Catania, Via S Sofia 78, 95123, Catania, Italy (polosa@unict.it). Author Affiliations: Ambulatorio di Diabetologia, Unità Operativa Complessa (UOC) Medicina Interna E JAMA Network Open. 2022;5(6):e2217709. doi:10.1001/jamanetworkopen.2022.17709 June 21, 2022 10/14 MA Network Open. 2022;5(6):e2217709. doi:10.1001/jamanetworkope JAMA Network Open \| Diabetes and Endocrinology Efficacy and Safety of Varenicline in Patients With Type 2 Diabetes D'Urgenza, Policlinico Universitario, Azienda Ospedaliero Universitaria (AOU) Policlinico-G. Rodolico-San Marco, Catania, Italy (Russo, Campagna, Polosa); Mossakowski Medical Research Institute, Polish Academy of Sciences, Warsaw, Poland (Walicka, Krysinski, Franek); Department of Internal Diseases, Endocrinology and Diabetology, Central Clinical Hospital of the Ministry of the Interior (MSWIA), Warsaw, Poland (Walicka, Krysinski, Franek); Dipartimento di Medicina Clinica e Sperimentale, Università Di Catania, Catania, Italy (Walicka, Polosa); Centro per la Prevenzione e Cura del Tabagismo, AOU Policlinico-G. Rodolico-San Marco, dell'Università Di Catania, Catania, Italy (Caponnetto, Maglia, Polosa); Institute for Biomedical Research and Innovation, National Research Council of Italy, Palermo, Italy (Cibella); Centro Diabetologico, UOC Andrologia Ed Endocrinologia, Policlinico Universitario, AOU, Catania, Italy (Alamo); UOC Medicina e Chirurgia d'Accettazione e Urgenza, University Teaching Hospital, G. Rodolico-San Marco, University of Catania, Catania, Italy (Campagna); Centro per il Diabete e l'Obesità, UOC Endocrinologia, Ospedale Garibaldi Nesima, Azienda Ospedaliera di Rilievo Nazionale e di Alta Specializzazione Garibaldi, Catania, Italy (Frittitta); Center of Excellence for the Acceleration of Harm Reduction, Dipartimento Di Medicina Clinica E Sperimentale, Università di Catania, Catania, Italy (Frittitta, Di Mauro, Polosa); Ambulatorio di Diabetologia, Ospedale V. Emanuele, AOU Policlinico-Vittorio Emanuele, Catania, Italy (Di Mauro); Unit of Infectious Diseases, Department of Clinical and Experimental Medicine, University of Messina, Messina, Italy (Caci); ECLAT Srl, Spin-off of the University of Catania, Catania, Italy (Polosa). Author Contributions: Drs Caponnetto and Cibella had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Concept and design: Russo, Campagna, Di Mauro, Polosa. Concept and design: Russo, Campagna, Di Mauro, Polosa. Acquisition, analysis, or interpretation of data: Walicka, Caponnetto, Cibella, Maglia, Alamo, Campagna, Frittitta, Caci, Krysinski, Franek, Polosa. Acquisition, analysis, or interpretation of data: Walicka, Caponnetto, Cibella, Maglia, Alamo, Campagna, Frittitta, Caci, Krysinski, Franek, Polosa. Drafting of the manuscript: Russo, Walicka, Caponnetto, Maglia, Alamo, Campagna, Di Mauro, Caci, Krysinski, Polosa. Drafting of the manuscript: Russo, Walicka, Caponnetto, Maglia, Alamo, Campagna, Di Mauro, Caci, Krysinski, Polosa. Critical revision of the manuscript for important intellectual content: Cibella, Frittitta, Franek, Polosa. Statistical analysis: Cibella. Critical revision of the manuscript for important intellectual content: Cibella, Frittitta, Franek, Polosa. Statistical analysis: Cibella. Obtained funding: Polosa. Obtained funding: Polosa. Obtained funding: Polosa. Administrative, technical, or material support: Russo, Caponnetto, Alamo, Campagna, Polosa Supervision: Russo, Frittitta, Di Mauro, Caci, Polosa. JAMA Network Open \| Diabetes and Endocrinology Conflict of Interest Disclosures: Dr Walicka reported receiving personal fees from AstraZeneca outside the submitted work. Dr Polosa reported being employed as a tenured professor and as medical director at the University of Catania (Italy); receiving lecture fees and research funding from Pfizer, GlaxoSmithKline, CV Therapeutics, NeuroSearch A/S, Sandoz, MSD, Boehringer Ingelheim, Novartis, Duska Therapeutics, and Forest Laboratories; receiving lecture fees from a number of European electronic cigarette industry and trade associations (including FIVAPE in France and FIESEL in Italy) that were directly donated to nonprofit vaper advocacy organizations; receiving grants from European Commission initiatives (U-BIOPRED and AIRPROM) and Integral Rheumatology & Immunology Specialists Network; serving as a consultant for Pfizer, Global Healthcare Alliance for Treatment of Tobacco Dependence, CV Therapeutics, Boehringer Ingelheim, Novartis, Duska Therapeutics, Electronic Cigarette Industry Trade Association, Arbi Group Srl, Health Diplomats, and Sermo Inc; serving on the medical and scientific advisory boards of Cordex Pharma Inc, CV Therapeutics, Duska Therapeutics Inc, Pfizer, and PharmaCielo; being founder of the Center for Tobacco Prevention and Treatment and the Center of Excellence for the Acceleration of Harm Reduction, both at the University of Catania; being involved in a patent application for an app tracker for smoking behavior, which was developed for ECLAT Srl; being an unpaid scientific advisor for Lega Italiana Anti Fumo, the Consumer Advocates for Smoke-free Alternatives, and the International Network of Nicotine Consumers Organizations; and being chair of the European Technical Committee for standardization on “requirements and test methods for emissions of electronic cigarettes.” No other disclosures were reported. Funding/Support: This study was supported by grant WS5086648 from GRAND (Global Research Award for Nicotine Dependence), an independently reviewed competitive grants program funded by Pfizer Inc. Role of the Funder/Sponsor: The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. Data Sharing Statement: See Supplement 3. Additional Contributions: D. Arcoria, MD, Diabetologica Clinic Center; V. Borzi, MD, Internal Medicine Vittorio Emanuele Hospital; M. Buscema, MD, Internal Medicine Cannizzaro Hospital; C. Gatta, MD, Internal Medicine Vittorio Emanuele Hospital; C. Leotta, MD, Internal Medicine Cannizzaro Hospital; E. Mondati, MD, Internal Medicine S. Marta Hospital; and G. Padova, MD, Centro per il Diabete e l'Obesità (UOC Endocrinologia, Ospedale JAMA Network Open. 2022;5(6):e2217709. doi:10.1001/jamanetworkopen.2022.17709 June 21, 2022 11/14 Downloaded From: https://jamanetwork.com/ on 06/21/2022 JAMA Network Open \| Diabetes and Endocrinology JAMA Network Open \| Diabetes and Endocrinology JAMA Network Open \| Diabetes and Endocrinology Efficacy and Safety of Varenicline in Patients With Type 2 Diabetes Garibaldi Nesima, assisted with patient recruitment. G. Di Vincenzo’s (MD) team at MD, Vittorio Emanuele Hospital assisted with laboratory analyses. R. Vigneri, MD; S. Squatrito, MD; and F. Purrello, MD, Centro per il Diabete e l'Obesità-UOC Endocrinologia, Ospedale Garibaldi Nesima, provided scientific advice. These individuals received no additional compensation, outside of their usual salary, for their contributions. Mike Coughlan edited the manuscript with funding from ECLAT Srl. 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Addiction. 2011;106(1):188-196. doi: 10.1111/j.1360-0443.2010.03136.x June 21, 2022 13/14 JAMA Network Open. 2022;5(6):e2217709. doi:10.1001/jamanetworkopen.2022.17709 Downloaded From: https://jamanetwork.com/ on 06/21/2022 Downloaded From: https://jamanetwork.com/ on 06/21/2022 SUPPLEMENT 1. Trial Protocol SUPPLEMENT 2. eTable 1. Study Schedule and Assessments eTable 2. Multiple Logistic Regression Model for CAR 9-24 Week eTable 3. Summary of Adverse Events Occurring in 5% of Either Treatment Group eTable 4. Multiple Logistic Regression Model for CAR 9-12 Week eTable 5. Multiple Logistic Regression Model for CAR 9-52 Week eTable 6. Metabolic and Cardiovascular Parameters Measured at Baseline and Week 12 SUPPLEMENT 3. Data Sharing Statement SUPPLEMENT 3. 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https://openalex.org/W3016956603	https://pathology.medresearch.in/index.php/jopm/article/download/95/188	English	null	Histopathological differentiation and incidence of soft tissue tumors: study of 140 cases	Pathology update	2,017	cc-by	3,252	ISSN 2456-1487 Research Article ISSN 2456-1487 Research Article July - September, 2017/ Vol 3/ Issue 3 ISSN 2456-1487 Research Article Abstract Background: The incidence of soft tissue tumors, especially the frequency of benign tumors relative to malignant ones, is never determined accurately. The fact that many benign tumors, such as lipomas and hemangiomas, do not undergo biopsy makes direct application of data from most hospital series invalid for the general population. Histologic grade represents the most important Prognostic factor for all soft tissue sarcomas, strongly associated with the advent of metastasis and patients survival. Objectives: the objective of the study was to find occurrence of soft tissue tumor and analyse various type and subtypes of soft tissue tumors. Methodology: a prospective study carried out at tertiary care teaching hospital. The operated specimens or biopsy material of soft tissue tumors received were studied in detailed by department of Histopathology. Histological diagnosis was achieved either by performing incisional or trucut needle biopsy and/or reviewing the slides when patient underwent biopsy elsewhere. Observations: The incidence were high in the age group of 31 to 40 years (36 cases, 25%), followed by 21 to 30 years (24 Cases, 17.1%) and 41 to 50(21 Cases, 15%) years. However the malignant tumor incidence was high at age of 51 to 60 years of age. Region wise involvement shows soft tissue tumors were found in head and neck region (29.2%), followed by genital region (25.7%) and upper limb (15.8%). Type wise distribution was lipomatous (28.5%), followed by smooth muscle tumor (21.5%), peripheral nerve sheath tumor (17.9%), Tumor of blood and lymph vessels (17.1%), Conclusion: benign soft tissue tumor are much common than the malignant tumors. Among benign tumors lipomatous tumors are common followed by smooth muscle tumor and other tumors. Key words: Soft tissue tumor, Histopatological examination, Lipomatous tumor Histopathological differentiation and incidence of soft tissue tumors: study of 140 cases Patel Y. M.1, Tandel H. K.2 1Dr. Yogita kumari Manilal Patel, 2Dr. Hetal kumari Kishor bhai Tandel, Tutor; both authors are affiliated with Department of Pathology, GMERS Medical College, Valsad, Gujarat, India. ddress for Correspondence: Dr. Hetal kumari Kishor bhai Tandel, Email:hetaltandel022@gmail.com Address for Correspondence: Dr. Hetal kumari Kishor bhai Tandel, Email:hetaltandel022@gmail.c Manuscript received: 10th August 2017 Reviewed: 20th August 2017 Author Corrected: 29th August 2017 Accepted for Publication: 4th September 2017 Materials and Methodology: Study design and setting:It was a prospective study carried out at B J Medical College and Civil Hospital from September 2014 to July 2015. Scientific and Human Research Ethical committee permission was taken before starting of the study. Inclusion and Exclusion criteria:Participants were explained about the purpose and objectives of the study before enrolling in the study. The participants who gave the written consent were included in the study. Statistical analysis done using Microsoft excel sheets. Research Article The operated specimens or biopsy material of soft tissue tumors received during the study period from surgery department. And they were studied in detailed by department of Histopathology. Only mesenchymal lesions originating in soft tissue were included in the study. Intra-abdominal and retroperitoneal lesions were also included when the lesions were not thought to originate in bowel or abdominal viscera. Lesions arising in the chest and abdominal walls were also included. Age and sex incidence, site of lesion, clinical features, gross and microscopic appearance were carefully studied as per the proforma. Histological diagnosis was achieved either by performing incisional or trucut needle biopsy and/or reviewing the slides when patient underwent biopsy elsewhere. Routinely in our institute Hematoxylin and Eosin staining is done. Special stain like Phosphotungstic acid haematoxylin (PTAH), trichrome stain, reticulin stain, alcian blue stain, and Periodic Acid Schiff (PAS) reaction were also used in diagnosis of histological types and subtypes whenever required. technique, which does not supersede or replace the traditional morphologic diagnosis [3]. Soft tissue masses present a challenge to the pathologist because of their extremely varied morphology and biologic behaviour [4]. So, the objective of the study was to find occurrence of soft tissue tumor and analyse various type and subtypes of soft tissue tumors. Available online at: www.pathologyreview.in 336 \| P a g e Introduction hemangiomas, do not undergo biopsy makes direct application of data from most hospital series invalid for the general population. Histologic grade represents the most important Prognostic factor for all soft tissue sarcomas, strongly associated with the advent of metastasis and patients survival [1,2]. Soft tissue tumors are defined as mesenchymal proliferations which occur in the extraskeletal nonepithelial tissues of the body, excluding the viscera, coverings of brain and lymphoreticular system. The annual incidence of soft tissue tumor is 1.4 per 100000 population. Soft tissue tumors are the fourth most common malignancy in children, after hematopoietic neoplasm, neural tumor and wilmstumor [1]. The incidence of soft tissue tumors, especially the frequency of benign tumors relative to malignant ones, is nearly impossible to determine accurately. Benign soft tissue tumors outnumber malignant tumors by a wide margin. The fact that many benign tumors, such as lipomas and The use of ancillary techniques like immuno- histochemistry, electron microscopy flow cytometry and cytogenetics, has increased insight into the tumor biology and has provided tools for greater diagnostic accuracy. Yet the foundation of these newer techniques rests upon the histologic diagnosis made on light microscopic evaluation of hematoxylin and eosin stained sections and use of special stains. It is critical to recognize immunohistochemistry as an adjunctive Manuscript received: 10th August 2017 Reviewed: 20th August 2017 Author Corrected: 29th August 2017 Accepted for Publication: 4th September 2017 Tropical Journal of Pathology & Microbiology 335 \| P a g e Available online at: www.pathologyreview.in 335 \| P a g e 335 \| P a g e Available online at: www.pathologyreview.in 33 July - September, 2017/ Vol 3/ Issue 3 ISSN 2456-1487 Jul Tropical Journal of Pathology & Microbiology Result Category Benign Intermediate Malignant 9 (6.43%) 0 (0.00%) 2 (1.43%) 18 (12.86%) 0 (0.00%) 1 (0.71%) 24 (17.14%) 0 (0.00%) 0 (0.00%) 33 (23.57%) 1(0.71%) 2 (1.43%) 19 (13.57%) 0 (0.00%) 2 (1.43%) 12 (8.57%) 1 (0.71%) 5 (3.57%) 10 (7.14%) 0 (0.00%) 1 (0.71%) 125 (89.29%) 2 (1.42%) 13 (9.29%) Total 11 (7.86%) 19 (13.57%) 24 (17.14%) 36 (25.71%) 21 (15.00%) 18 (12.86%) 11 (7.86%) 140 (100%) Table- 2: Age wise distribution of nature of tumors 2: Age wise distribution of nature of tumors. Table- 3: Gender wise distribution of nature of tumors 3: Gender wise distribution of nature of tumors. Table- 3: Gender wise distribution of nature of tumors Gender Benign Male 73 (52.14%) Female 52 (37.14%) Total 125 (89.29%) M:F ratio 1.4:1 3: Gender wise distribution of nature of tumors. Result Over a period of one year and 3 months total 140 cases of soft tissue tumor could be included in the study. Among them majority of tumor [125 (89%)] were benign in nature. Whereas 9% of tumor were malignant and 2% were intermediate in nature [Table 1] In this study, age of study subjects were ranging from 0 to 70 years. Peak incidence of the tumors were in the age group of 31 to 40 years (36 cases, 25%), followed by 21 to 30 years (24 Cases, 17.1%) and 41 to 50(21 Cases, 15%) years. Incidences of tumors were found decreased in both the extreme of age group which are in the 0 to 10 years (11 Cases, 7.9%) and 61 to 70 years (11 Cases, 7.9%). [Table 2] However the malignant tumorincidence increase as the advances of the age. And it shows highest at age of 51 to 60 years of age [5 cases (3.57%)]. Gender wise distribution shows out of total 140, nearly 58% were male and 42% were female with Male: Female ratio was 1.37:1. [Table 3] Nature wise distribution shows that benign, intermediate and malignant tumors were similarly distributed in male and female. Table-1: Distribution of tumors according to nature. Category Frequency Percentage Benign 125 89.3 % Intermediate 2 1.4 % Malignant 13 9.3 % Total 140 100 % Table-1: Distribution of tumors according to nature. Table-1: Distribution of tumors according to nature. Available online at: www.pathologyreview.in 336 \| P a g e July - September, 2017/ Vol 3/ Issue 3 September, 2017/ Vol 3/ Issue 3 September, 2017/ Vol 3/ Issue 3 ISSN 2456-1487 Research Article Table- 2: Age wise distribution of nature of tumors Age (In completed years) Benign 0 – 10 9 (6.43%) 11 – 20 18 (12.86%) 21 – 30 24 (17.14%) 31 – 40 33 (23.57%) 41 – 50 19 (13.57%) 51 – 60 12 (8.57%) 61 – 70 10 (7.14%) Total 125 (89.29%) 2: Age wise distribution of nature of tumors. Tropical Journal of Pathology & Microbiology Tropical Journal of Pathology & Microbiology Discussion 1 showing a male predominance. These findings are in good correlation with most of the study reported in literature of Mirza et al(2005) [5], Kransdorf et al 1995 [6,7] and Enzinger and Weiss [8,9,10]. In the study of Mirza et al, there were 3 cases of liposarcomas accounting for 12.5% of all malignant soft tissue tumors. Whereas in present study there were 2 cases of liposarcomas accounting for 13.3% of all malignant soft tissue tumors. Soft tissue can be defined as non-epithelial, extra- skeletal tissues of the body exclusive of the reticulo- endothelial system, glia and supporting tissues of various parenchymal organs. It is represented by the voluntary muscles, fat and fibrous tissue, along with the vessels serving these tissues. By convention, it also includes peripheral nervous system. Total 140 cases of soft tissue tumors were studied over a period of one year in this study. These finding are found similar with the studies conducted by Kransdorf et al [6]. In the literature of Weiss SW regarding soft tissue sarcomas, liposarcomas comprised 21% of all sarcomas. So the finding of this study is comparable to the above mentioned studies. Fibrous tumors comprised fifth most common tumor among all soft tissue tumors in present study. Fibrous tumors were diagnosed in 11 cases that comprised 7.9% of all soft tissue tumors. The sex and site of fibrous tumors in the present study are comparable with those studies of the Mirza et al (2005) and of Kransdorf’s study. Hemangiomas were second most common among benign tumors constituting 20 cases accounting for 17.69% of all benign soft tissue tumors. The incidence of benign tumor in present study was 89% while in the study by Mirza et al (2005) [57/5] the incidence rate of Benign tumor was 82 %. The incidence rate of malignant tumors was less (10.7%) in present study as compared to the study by Mirza et al (18%) [5]. The difference found in the result is might be due to recent newer advance technique of diagnosis, tumor can be detected at very early stage. In the study of Mirza et al the ratio of Benign : Malignant is 4.70 : 1, where as in present study the ratio found 8.3 : 1, which is higher as compared to previous study, as due to newer technique of diagnosis the tumor can be detected in benign stage [5]. Research Article Research Article In this study, around one third of the soft tissue tumors were found in head and neck region (29.2%), followed by genital region (25.7%) and upper limb (15.8%). Soft tissue tumors were also found in other regions like lower limb, Back, CNS, GIT, Abdomen but in lower incidence [figure 1]. Benign lesions commonly found in Head and neck region, followed by genital region, upper limb and back. Malignant soft tissue lesions werecommonly seen in GIT followed by upper limb and head and neck. Majority of the soft tissue tumors were lipomatous (28.5%) in nature, followed by smooth muscle tumor (21.5%), peripheral nerve sheath tumor (17.9%), Tumor of blood and lymph vessels (17.1%), Fibroblastic/ myofibroblastictumors (7.9%), rest were from the other types of the soft tissue tumor. Among benign tumors lipomatous tumor and leiomyomas were the most common tumors. While other benign tumors were tumor from peripheral nerve sheath and from blood and lymph vessels. The common malignant tumors were stromal tumors and Fibroblastic and myofibroblastictumors. Two patient found with intermediate tumor had epithelioid hemangioendothelioma. [Table 4] Available online at: www.pathologyreview.in 338 \| P a g e Result Category Benign Intermediate Malignant 73 (52.14%) 1 (0.71%) 7 (5.00%) 52 (37.14%) 1 (0.71%) 6 (4.29%) 125 (89.29%) 2 (1.42%) 13 (9.29%) 1:1 1.17:1 Total 81 (57.86%) 59 (42.14%) 140 (100%) 1.37:1 Table- 4: Gender wise and category wise distribution of histopathological type of tumor Type of tumor Lipomatous Tumor Smooth muscle tumor Peripheral Nerve sheath tumor Tumor of blood and lymph vessel Fibroblastic and myofibroblastic tumor Stromal tumor Perivascular Tumors Skeletal Muscle Tumors Extra skeletal Osseous And Cartilaginous Tumors Total Figure-1: Anatomical location 4: Gender wise and category wise distribution of histopathological type of tumor Gender Total Category Male Female Benign Intermediate 15 25 40 38 0 30 0 30 30 0 14 11 25 25 0 5 19 24 22 2 tumor 6 5 11 7 0 2 2 4 0 0 0 2 2 2 0 1 1 2 0 0 2 0 2 1 0 75 65 140 125 2 1: Anatomical location of soft tissue tumors according to category 4: Gender wise and category wise distribution of histopathological type of tumor Category Total Intermediate Malignant 2 40 0 30 0 25 0 24 4 11 4 4 0 2 2 2 1 2 13 140 of soft tissue tumors according to category Table- 4: Gender wise and category wise distribution of histopathological type of tumor Type of tumor Lipomatous Tumor Smooth muscle tumor Peripheral Nerve sheath tumor Tumor of blood and lymph vessel Fibroblastic and myofibroblastic tumor Stromal tumor Perivascular Tumors Skeletal Muscle Tumors Extra skeletal Osseous And Cartilaginous Tumors Total Figure-1: Anatomical location 4: Gender wise and category wise distribution of histopathological type of tumor Gender Total Category Male Female Benign Intermediate 15 25 40 38 0 30 0 30 30 0 14 11 25 25 0 5 19 24 22 2 tumor 6 5 11 7 0 2 2 4 0 0 0 2 2 2 0 1 1 2 0 0 2 0 2 1 0 75 65 140 125 2 1: Anatomical location of soft tissue tumors according to category 4: Gender wise and category wise distribution of histopathological type of tumor Category Total Intermediate Malignant 2 40 0 30 0 25 0 24 4 11 4 4 0 2 2 2 1 2 13 140 of soft tissue tumors according to category Table- 4: Gender wise and category wise distribution of histopathological type of tumor 4: Gender wise and category wise distribution of histopathological type of tumor 4: Gender wise and category wise distribution of histopathological type of tumor Figure-1: Anatomical location 1: Anatomical location of soft tissue tumors according to category of soft tissue tumors according to category Tropical Journal of Pathology & Microbiology Tropical Journal of Pathology & Microbiology Available online at: www.pathologyreview.in www.pathologyreview.in 337 \| P a g e Available online at: www.pathologyreview.in www.pathologyreview.in 337 \| P a g e July - September, 2017/ Vol 3/ Issue 3 ISSN 2456-1487 References 1. Joyner CJ, Triffitt J, Puddle B, et al: Development of a monoclonal antibody to the aP2 protein to identify adipocyte precursors in tumors of adipose differentiation. Pathol Res Pract 1999;195:461. 9. Weiss SW, Goldblum JR. Benign lipomatous tumors. Chapter-16 In: Enzinger and Weiss’s Soft Tissue Tumors. 4th Edition, St. Louis: Mosby, 2001: 571- 639. 2. Nnodim JO. Development of adipose tissues. Anat Rec 1987; 219:331. 10. Weiss SW, Goldblum JR. Liposarcoma. Ch Weiss SW, Goldblum JR. Liposarcoma. Chapter-17 In: Enzinger and Weiss’s Soft Tissue Tumors. 4th Edition, St. Louis: Mosby, 2001: 641-693. 3. Brooks JJ, Perosio PM: Adipose tissue. Histology for pathologists, 2nd edn. Philadelphia: Lippincott- Raven; 1997:167. 4. Ross R. The smooth muscle cell. II. Growth of smooth muscle in culture and formation of elastic fibers. J Cell Biol. 1971 Jul;50(1):172-86. 4. Ross R. The smooth muscle cell. II. Growth of smooth muscle in culture and formation of elastic fibers. J Cell Biol. 1971 Jul;50(1):172-86. Funding: Nil, Conflict of interest: None initiated, Permission from IRB: Yes 8. Weiss SW, Enzinger FM. Myxoid variant of malignant fibrous histiocytoma. Cancer. 1977 Apr; 39 (4): 1672-85. Research Article 4.70:1. This is due to the fact that many benign tumors such as lipoma and hemangioma do not undergo biopsy as compared to sarcoma, which come to medical attention quite early [11]. However, in Kransdorf study the incidence of malignant tumorswere high which might be because of thereffered cases from peripheral centres [7]. In conclusion, in the present study benign tumors comprised 89% and malignant tumor comprised 10.7% among all tumors and benign tumors are common in occurred in second and third decade of life. In the present study majority of malignant soft tissue tumors occurred in fifth decade of life. 5. Baig MA. Histological study of soft tissue tumor (Three Year Study). International Journal of Science and Research (IJSR) 2005: 1039 – 49. 6. Kransdorf MJ. Malignant soft-tissue tumors in a large referral population: distribution of diagnoses by age, sex, and location. AJR Am J Roentgenol. 1995 Jan;164 (1):129-34. 7. Kransdorf MJ. Benign soft-tissue tumors in a large referral population: distribution of specific diagnoses by age, sex, and location. AJR Am J Roentgenol. 1995 Feb; 164(2):395-402. Patel Y. M, Tandel H. K. Histopathological differentiation and incidence of soft tissue tumors: study of 140 cases. Trop J Path Micro 2017;3(3):335-339.doi: 10.17511/jopm.2017.i3.19. 11. Ross R. The smooth muscle cell. II. Growth of smooth muscle in culture and formation of elastic fibers. J Cell Biol. 1971 Jul;50(1):172-86. Tropical Journal of Pathology & Microbiology Discussion Average age of occurrence of benign tumor in the study by Mirza et al was 29.2 years, which is similar to the present study (32.1 years). Whereas the average age of incidence of malignant tumor found highly increased in present study which is 55.6 years as compared to previous study of Mirza et al it was 37.8 years. The ratio of M: F found of benign and malignant is similar in the study of Mirza et al and in the present study [5]. These findings are also very similar with the studies reported by Kransdorf. The average age of hemangiomas in this study was 31.16 years, which is little higher as compared to studies reported Mirza et al and Kransdorf. This is because; in this study the age range varied from 10 to 80 years. [11]. The sex and site distribution were similar to the literature given in Enzinger and Weiss (2001). [10] In the present study benign-malignant ratio is 8.3: 1 while in the study of Mirza et al, there were 113 benign tumors and 24 malignant tumors with a benignmalignant ratio of In the present study adipose tissue tumors constituted, the commonest soft tissue tumor accounting for 50 % of all soft tissue tumors. The male to female ratio of 1.34: Tropical Journal of Pathology & Microbiology Available online at: www.pathologyreview.in 338 \| P a g e 338 \| P a g e 338 \| P a g e Available online at: www.pathologyreview.in 33 July - September, 2017/ Vol 3/ Issue 3 ISSN 2456-1487 July Available online at: www.pathologyreview.in 339 \| P a g e How to cite this article? Patel Y. M, Tandel H. K. Histopathological differentiation and incidence of soft tissue tumors: study of 140 cases. Trop J Path Micro 2017;3(3):335-339.doi: 10.17511/jopm.2017.i3.19. Tropical Journal of Pathology & Microbiology 339 \| P a g e Available online at: www.pathologyreview.in 339 \| P a g e
https://openalex.org/W4244823281	https://zenodo.org/records/3392184/files/13554__1_222709_LE_333481.pdf	Dutch	null	Binnengekomen boeken	MAB	1,986	cc-by	527	Binnengekomen boeken Onroerend-goedbelastingen: hoe en wat Kluwer Belastingwijzers nr. 18 Anneke Bangma-Beenhakker en Jan Paul Kruimel Uitgeverij Kluwer B.V. te Deventer Prijs f 24,50 Geld en financiering: een toenemende ver vlechting Rede uitgesproken door Dr. J. J. Sijben bij het aanvaarden van het ambt van gewoon hoogleraar aan de Katholieke Hogeschool Til burg Uitgegeven door Tilburg University Press te Tilburg Prijs / 8,60 Omgeving, doelstellingen en organisatie; vi sies uit theorie en praktijk Bundel aangeboden aan Prof. Dr. H. O. Goldschmidt t.g.v. zijn emeritaat Redactie Dr. J. A. C. de Haan/Prof. Drs. J. A. M. Oonincx/Prof. Dr. P. Ribbers Uitgeverij Van Gorcum & Comp B.V. te Maas tricht Prijs f 65,- Introductie tot het Nederlands belastingrecht (3e geheel herziene druk) Mr. A. M. van Lieshout, m.m.v. Mr. A. H. R. M. Denie en Dr. B. J. M. Terra Uitgeverij Gouda Quint B.V. te Arnhem Prijs f 52,50 Handelsregister, Verenigingen- en Stichtin genregister Serie Praktijkhandleidingen Mr. W. R. Veldhuyzen Uitgeverij W. E. J. Tjeenk Willink B.V. te Zwolle Prijs ƒ 49,50 Onroerend-goedbelastingen: hoe en wat Kl B l i ij 18 Voorzien van informatie. Methoden voor in- formatiebeleidsvorming en informatieplan ning P f h if D J A M Th g g Kluwer Belastingwijzers nr. 18 A k B B h kk g Proefschrift van Drs. J. A. M. Theeuwes ter verkrijging van de graad van doctor in de technische wetenschappen aan de Technische Hogeschool Eindhoven op 30 mei 1986. Theorie van de kostencalculatie Prof. Dr. K. A M. Bogaert Uitgeverij Wolters-Noordhoff B.V. te Gronin gen Prijs f 42,50 Theorie van de kostencalculatie Prof. Dr. K. A M. Bogaert Uitgeverij Wolters-Noordhoff B.V. te Gronin gen Prijs f 42,50 Gids voor Belastingstudie 1986 (12e druk) J. A. Rouwenhorst en J. Meulenbeld Uitgeverij Wolters-Noordhoff B.V. te Gronin gen Prijs ƒ 64,25 gPrijs ƒ 64,25 Introductie tot het Nederlands belastingrecht (3e geheel herziene druk) Mr. A. M. van Lieshout, m.m.v. Mr. A. H. R. M. Denie en Dr. B. J. M. Terra Uitgeverij Gouda Quint B.V. te Arnhem Prijs f 52,50 Computerboekhouden Drs. H. P. A. J. Langendijk Uitgeverij Wolters-Noordhoff B.V. te Gronin gen Prijs ƒ 19,75 Computerboekhouden Drs. H. P. A. J. Langendijk Uitgeverij Wolters-Noordhoff B.V. te Gronin gen Prijs ƒ 19,75 ( g ) Mr. A. M. van Lieshout, m.m.v. Mr. A. H. R. M. Denie en Dr. B. J. M. Terra Uitgeverij Gouda Quint B.V. Binnengekomen boeken te Arnhem Prijs f 52,50 Handelsregister, Verenigingen- en Stichtin genregister Serie Praktijkhandleidingen Mr. W. R. Veldhuyzen Uitgeverij W. E. J. Tjeenk Willink B.V. te Zwolle Prijs ƒ 49,50 gPrijs ƒ 19,75 De vennootschap onder firma. De heffing van inkomstenbelasting bij oprichting en ontbin ding van de firma en bij toe- en uittreden van firmanten (5e herziene druk) Fiscale Monografieën nr. 10 Mr. J. Spaanstra/Prof. Mr. J. F. M. Giele/Dr. W. A. Vermeend Uitgeverij Kluwer B.V. te Deventer Prijs f 59,50 De vennootschap onder firma. De heffing van inkomstenbelasting bij oprichting en ontbin ding van de firma en bij toe- en uittreden van firmanten (5e herziene druk) Fiscale Monografieën nr. 10 Mr. J. Spaanstra/Prof. Mr. J. F. M. Giele/Dr. W. A. Vermeend Uitgeverij Kluwer B.V. te Deventer Prijs f 59,50 11-507 11-507
https://openalex.org/W4297939146	https://zenodo.org/record/7121310/files/A%20bibliometric%20review%20of%20sustainable%20finance%20setting%20the%20research%20agenda.pdf	English	null	A bibliometric review of sustainable finance: setting the research agenda	Zenodo (CERN European Organization for Nuclear Research)	2,021	cc-by	320	1 University of Cagliari, Italy, r.fatima@studenti.unica.it. MIRDEC-GLOBECOS Istanbul 2021 - International Academic Conference on Economics, Business and Contemporary Issues in Social Science 29-30 November 2021, Istanbul, Turkey Masters International Research & Development Center Global Community of Social Science MIRDEC-GLOBECOS Istanbul 2021 - International Academic Conference on Economics, Business and Contemporary Issues in Social Science 29-30 November 2021, Istanbul, Turkey Masters International Research & Development Center Global Community of Social Science y g y 2 Bucharest University of Economic Studies, Romania, iustina.boitan@fin.ase.ro. 3 University of Cagliari, Italy, rosella.care@unica.it. y g y 2 Bucharest University of Economic Studies, Romania, iu Keywords: Sustainable Finance, Sustainable Development, Bibliometrics Abstract The emerging environmental, social and economic pressures highlighted the need for long-term approaches to bring sustainable development. Sustainable finance emerged as a novel approaches that actively contribute to sustainable development. However, it is often considered as an umbrella term that encompasses different financial practices, tools, and instruments. All the progress and success in the field of sustainable finance to date notwithstanding, it is thus essential to develop a standardized conceptual framework to clarify the ambiguity among the core construct. Based on this consideration, this study applies a bibliometric approach to provide a robust roadmap that delineates the sustainable finance conceptual landscape. The concrete outcomes of this research elucidate the origin and evolution of the sustainable finance concept and provide a contemporary review of the related tools, approaches, and instruments. Simultaneously, this study also depicts the precise boundaries among the core elements of sustainable finance. The main contribution of this study is thus to provide researchers, organizations, and the society a better understanding of all the aspects of sustainable finance that are useful for sustainable development in the financial world. Further, this research uncovers the underexplored areas within the sustainable finance paradigm to which researchers and organizations should channel their attention. Keywords: Sustainable Finance, Sustainable Development, Bibliometrics JEL Codes: G01, Q01 JEL Codes: G01, Q01 94
https://openalex.org/W2935363975	https://tidsskriftet-ip.no/index.php/intpol/article/download/1626/3124	Norwegian	null	Forsvar vår dyd, men kom oss ikke for nær. Norge og det militære samarbeidet i NATO	Internasjonal politikk	2,019	cc-by	4,969	©2019 Paal Sigurd Hilde. This is an Open Access article distributed under the terms of the Creative Commons Attri- bution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and re- distribute the material in any medium or format and to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license. Citation: Paal Sigurd Hilde (2019). Forsvar vår dyd, men kom oss ikke for nær. Norge og det militære samarbeidet i NATO. Internasjonal Politikk, 77(1): 60–70. http://dx.doi.org/10.23865/intpol.v77.1626 *Kontaktinformasjon: Paal Sigurd Hilde, e-post: philde@ifs.mil.no Fokus: Nato 70 Fokus: Nato 70 Forsvar vår dyd, men kom oss ikke for nær. Norge og det militære samarbeidet i NATO Paal Sigurd Hilde Institutt for Forsvarsstudier, Forsvarets høgskole Årgang 77, Nummer 1, side 60-70, 2019, ISSN 1891-1757, www.tidsskriftet-ip.no, Publisert april 2019 Årgang 77, Nummer 1, side 60-70, 2019, ISSN 1891-1757, www.tidsskriftet-ip.no, Publisert april 2019 Sammendrag Artikkelen tar utgangspunkt i Johan Jørgen Holsts og Rolf Tamnes’ klassiske kategoriseringer og gir et perspektiv på norsk sikkerhetspolitikk som et forsøk på å finne en balanse mellom ulike behov: støtte fra vest, lavspenning med øst og innenrikspolitisk oppslutning. Artikkelen retter oppmerksomheten mot to områder av det militære samarbeidet i NATO som har vært sentrale for Norge gjennom 70 års medlemskap: NATOs kommandostruktur og alliert tilstedevæ relse i og planer for forsterkning av landet. Områdene har gitt tydelige utrykk til den norske balansegangen og vært viktige for utviklingen av denne. Overordnet har norsk sikkerhetspolitikk gjennom en skiftende internasjonal kontekst siden 1949 beveget seg mot mer integrasjon og avskrekking og mindre beroligelse og avskjerming. Et større steg i denne retning kom i 1995, mindre i 1951, 1977 og 2016. Selv om balansepunktet har blitt forskjøvet, er imidlertid norske myndig heters vektlegging av å finne en balanse fortsatt relevant og levende. Nøkkelord: sikkerhetspolitikk • kommandostruktur • alliert øving og trening • allierte forsterkninger • selvpålagte restriksjoner Erfaringene fra den 9. april 1940 og den påfølgende krigen og okkupasjonen sto sen tralt da Norge ni år senere, den 4. april 1949, gikk inn i Atlanterhavspakten. Mellom krigstidens tro på at nøytralitet og en geografisk perifer posisjon ville skjerme landet fra krigene på kontinentet, hadde vist seg ikke å holde. Norge ville bli involvert i en eventuell ny stormaktskrig og trengte sterke allierte som kunne komme landet 60 orsvar vår dyd, men kom oss ikke for nær. Norge og det militære samarbeidet i NAT til unnsetning. Gitt valget mellom et demokratisk vest og totalitært øst var Norges undertegnelse av Atlanterhavspakten et åpenbart utfall. Samtidig var det særlig to forhold som gjorde utfallet mindre selvfølgelig og Norge forbeholden i sitt medlemskap. Det første var innenrikspolitisk og politisk- ideologisk. Det var knyttet delvis til «holdningene fra nøytralitetstiden» som var sær lig sterke hos de såkalte nasjonalkonservative, og delvis til «sympati for det sovjetiske samfunnssystem» og skepsis mot den vestlige oppdemmingspolitikken i arbeiderbe vegelsen (Eriksen & Pharo, 1997, s. 31; Sverdrup, 1996, s. 202). Det andre forholdet var det realpolitiske målet om lavspenning i relasjonene med Sovjetunionen, som Norge fra september 1944 delte grense med i nord. Målet var motivert i engstelsen for sovjetiske intensjoner og territorielle krav mot Norge. Formet i skjæringspunktet mellom behovet for støtte fra vest, lavspenning med øst og innenrikspolitiske hensyn ble norsk sikkerhetspolitikk en balansepolitikk – et politisk kompromiss. 1 Etter slutten av den kalde krigen kom en ny balansegang til som komplementerte den tradis jonelle: En mellom tradisjonelle, kollektive forsvarsoppgaver og krisehåndteringsoperasjoner uten for NATOs område (se Hilde & Widerberg, 2014). Den er ikke så viktig for denne artikkelen. Sammendrag Overfor Sovjetunionen besto balansegangen av avskrekking og beroligelse, og overfor Alliansen og allierte av integrasjon og avskjerming, i henholds vis Johan Jørgen Holsts (1966) og Rolf Tamnes’ (1987) klassiske kategoriseringer.1 Forsvarsminister Jens Christian Hauge så alt våren 1949 dualismen i den norske holdningen og oppsummerte den med ordene i tittelen: «Forsvar vår dyd, men kom oss ikke for nær» (Eriksen & Pharo, 1997, s. 85). Balansegangen har vært en dialektikk – et samspill – mellom integrasjons- og avskjermingstiltak, der norske regjeringer gjennom sju tiår i NATO har søkt – og ofte strevet med – å ivareta de ulike hensynene. Avskjermingstiltak har gjerne kompen sert for integrasjonsvedtak, og tatt form av selvpålagte restriksjoner. Basepolitikken, begrensingene på alliert aktivitet i nord og atompolitikken har vært de viktigste slike restriksjonene. Gitt at de selvpålagte restriksjonene var og er nettopp det – selvpålagte – har norske myndigheter kunnet justere og fortolke dem. Norske myndigheter har aldri gitt en inkluderende beskrivelse, noe som har gitt handlingsrom. En skiftende sikkerhetspolitisk kontekst og endringer i forsvarspolitiske behov har over 70 år ført til flere justeringer og fortolkninger. Denne artikkelen retter oppmerksomheten mot det militære samarbeidet i NATO og hvordan Norges balansegang har fått uttrykk på to sentrale områder: NATOs kommandostruktur og alliert tilstedeværelse i og planer for forsterkning av Norge. Områdene representerer to av de tydeligste utrykkene for den norske balansegangen og har vært viktige for utviklingen av denne. Som vi skal se, har norske interesser knyttet til NATOs kommandostruktur og alliert tilstedeværelse og forsterkning lig get til grunn for mange av endringene norske regjeringer har foretatt i de selvpålagte restriksjonene. 61 Paal Sigurd Hilde Paal Sigurd Hilde Norge og NATOs kommandostruktur I USA var det i 1949–50 liten interesse for en institusjonalisering av NATO. Med norske øyne var dette en fordel. Verken utenriksminister Halvard Lange eller for svarsminister Hauge ønsket noe integrert militært samarbeid under ledelse av en alliert generalstab (Eriksen & Pharo, 1997, s. 33). Atlanterhavspakten skulle være en tradisjonell militær allianse. Koreakrigen ble en katalysator for utviklingen av NATO og endret både USAs og Norges syn. I september 1950 besluttet de allierte å opprette en felles kommando under amerikansk ledelse. Den norske skepsisen ble omvandlet til en aktiv holdning. To hovedmål utkrystalliserte seg; mål som tydelig reflekterte den norske balanse gangen. På den ene siden førte Norge en invitasjonspolitikk der målet var å bruke kommandostrukturen til å knytte alliansen og allierte til forsvaret av landet. På den andre vektla norske myndigheter avskjerming gjennom å sikre nasjonal kontroll over, eller størst mulig nasjonal påvirkning i NATOs kommandolinje, og derigjennom på planverk og eventuelle operasjoner i Norge og norske nærområder. De konkrete uttrykkene disse målene har fått frem til dag, og i hvilken grad Norge har lykkes med å få gjennomslag, har variert. Kjernen i den norske målsettingen har imidlertid ligget fast, noe vi tydelig kan spore i dagens politikk. 2 For Øverstkommanderende i Sør-Norge gjaldt dette fra 1981. Integrasjon Målet om å binde allierte til forsvaret av Norge lå bak det norske engasjementet for å få opprettet et NATO-hovedkvarter med tung amerikansk og britisk deltakelse i Norge. Forsvarsminister Hauge vektla at et slikt hovedkvarter ville sikre bedre kunn skap om norske forhold hos allierte militære sjefer; en kunnskap de kunne videre formidle i kommandokjeden. Det ville også gi norske myndigheter enklere tilgang til de militære sjefene (Stortinget, 1951b, s. 3). Viktigst var imidlertid at den direkte involveringen av amerikanere og briter ville fungere som «en pant på viljen» til de to landene, slik utenriksminister Lange formulerte det (Stortinget, 1951a, s. 8). Det ville bidra til å sikre at de kom Norge til unnsetning. Norske myndigheter ønsket en regional kommando i Norge, men fryktet sam tidig at dersom nordregionen ble begrenset til bare norsk territorium, kunne den lett bli nedprioritert. Norge søkte derfor å knytte forsvaret av Norge til forsvaret av kontinentet. Utfallet ble igjen positivt med norske øyne: Nordkommandoen (Allied Forces Northern Europe, AFNORTH) fikk til slutt ansvaret for Norge, Danmark og de danske stredene, samt den nordligste tyske delstaten Schleswig-Holstein (Pedlow, 2010, s. 7). Gitt betydningen av havområdene utenfor Norge for forsvaret av landet, førte Norge en aktiv politikk også overfor NATOs Atlanterhavskommando (Allied Command Atlantic, ACLANT). Norge søkte aktivt å involvere seg i ACLANT, og å involvere ACLANT i forsvaret av Norge. Et viktig mål var å sikre et best mulig 62 Forsvar vår dyd, men kom oss ikke for nær. Norge og det militære samarbeidet i NATO samarbeid mellom ACLANT og Europakommandoen (Allied Command Europe, ACE). Én grunn til dette var at ACLANT i krig hadde kommandoen over marine infanteriavdelinger som mot slutten av den kalde krigen hadde Norge som prioritert innsatsområde. Brudd og kontinuitet Slutten av den kalde krigen og den sikkerhetspolitiske utviklingen i tiårene siden har medført store endringer også for NATOs kommandostruktur (se Hilde, 2018). Utviklingen har derigjennom hatt stor betydning for de tradisjonelle norske målene som ble utfordret av endringene i NATOs oppgaver. Integrasjonsmålet var det før ste som kom under press. Allerede i 1994 ble Nordkommandoen lagt ned og fra 1994–99 var Norge en del av ansvarsområdet for NATOs nordvestlige kommando lokalisert i Storbritannia. Norge hadde ikke lenger den samme, fysiske «pant på viljen» til sine viktigste allierte. Koblingen til kontinentet forsvant også under nordvestkom mandoen, men kom tilbake i 1999–2003 da Norge ble del av en ny Regionkom mando nord i Nederland. Også avskjermingstiltakene ble over tid stadig vanskeligere å opprettholde. Kravene om norsk samtykke og norsk sjef i linjen ble videreført ved at de i 1994 ble stilt direkte til sjefene for Europa- og Atlanterhavskommandoen. Tross dette viste det seg raskt å være vanskelig å bevare en norsk sjef i kommandolinjen. På 1990-tallet lykkes Norge med å opprettholde norske hovedkvarter i kommandokje den. Den faktiske rollen disse ville ha i krise og krig, ble imidlertid etter hvert svært begrenset (Børresen, Gjeseth & Tamnes, 2004, s. 148). Etter angrepet på USA den 11. september 2001 akselererte omstillingen av NATO. I en ny, omfattende omstil ling av kommandostrukturen ble det gjenværende NATO-hovedkvarteret i Norge foreslått nedlagt. Norske myndigheter innså at målet om en norsk sjef i linjen ikke lenger var realistisk og oppga ambisjonen. Delvis av denne grunn, men også gitt andre endringer, ble restriksjonene satt i 1953/67 og 1970 lite hensiktsmessige som sådan. De ble derfor opphevet i 2006. De tradisjonelle norske målene fikk imidlertid en renessanse da den sik kerhetspolitiske situasjonen endret seg i Europa. Den russiske intervensjonen i Georgia i 2008 og særlig anneksjonen av Krim og intervensjonen i Ukraina i 2014 var viktige vendepunkt. I omstillingene av kommandostrukturen som fulgte i 2010–11 og 2017–18, arbeidet Norge for gjeninnføring av regionalt ansvar i kommandostrukturen; et ansvar som gradvis hadde blitt fjernet (Børresen, Gje seth & Tamnes, 2004, s. 148). Hensikten var prinsipielt den samme som da Hauge kjempet for Nordkommandoen: Å sikre bedre kunnskap om utviklingen i norske nærområder, slik at kommandostrukturen bedre og raskere kan reagere på utfordringer der. Norske myndigheter søkte videre å gi nasjonale hovedkvar ter en større rolle. Avskjerming Invitasjon og integrasjon var speilet av avskjerming. Det første konkrete uttrykket kom i form av svaret på en sovjetisk note vinteren 1949. Noten stilte spørsmålet om Norge ville forplikte seg til å opprette militære fly- og flåtebaser, implisitt amerikanske baser, dersom landet ble med i Atlanterhavspakten. Det norske svaret ble kjent som baseerklæringen og var at Norge ikke ville tillate «baser for fremmede makters strids krefter på norsk territorium så lenge Norge ikke er angrepet eller utsatt for trusler om angrep.» (Holst, 1967, s. 66) Beslutningen om tettere å integrere «fellesforsvaret» i Europa gjennom en felles kommandostruktur, skapte et behov for å tydeliggjøre at basepolitikken ikke var til hinder for en viss alliert tilstedeværelse også i fredstid. I februar 1951 presiserte for svarsminister Hauge derfor basepolitikken i en redegjørelse for Stortinget (1951c: 286). Hauge nevnte ikke hovedkvarter spesifikt da det ennå ikke var klart at en kom mando ville bli opprettet i Norge. Basepolitikken ble tross det fra 1951 «forstått og praktisert slik at» den ikke hindret «anlegg for kommando, kontroll og kommunika sjon», slik Forsvarsdepartementet (1977: 4) senere understreket. Norsk avskjerming overfor kommandostrukturen tok dermed en annen form. Målet var som nevnt var å sikre norsk kontroll eller størst mulig norsk påvirkning. Norske myndigheter søkte å oppnå dette på to måter. Den første var direkte å avgrense Nordkommandoens handlingsrom. I 1953 satte norske myndigheter krav til sjefen for AFNORTH om norsk samtykke for beslutninger om å bombe norsk territorium, sende norske soldater utenfor kommandoens ansvarsområde og å iverk sette operasjoner som avvek vesentlig fra godkjente planer (Skogrand, 2004, s. 201). Ordningen ble stadfestet i 1967, og i 1970 ble tilsvarende begrensinger satt for avgi velse av kommando over norske styrker til sjefen for allierte sjøstridskrefter øst i Atlanterhavet (Eastern Atlantic Area, EASTLANT). Den andre måten var å sikre norsk kontroll gjennom å ha norske offiserer i kommandokjeden. De norske kravene av 1953/67 og 1970 inneholdt en betin gelse om at operasjoner i Norge skulle ledes av en norsk sjef (Børresen, Gjeseth & Tamnes, 2004, s. 55). Hovedkvarterene underlagt AFNORTH var på linje med dette norske hovedkvarter ledet av en norsk sjef som hadde en rolle både i NATOs og nasjonal kommandostruktur. Tilsvarende fikk de norske hovedkvarterene etter 1970 ansvar for operasjoner med maritime fly og ubåter i havet utenfor Norge på vegne av Atlanterhavskommandoen.2 63 Paal Sigurd Hilde Paal Sigurd Hilde Brudd og kontinuitet Også her var hensikten tradisjonell, selv om formen var mer indirekte: å gi Norge innflytelse på planleggingen for og utøvelsen av eventu elle operasjoner i Norge og norske områder. I motsetning til under den kalde krigen har Norge ikke søkt å innføre formaliserte begrensinger. I god tradisjon har norske myndigheter også tatt til orde for å styrke den maritime delen av kommandostrukturen. 64 Forsvar vår dyd, men kom oss ikke for nær. Norge og det militære samarbeidet i NATO Allierte forsterkninger, øving og trening Parallelt med innsatsen knyttet til kommandostrukturen søkte norske myndigheter å sikre at allierte styrker kom landet til unnsetning i krise og krig. Norge balanserte avskjermingen i basepolitikken med en invitasjonspolitikk for å sikre forpliktelser om forsterkninger både i NATOs planverk og fra allierte avsenderland. Videre vektla norske myndigheter at styrkene måtte forberedes for innsats i krig gjennom trening og øving i fred. Forhåndslagring av materiell for å sikre rask overføring til Norge, var et annet grep som over tid fikk økt betydning. Slutten av den kalde krigen begrenset raskt hva Norge kunne oppnå gitt at NATOs og alliertes oppmerksomhet vendte seg mot nye oppgaver. Det ledet Norge til å justere balansen mellom invitasjon og avskjerming. Som med kommandostruk turen har den endrede sikkerhetspolitiske situasjonen i det siste tiåret ført til en renessanse for den tradisjonelle norske tilnærmingen. Avskjerming Våren 1949 gjorde forsvarsminister Hauge det klart at Norge ville begrense alliert aktivitet nord i Norge generelt, og i Finnmark og på Svalbard spesielt. Som med basepolitikken var begrensningen ment å kompensere for medlemskapet i Atlanter havstraktaten ved å sikre at «provokasjonsmomentet» overfor naboen i øst ble minst mulig (Eriksen & Pharo, 1997, s. 32–33). Ut over 1950- og 60-tallet formulerte norske myndigheter en rekke konkrete restriksjoner på aktivitet på norsk landterri torium og på luft- og sjøaktivitet som berørte Norge (for detaljer se Moen, 1998). For aktivitet til havs og i luften ble den 24. østlige lengdegrad, som går like øst for Hammerfest, en viktig grense. Den norske balanseringen av integrasjon med avskjerming av alliert tilstede værelse og aktivitet ble justert gjentatte ganger. Hauge presiserte i februar 1951 at basepolitikken ikke var til hinder for «militære anlegg» som skulle sikre at Norge kunne «ta imot og underholde på effektivt vis allierte stridskrefter som overføres til Norge», eller for «allierte samøvinger eller […] kortvarige besøk» (Stortinget, 1951c, s. 286). Mens Norge avviste mer omfattende amerikanske planer om stasjonering av flystyrker, ga presiseringen rom for å godta SAC-installasjonene. Den første store NATO-øvelsen som inkluderte Norge, øvelse Mainbrace i 1952, ble på den annen side underlagt klare restriksjoner. Allierte sjøstyrker fikk for eksempel ikke operere nord av Narvik (68° nord) (Skogrand, 2004, s. 196). Også senere konkretiseringer og justeringer var hendelsesdrevne. Det mest kjente eksempelet er etterretningsflyet U-2 som ble skutt ned i 1960 på vei fra Karachi i Pakistan til Bodø (Tamnes, 1991, s. 116–136). Hendelsen førte til en inn skjerping av regelen fastsatt første gang i 1955 om alliert fly- og sjøaktivitet øst for 24° lengdegrad. En forespørsel om å øve AMF-styrken i Finnmark i 1964 førte til svarende til en tydeliggjøring av at allierte bakkestyrker ikke kunne trene og øve i Finnmark. Over tid utviklet de selvpålagte restriksjonene seg til et detaljert sett med regler for hvor mange soldater og fly som kunne oppholde seg i Norge til enhver tid, hvor lenge og hvor de kunne oppholde seg, samt hvilke typer fly og avdelinger som ikke var velkomne (Børresen, Gjeseth & Tamnes, 2004, s. 58). På 1970-tallet kom spørsmål opp om forhåndslagring av tyngre utstyr for allierte bakkestyrker knyttet til deres planlagte bruk i Norge. Tidligere avtaler hadde stort sett omfattet drivstoff, ammunisjon og reservedeler for fly og fartøy. Integrasjon Mens målet om å få et alliert hovedkvarter til Norge ble oppnådd relativt rask, tok det lang tid før norske myndigheter fikk konkrete forpliktelser om forsterkninger. I de første årene av den kalde krigen ble tilgjengelige styrker prioritert til kontinentet. Norge lå i et kontinentalt perspektiv ikke i fremste linje. Planene antok videre at der som Norge ble angrepet, så var det umulig å forsvare mer enn en liten del av landet (Gjeseth, 2012, s. 95–97). Norge kunne følgelig ikke regne med allierte forsterknin ger av særlig omfang. I denne fasen var det i hovedsak det amerikanske luftforsvaret som viste interesse. Formålet var dels å bruke flyplasser i Sør-Norge for operasjoner over og mot kontinentet og dels å støtte operasjoner med strategiske bombefly. Ordnin gen med Strategic Air Command (SAC) som varte frem til slutten av 1950-tallet, inkluderte opprettelsen av sambandsstasjoner på Sola, Gardermoen og senere Bodø (Skogrand & Tamnes, 2001, s. 75–95). Interessen illustrerer at det foruten norske invitasjoner også tidvis har vært påtrykk fra allierte. Det er det eksempler på gjen nom hele perioden frem til i dag. Styrkingen av NATOs konvensjonelle styrkestruktur ga over tid større rom for også å forsterke flankene. På 1960-tallet fikk flere avdelinger nordflanken som mulig innsatsområde. De var imidlertid stort sett små og lett utstyrt og hadde begren set militær verdi. En var Europakommandoens mobile styrke (ACE Mobile Force, AMF) som ble opprettet i 1960–61. AMF var en liten, flernasjonal avdeling som skulle settes inn på flankene for å avskrekke ved å vise NATOs flaggborg, fremfor å vinne slag (Skogrand, 2004, s. 185). Mot slutten av 1970- og 80-tallet endret imidlertid situasjonen seg av flere årsaker. Den økende strategiske betydningen av Nordflåten på Kola, amerikansk sjømilitær opprustning og innføringen av en ny amerikansk maritim strategi som vektla å angripe Nordflåten i nord, var viktigst. Mot slutten av 1980-tallet, da omfanget 65 Paal Sigurd Hilde av forsterkninger med Norge som høyest prioritert innsatsområde var på sitt største, omfattet de omlag 24 tusen soldater og 200–300 fly (Tamnes & Eriksen, 1999, s. 19). Marineinfanteriavdelinger fra Storbritannia, Nederland og ikke minst USA var vik tige, hvorav det amerikanske marinekorpsets Andre ekspedisjonsstyrke (II Marine Expeditionary Force, II MEF) var den mest slagkraftige. Brudd og kontinuitet Den kalde krigens slutt førte i løpet av få år til store endringer i NATOs og allierte forsvars- og forsterkningsplaner, og i alliert trening, øving og annen aktivitet. Det omfattende og konkrete forsterkningsplanverket som omfattet Alliansen som sådan, ble erstattet av et løsere og mer fleksibelt planverk for flankene. Allierte forsvar ble redusert i størrelse, nye oppgaver kom til og interessen falt for både å øremerke styr ker for innsats i Norge og å forhåndslagre materiell (Børresen, Gjeseth & Tamnes, 2004, s. 151–153). Norske myndigheter arbeidet iherdig for å bevare forpliktelser til Norge i NATO og fra allierte. Dette omfattet forsterkningsplanverket, men også fortsatt alliert tre ning og øving i Norge. NATO hadde på 1960-tallet etablert øvelses-serier som jevn lig ble avholdt i Norge frem til 1990-tallet (Skogrand, 2004, s. 186). AMF-styrken hadde Express-serien og ACLANT Teamwork-serien. Også andre maritime øvelser, som Ocean Safari-serien fra 1975, var av stor betydning for Norge (Børresen, Gje seth & Tamnes, 2004, s. 101). Omfanget av øvelsene økte kraftig på 1980-tallet. På 1990-tallet var trenden motsatt og den ble forsterket ytterligere etter den 11. sep tember 2001. Både antallet NATO-ledede øvelser og interessen og ressursene for å delta i dem falt. For å kompensere opprettet Norge nasjonale øvelser dit allierte ble invitert, herunder Joint Winter fra 2000–2004 og Cold Response fra 2006. Tross dette fortsatte den negative trenden på 2000-tallet (Tveiten, 2016, s. 39–41). Ønsket om å opprettholde alliert trening og øving i Norge var en hoved- beveggrunn for justeringen av de selvpålagte restriksjonene i 1995. Regjeringen ville «legge forholdene til rette for» at det fortsatt var «attraktivt for allierte styrker å øve i Norge» (Forsvarsdepartementet, 1995, s. 23). Rustningskontrollavtaler og samar beid med NATO-partnere var andre argumenter for hvorfor de selvpålagte restrik sjonene delvis hadde blitt uhensiktsmessige. Endringene innbar at restriksjonene nå gjaldt «all utenlandsk militær virksomhet i Norge» og ikke bare alliert, at de mange, konkrete reglene ble erstattet av generelle retningslinjer, samt at «de særlige begren singene for Finnmark [ble] gjort lempeligere og lettere å praktisere.» (ibid.) Endringen i den sikkerhetspolitiske situasjonen på slutten av 2000-tallet skapte som nevnt en renessanse for den tradisjonelle norske tilnærmingen. Etter 2014 for sterket norske myndigheter sin innsats for å sikre norske interesser i utarbeidelsen av NATOs planverk og å legge til rette for allierte forsterkninger til Norge. Avskjerming Det var nok en viktig grunn til at Forsvarsdepartementet (1977, s. 4) presiserte at de selvpålagte restriksjonene ikke hindret etablering av «lagre av ammunisjon og utstyr, forsyninger 66 Forsvar vår dyd, men kom oss ikke for nær. Norge og det militære samarbeidet i NATO o.l. for allierte styrker.» Presiseringen åpnet for en omfattende avtale i 1981 om for håndslagring for det amerikanske marinekorpset (Stortinget, 1980). Også her var det en balanse, imidlertid. Den opprinnelige anbefalingen var at lagrene skulle leg ges i Nord-Norge, men etter en heftig debatt falt valget på Trøndelag (Tamnes & Eriksen, 1999, s. 24). Brudd og kontinuitet Interessen for forhåndslagring av utstyr har økt til gamle høyder, og det samme har interessen for å delta på øvelser. Øvelsen Trident Juncture 2018 var et tydelig uttrykk for det. 67 Paal Sigurd Hilde Paal Sigurd Hilde Dagens ambisjoner knyttet til II MEF har flere likhetstrekk med planene på slutten av den kalde krigen. Den nye given i invitasjonspolitikken har ikke blitt balansert av nye avskjer mingstiltak. Reglene som ble løsnet på i 1995, har ikke blitt strammet inn igjen, men derimot løsnet ytterligere. Det tydeligste eksempelet er beslutningen i 2016 om å etablere en roterende tilstedeværelse av Marinekorpset; en beslutning som hadde bakgrunn i et amerikansk initiativ (Aftenposten, 2016). Jevnlig og hyppig øving og trening i Norge har funnet sted i flere tiår. Britiske Royal Marines’ årlige Exercise Clockwork feiret femtiårsjubileum i januar 2019 (ABC Nyheter, 2019). Til stedeværelsen av deres amerikanske kollegaer har imidlertid et enda mer permanent preg, i form av en kontinuerlig tilstedeværelse av stridsstyrker, som er ny i norsk etterkrigshistorie. Oppsummering Beslutningen om å gå inn i NATO og det integrerte militære samarbeidet var utgangspunktet for Norges sikkerhetspolitiske balansegang. Hensynet til å berolige Sovjetunionen, samt sikre støtte i hjemlig opinion, var hovedargumentene for de selvpålagte restriksjonene. Behovet for å sikre troverdig avskrekking tross restriksjo nene var igjen grunnlag for invitasjonspolitikken. Slik ser norske myndigheter det fortsatt. Som regjeringen vektla i 2018: «Alliert forsterkning for å forsvare Norge må være troverdig og bærekraftig. [… Det] er en forutsetning for basepolitikken, som nettopp innebærer et avkall på […] beredskap» (regjeringen.no, 2018). Som fremstillingen over viser, er det stor grad av kontinuitet i den norske balan segangen. Dagens tilnærming er i prinsipp den samme som under den kalde krigen. Vi finner imidlertid også brudd som i hovedsak har blitt utløst av endrede rammer. Slutten av den kalde krigen innebar den mest omfattende endringen, men etable ringen av NATOs organisasjon på 1950-tallet, den 11. september 2001 og den nye spenningen i Europa særlig etter 2014, var alle betydningsfulle. Endringene i de sikkerhetspolitiske rammene har tvunget frem, eller åpnet for, justeringer av balanse gangen for å sikre at de rådende oppfatningene om behovet for å sikre alliert støtte, å ta hensynet til naboen i øst og å opprettholde støtte i hjemlig opinion ble ivaretatt. I perioder der norske interesser har stemt overens med våre alliertes, som på 1970- og 80-tallet og i perioden etter 2014, har oppgaven med å finne en ny balanse vært relativt smertefri. I perioder der Norge har svømt mot strømmen i NATO, som på 1990-tallet, har den vært krevende. Bruddene har tatt form av justeringer av de konkrete selvpålagte restriksjonene på den ene siden og gjerne en forsterket invitasjonspolitikk på den andre. Også her er det en kontinuitet. Presiseringene i 1951 og 1977 åpnet for integrasjon ved å tyde liggjøre hva som ikke var ekskludert av de selvpålagte restriksjonene. Det samme, om enn på en mindre formell måte, gjorde regjeringens vektlegging av at Mari nekorpsets tilstedeværelse er «godt innenfor etablerte retningslinjer og ikke minst 68 orsvar vår dyd, men kom oss ikke for nær. Norge og det militære samarbeidet i NAT basepolitikken», slik utenriksminister Ine Eriksen Søreide formulerte det (nrk.no, 2018). Sammen med den mer omfattende justeringen av restriksjonene i 1995 avtegner det seg en gradvis glidning mot mindre avskjerming og mer integrasjon; med andre ord mot mer avskrekking og mindre beroligelse. Om forfatteren Paal Sigurd Hilde er førsteamanuensis ved Forsvarets høgskole, Institutt for for- svarsstudier. Oppsummering Forklaringen er nok i stor grad knyttet til endringer i oppfatninger om alliert interesse og støtte, trussel fra øst, og politiske synspunkter i Norge. Om historien er en læremester, kan den økte allierte interessen for og aktivi teten i Norge i de siste årene – den økte integrasjonen – fort bli søkt balansert av avskjermingstiltak, for eksempel ved et regjeringsskifte. Lite tyder på at Jens Chris tian Hauges ord om å forsvare vår dyd, men ikke trå oss for nær vil bli avleggs i norsk sikkerhetspolitikk i nær fremtid. Paal Sigurd Hilde Paal Sigurd Hilde Stortinget (1951b). Referat fra Den utvidede utenriks- og konstitusjonskomite, 29. juni, stortinget.no. Stortinget (1951c). Stortingstidende inneholdende femognittiende ordentlige Stortings forhandlinger 1951, st no. Stortinget (1980). Innst. S. nr. 139 (1980–81). Om samtykke til undertegning av en rammeavtale mello og Amerikas Forente Stater vedrørende alliert militær forhåndslagring i og forsterkning av Norge, storti g ( ) ( ) y g g og Amerikas Forente Stater vedrørende alliert militær forhåndslagring i og forsterkning av Norge, stortinget.no Sverdrup, J. (1996). 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Frankfurt am Main: Peter Lang. Holst, J. J. (1966). Norsk sikkerhetspolitikk i strategisk perspektiv. I Internasjonal Politikk, årg. 24 463–490. Holst, J. J. (1967). Norsk sikkerhetspolitikk i strategisk perspektiv. Bind II: Dokumentasjon. Oslo: NUPI. Moen, K. E. (1998). Selvpålagte restriksjoner i nord. Oslo: Forsvarsstudier, nr. 5. NRK (2018, 12. juni). Norge åpner for økt og forlenget amerikansk militær tilstedeværelse. Hentet fra https:// www.nrk.no/troms/norge-apner-for-okt-og-forlenget-amerikansk-militaer-tilstedevaerelse-1.1408 0860. G. W. (2010). The evolution of NATO’s command structure, 1951–2000. SHAPE. Hentet fra https:// pe.nato.int/resources/21/evolution%20of%20nato%20cmd%20structure%201951-2009.pdf. gen.no (2018, 13. juni). Sammen er vi sterke. Hentet fra https://www.regjeringen.no/no/aktuelt/ men_sterke/id2604283/. Skogrand, K. & Tamnes, R. (2001). Fryktens likevekt. Atombomben, Norge og verden 1945–1970. Oslo: Tiden Norsk Forlag. Skogrand, K. (2004). Norsk forsvarshistorie: Alliert i krig og fred 1940–1970. Bind 4. Bergen: Eide forlag. Stortinget (1951a). Referat fra Den utvidede utenriks- og konstitusjonskomite og Militærkomiteen, 24. april, stortinget.no. 69 Abstract in English The article builds on the classical interpretations of Norwegian security policy made by Johan Jørgen Holst and Rolf Tamnes. It portrays Norwegian secu rity policy as a balancing act between disparate aims: securing support from allies, fostering low tensions with the Soviet Union/Russia and maintaining domestic support. The article analyses the Norwegian approach to two areas of military cooperation in NATO that have been particularly important to Norway throughout 70 years of NATO membership: the NATO command structure, and allied presence in and reinforcement of Norway. The two areas have both been key arenas where Norway has performed its balancing act, and important for the development of Norwegian policy. Overall, in the changing international security environment since 1949, Norwegian security policy has shifted towards more “integration” with NATO and “deterrence” of the Soviet Union/Russia, and less “assurance” of the Eastern neighbour and “screening” (limiting) of Allied presence and activity in Norway – to use Holst’s and Tamnes’ terms. A significant step in this direction was made in 1995 and smaller ones in 1951, 1977 and 2016. Though the balancing point has thus shifted, the Norwegian emphasis on balancing is still relevant and very much alive. Keywords: security policy • NATO command structure • training and exercises • allied reinforcements and self-imposed restrictions 70
https://openalex.org/W4387694924	https://upcommons.upc.edu/bitstream/2117/334950/1/TMAOT1de1.pdf	es		Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: perspectiva del agricultor en el noroeste de México	null	2,023	public-domain	44,180	Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: perspectiva del agricultor en el noroeste de México Miguel Angel Orduño Torres ADVERTIMENT La consulta d’aquesta tesi queda condicionada a l’acceptació de les següents condicions d'ús: La difusió d’aquesta tesi per mitjà del repositori institucional UPCommons (http://upcommons.upc.edu/tesis) i el repositori cooperatiu TDX ( h t t p : / / w w w . t d x . c a t / ) ha estat autoritzada pels titulars dels drets de propietat intel·lectual únicament per a usos privats emmarcats en activitats d’investigació i docència. 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Introducing its content in a window or frame foreign to the UPCommons service is not authorized (framing). These rights affect to the presentation summary of the thesis as well as to its contents. In the using or citation of parts of the thesis it’s obliged to indicate the name of the author. Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. Tesis por compendio de publicaciones Por MIGUEL ANGEL ORDUÑO TORRES DIRECTOR ZEIN KALLAS CALOT, PhD. INSTITUT DE SOSTENIBILITAT PROGRAMA DE DOCTORADO EN SOSTENIBILIDAD UNIVERSITAT POLITÈCNICA DE CATALUNYA Septiembre 2020 Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. Tesis por compendio de publicaciones Por MIGUEL ANGEL ORDUÑO TORRES DIRECTOR ZEIN KALLAS CALOT, PhD. INSTITUT DE SOSTENIBILITAT PROGRAMA DE DOCTORADO EN SOSTENIBILIDAD UNIVERSITAT POLITÈCNICA DE CATALUNYA Septiembre 2020 Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. Curs acadèmic: 2019-2020 Acta de qualificació de tesi doctoral Nom i cognoms: Miguel Angel Orduño Torres Programa de doctorat: Sostenibilitat Unitat estructural responsable del programa: Institut de Sostenibilitat Resolució del Tribunal Reunit el Tribunal designat a l'efecte, el doctorand / la doctoranda exposa el tema de la seva tesi doctoral titulada __________________________________________________________________________________________ _________________________________________________________________________________________. Acabada la lectura i després de donar resposta a les qüestions formulades pels membres titulars del tribunal, aquest atorga la qualificació: NO APTE APROVAT NOTABLE (Nom, cognoms i signatura) (Nom, cognoms i signatura) President/a Secretari/ària EXCEL·LENT (Nom, cognoms i signatura) (Nom, cognoms i signatura) (Nom, cognoms i signatura) Vocal Vocal Vocal ______________________, _______ d'/de __________________ de _______________ El resultat de l’escrutini dels vots emesos pels membres titulars del tribunal, efectuat per la Comissió Permanent de l’Escola de Doctorat, atorga la MENCIÓ CUM LAUDE: SÍ NO (Nom, cognoms i signatura) (Nom, cognoms i signatura) President/a de la Comissió Permanent de l’Escola de Doctorat Secretari/ària de la Comissió Permanent de l’Escola de Doctorat Barcelona, _______ d'/de ____________________ de _________ Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. AGRADECIMIENTOS Para la realización de este trabajo quiero agradecer especialmente a mi familia, por la paciencia que me han tenido a lo largo de estos años. Eternamente a mi campeón, mi niño Maximiliano! Quiero destacar el apoyo y el agradecimiento infinito a Zein Kallas Calot, por su inestimable ayuda al dirigir mi tesis, con suma paciencia y dedicación. También quiero agradecer a todos los que contribuyeron a la culminación de esta tesis. Gracias a cada uno de vosotros. i Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. ABSTRACT Climate change is the most recurrent natural process on a global scale and one of the greatest challenges of our times; due to the close relationship between society, productive activities and economic development. One of the productive activities most vulnerable to climate change is agriculture, which is threatened in the quality and yield of crops and with it, food security worldwide. Climate variability increases uncertainty in production, affecting the behavior and decisions of farmers. In this thesis, we analyzed farmers’ preferences towards the mitigation and adaptation actions to climate change in the northwestern region of Mexico. A Specific emphasis was carried out on farmers’ technical efficiency, risk and environmental attitudes. The -irrigation district (076) of Valle del Carrizo in the north of the state of Sinaloa, Mexico was taken as a case study. The data was collected through a face-to-face survey, conducted with a group of 370 farmers stratified by farm size, gender and age. Firstly, farmers’ risk attitudes were analyzed using the hypothetical Multiple Price List (MPL) Method known as lotteries. This risk behavior was posteriorly related to farmer’ environmental attitude using the New Ecological Paradigm (NEP). Farmers’ perceptions towards climate change and their socioeconomic were also highlighted. The results showed a risk level of 0.32, according to the constant relative risk aversion coefficient (CRRA), placing farmers in the region as a risk-averse group. The heterogeneity analysis showed that farmers who received economic support and used it in structural investment at the farm level were more risk-tolerant; respect to the gender, women were more risk-tolerant than their counterparts (risk tolerant: 61% women and 39% men). Farmers “above 60 years of age” had a lower risk aversion on average, compared to farmers in the range of 41 to 60 years’ old, who were more conservative, with a higher level of risk aversion. Secondly farmers' preferences towards climate change mitigation and adaptation actions were analyzed using the Analytical Hierarchy Process (AHP) methodology. Preferences were related to their stated risk attitudes, environmental beliefs and characteristics. The results showed that farmers' environmental beliefs and perceptions of farmers such as “the use of less polluting machinery” and “investment in improving irrigation infrastructure” were the most preferred climate change mitigation and adaptation actions for farmers from the study region. The environmental opinions analyzed allowed to identify farmers’ ecocentric and anthropocentric attitudes, highlighting the commitment of most farmers to the sustainable use of natural resources. Thirdly, the technical efficiency (TE) of agricultural producers was analyzed using the Stochastic Frontier (SF) method. The results showed the average efficiency levels (57%) for three identified groups of farmers: high TE (15% of the farmers), average TE (72%) and low TE (13%). The level of efficiency was related to their risk attitudes, their preferences on climate change mitigation and adaptation actions, and their perception of climate change. The results showed a relationship between the preferred adaptation actions against climate change. Farmers who showed greater TE preferred the action "change crops", while less efficient farmers preferred to "invest in irrigation infrastructure". ii Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. As part of the findings of this research, we found that it is necessary to involve farmers in making agricultural public policy decisions, making them aware of the effects of climate change on production in accordance with the declarations of sustainable development related to the environment. Key words: stated risk, farmers’ preferences, climate change, adaptation, mitigation, technical efficiency, environmental attitudes, sustainability. iii Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. RESUMEN El cambio climático es el proceso natural más recurrente a escala mundial y uno de los mayores desafíos de nuestro tiempo; debido a la estrecha relación entre sociedad, actividades productivas y desarrollo económico. Una de las actividades productivas más vulnerables al cambio climático es la agricultura, que se ve amenazada en la calidad y rendimiento de los cultivos y con ella, la seguridad alimentaria a nivel mundial. La variabilidad climática aumenta la incertidumbre en la producción, afectando el comportamiento y las decisiones de los agricultores. En esta tesis analizamos las preferencias de los agricultores hacia las acciones de mitigación y adaptación al cambio climático en la región noroeste de México. Se hizo especial hincapié en la eficiencia técnica, el riesgo y las actitudes medioambientales de los agricultores. Se tomó como estudio de caso el distrito de riego (076) del Valle del Carrizo en el norte del estado de Sinaloa, México. Los datos se recopilaron a través de una encuesta cara a cara, realizada con un grupo de 370 agricultores estratificados por tamaño de finca, género y edad. En primer lugar, se analizaron las actitudes frente al riesgo de los agricultores mediante el método hipotético de lista de precios múltiples (MPL) conocido como loterías. Este comportamiento de riesgo se relacionó posteriormente con la actitud ambiental de los agricultores utilizando el Nuevo Paradigma Ecológico (NEP). También se destacaron las percepciones de los agricultores sobre el cambio climático y su situación socioeconómica. Los resultados mostraron un nivel de riesgo de 0.32, de acuerdo con el coeficiente de aversión al riesgo relativo constante (CRRA), colocando a los agricultores de la región como un grupo adverso al riesgo. El análisis de heterogeneidad mostró que los agricultores que recibieron apoyo económico y lo utilizaron en inversiones estructurales a nivel de finca eran más tolerantes al riesgo; Respecto al género, las mujeres fueron más tolerantes al riesgo que sus contrapartes (tolerantes al riesgo: 61% mujeres y 39% hombres). Los agricultores "mayores de 60 años" tenían una menor aversión al riesgo en promedio, en comparación con los agricultores en el rango de 41 a 60 años, que eran más conservadores, con un mayor nivel de aversión al riesgo. En segundo lugar, se analizaron las preferencias de los agricultores hacia las acciones de mitigación y adaptación al cambio climático utilizando la metodología del Proceso de Jerarquía Analítica (AHP). Las preferencias fueron relacionadas con sus actitudes de riesgo declaradas, creencias y actitudes ambientales. Los resultados mostraron que las creencias y percepciones ambientales de los agricultores como “el uso de maquinaria menos contaminante” y “la inversión en mejora de la infraestructura de riego” fueron las acciones de adaptación y mitigación del cambio climático de mayor preferencia para los agricultores de la región de estudio. Las opiniones ambientales analizadas permitieron identificar las actitudes ecocéntricas y antropocéntricas de los agricultores, destacando el compromiso de la mayoría de los agricultores con el uso sostenible de los recursos naturales. En tercer lugar, se analizó la eficiencia técnica (TE) de los productores agrícolas utilizando el método de Frontera Estocástica (SF). Los resultados mostraron los niveles de eficiencia promedio (57%) para tres grupos identificados de agricultores: TE alto (15% de los agricultores), TE medio (72%) y TE bajo (13%). El nivel de eficiencia se relacionó con sus actitudes de riesgo, sus preferencias en las acciones de mitigación y adaptación al cambio climático y su percepción del cambio climático. Los resultados mostraron una relación entre las acciones de adaptación preferidas frente al cambio climático. Los agricultores que mostraron mayor TE prefirieron la acción "cambiar cultivos", mientras que los agricultores menos eficientes prefirieron "invertir en infraestructura de riego". iv Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. Como parte de los hallazgos de esta investigación, encontramos que es necesario involucrar a los agricultores en la toma de decisiones de política pública agrícola, sensibilizándolos sobre los efectos del cambio climático en la producción de acuerdo con las declaraciones de desarrollo sostenible relacionadas con el medio ambiente. Palabras clave: riesgo declarado, preferencias de los agricultores, cambio climático, adaptación, mitigación, actitudes ambientales, sostenibilidad. v Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. Contenido AGRADECIMIENTOS ..................................................................................................................................................... i ABSTRACT ........................................................................................................................ ¡Error! Marcador no definido. RESUMEN ....................................................................................................................................................................... iv CAPÍTULO 1 INTRODUCCIÓN Y ANTECEDENTES .............................................................................................. 1 1.1 Introducción ..................................................................................................................................................... 2 1.2 Objetivos principales ........................................................................................................................................ 5 1.3 Enfoque metodológico y estructura de la tesis ................................................................................................ 7 1.4 Bibliografía ..................................................................................................................................................... 10 CAPÍTULO 2 RESULTADOS Y CONCLUSIONES .................................................................................................. 14 2.1 Resultados Globales ....................................................................................................................................... 15 2.2 Conclusiones................................................................................................................................................... 22 2.3 Bibliografía ..................................................................................................................................................... 26 CAPÍTULO 3 LAS PUBLICACIONES ....................................................................................................................... 29 3.1. Primera publicación: Analysis of Farmers’ Stated Risk Using Lotteries and Their Perceptions of Climate Change in the Northwest of Mexico ........................................................................................................................... 30 3.2. Segunda Publicación: Farmers’ environmental perceptions and preferences regarding climate change adaptation and mitigation actions – towards a sustainable agricultural system in México...................................... 31 3.3. Tercera Publicación: Is Technical Eﬃciency Aﬀected by Farmers’ Preference for Mitigation and Adaptation Actions against Climate Change? A Case Study in Northwest Mexico .................................................. 32 vi Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. CAPÍTULO INTRODUCCIÓN Y ANTECEDENTES Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. 1.1 Introducción Existen múltiples evidencias que indican que la agricultura siempre ha sido un pilar económico y social fundamental en todo el mundo, sin embargo, el sector agrícola es un importante emisor de gases de efecto invernadero (GEI), que afectan el cambio climático (CC) (Yue, et al., 2017) (Rivera y Di Paola, 2013). El cambio climático es uno de los mayores desafíos de nuestros tiempos. Este fenómeno (CC) se refiere a la variación a largo plazo del clima, generado por causas naturales o acciones humanas que distorsionan los parámetros climáticos, como la temperatura y la precipitación (MAPAMA, 2018). El cambio climático según la Convención Marco sobre Cambio Climático se refiere a un cambio en el clima que se atribuye directa o indirectamente a la actividad humana que altera la composición de la atmósfera global y se produce además de la variabilidad natural del clima observado durante períodos de tiempo comparables. (IPCC, 2014). La relación entre la sociedad, la agricultura y el desarrollo económico en las zonas rurales está estrechamente vinculada a las consecuencias del cambio climático (Valladolid, 2017). Hay escenarios climáticos que enfatizan que un mayor aumento de la temperatura es predecible en tierra y en el mar, lo que tendrá consecuencias en las actividades agrícolas (Galindo, et al., 2014). Es importante destacar, que la agricultura en las regiones de África y América Latina es más vulnerable al cambio climático debido a su posición geográfica en el mundo (Cline, 2007; Bermúdez, 2000 y Ortiz, 2012). Asimismo, la relación entre el cambio climático y la agricultura es compleja, dado que esta última depende de la variación de la temperatura y los niveles de precipitación, así como de los aumentos de factores extremos, tales como; sequías, huracanes, inundaciones, incendios forestales, etc. (Vargas, 2007). El clima es el componente más importante en la práctica de actividades agrícolas y está afectando continuamente las decisiones de los agricultores, principalmente al incrementar la incertidumbre en la producción (Rivera y Di Paola, 2013). 2 Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. Por otro lado, las percepciones son un elemento clave de los sistemas sociales que influyen en las actitudes y respuestas al cambio climático, impactando en la toma de decisiones y la gobernanza a corto y largo plazo. (Soubry, et al., 2020). Por lo tanto, las percepciones de los agricultores sobre el cambio climático desempeñan un papel cada vez más importante en la producción agrícola y en las decisiones de los agricultores a nivel de finca (Bonatti, 2011; Makuvaro, et al., 2018 y Zhai, et al., 2018). Es importante señalar que se han implementado diversas acciones de mitigación y adaptación al cambio climático en los últimos veinte años (Ortiz, 2012). Las acciones de adaptación incluyen cambios en el cultivo y / o cultivos alternativos y mayores inversiones en infraestructura de riego (Galindo, et al., 2014). Además, varios estudios sugieren que las prácticas agrícolas tradicionales representan una herramienta para la adaptación, mediante el uso de variedades tolerantes a la sequía, policultivos, deshierbe oportuno, recolección de plantas silvestres, entre otras técnicas. Estas medidas buscan fortalecer la agricultura moderna a través de prácticas sostenibles para ajustar las experiencias agrícolas, que a su vez permitirán la conservación de la agricultura tradicional. (Altieri y Nicholls, 2008) (Galindo, et al., 2014) Existe evidencia de que los recursos hídricos son vulnerables al cambio climático y que las consecuencias para la sociedad y los ecosistemas dependen de las medidas de adaptación. (Adger, 2007). El estrés hídrico y una demanda exacerbada de agua para el riego son, en resumen, los impactos más probables a escala mundial, en el sector agrícola. Por otro lado, el Panel Intergubernamental sobre Cambio Climático (IPCC) define la mitigación como la "intervención antropogénica para reducir las fuentes o mejorar los sumideros de gases de efecto invernadero". Mitigar el cambio climático no es solo una tarea para los agentes de la cadena productiva o los agricultores. Es una tarea conjunta que debe comenzar con las instituciones públicas y su 3 Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. participación en ella. Las políticas agrarias que promueven una agricultura sostenible y menos intensiva conducirían a mitigar el cambio climático (Rivera y Di Paola, 2013) La fertilización representa un aspecto destacado en la mitigación del cambio climático, en términos de sus repercusiones negativas que afectan la productividad de los cultivos. Sin embargo, todavía hay dudas sobre los posibles beneficios, dado que las múltiples interacciones y los factores de estrés no se incorporan a los modelos actuales. (Ocampo, 2011) Un aspecto importante para destacar es el concepto de "vulnerabilidad de los cultivos", ya que, al ser diferentes en cada región, las tecnologías aplicadas también cambian; dependiendo de la inestabilidad climática natural debido a alteraciones en los regímenes de lluvia y viento y la incidencia de eventos extremos (Ocampo, 2011). La alta vulnerabilidad de la región latinoamericana tiene una baja capacidad de adaptación a los desastres naturales consecuencia del cambio climático (Honty, 2007), debido a la escasa disposición de recursos económicos, materiales, científicos y tecnológicos para compensar los costos asociados con la adaptación. Cabe señalar que la baja capacidad de reacción política paga muy poco las implicaciones del cambio climático (Honty, 2007). Esta vulnerabilidad se entiende como la probabilidad de que una comunidad sufra daños materiales y humanos ante desastres naturales según la CEPAL y el BID, dependiendo de la fragilidad de su infraestructura, vivienda, actividades productivas, nivel de organización, sistemas de alerta, progreso político e institucional, es por eso por lo que la agricultura es un sector clave, donde la variabilidad del clima se traduce en el impacto en la alimentación, el estilo de vida, los ingresos y la seguridad alimentaria. Existen múltiples factores que afectan la vulnerabilidad en las zonas rurales, sin embargo, el clima no parece ser el principal (Alfaro, 2017). En el caso de México, dado su espacio geográfico, sus escenarios climáticos y su orografía, ocurren eventos hidrometeorológicos extremos con graves consecuencias, donde el territorio más afectado suele ser la zona costera, las zonas de inundación y las laderas de las montañas. 4 Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. Además de lo anterior, se espera que la sequía meteorológica crezca en ciertas regiones; se han presentado cambios en el 50% del entorno forestal (Zamora, 2015). Además, se prevé que la agricultura de secano se reducirá severamente. Un punto que se considera relevante es que el gobierno mexicano acepta la existencia del cambio climático en la geografía nacional; por lo que considera necesario aplicar acciones que le permitan adaptarse en sus diferentes regiones. (Zamora, 2015) En México, las Áreas Naturales Protegidas (ANP) han ganado notoriedad porque la adaptación al cambio climático de sus ecosistemas y poblaciones contribuye a la mitigación de los efectos adversos del mismo (Zamora, 2015) 1.2 Objetivos principales Tomando como base lo anterior, para llevar a cabo el análisis de la agricultura, fue tomado como caso de estudio, la región agrícola que comprende el distrito de riego 076 del Valle del Carrizo, el cual, se encuentra ubicado en el norte del estado de Sinaloa, México, y que, desde la perspectiva del agricultor, sus preferencias de adaptación y mitigación a través del análisis del riesgo declarado y su nivel de eficiencia técnica, se proponen los siguientes objetivos: 1. Analizar las actitudes de riesgo declaradas de los agricultores en una región agrícola del noroeste de México, utilizando el método de listas de precios múltiples (MPL), conocido como "Loterías", como un método alternativo de obtención de riesgo que pertenece al enfoque de utilidad esperada. Adicionalmente se busca analizar la heterogeneidad del nivel de riesgo declarado frente a las opiniones medioambientales y las percepciones del cambio climático de los agricultores. 2. Identificar la importancia relativa de varias acciones de adaptación y mitigación del cambio climático relacionadas con las actividades agrícolas en una región marginal de México para 5 Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. brindar argumentos a los responsables de la formulación de políticas al priorizar soluciones que contribuyan a la sostenibilidad de sus sistemas agrícolas. 3. Analizar si la eficiencia técnica se ve afectada por la preferencia de los agricultores por las medidas de adaptación y mitigación al cambio climático en México, mediante el uso del método de frontera de producción estocástica paramétrica (SPF) para identificar su relación con las preferencias declaradas de los agricultores por varias medidas de adaptación y mitigación del cambio climático, utilizando el método del proceso de jerarquía analítica (AHP). Asimismo, identificar la dirección y la magnitud de la relación entre Eficiencia Técnica (ET) y las preferencias de los agricultores. Esta tesis contribuye a la literatura existente sobre las actitudes de riesgo de los agricultores, al aportar una nueva perspectiva utilizando el método Listas de Precios Múltiples (MPL) en México. De igual manera, a nivel metodológico, este documento también contribuye a los escasos estudios que utilizan el método de Proceso de Jerarquía Analítica (AHP), para determinar las preferencias de adaptación y mitigación de los agricultores, con respecto al cambio climático. Nuestro estudio verifico la validez y la idoneidad de esta técnica para comprender las necesidades y prioridades de los agricultores. Por otro lado, en esta investigación, se utilizó el método de frontera estocástica para medir el nivel de eficiencia técnica (ET) de los agricultores de la región y su relación con las preferencias sobre acciones de adaptación y mitigación del cambio climático, el nivel de riesgo declarado y sus actitudes ambientales. Para lograr los objetivos anteriormente planteados, se ha propuesto un enfoque metodológico basado en la realización de encuestas cara a cara, cuyo tamaño de muestra se determinó en base a la fórmula de poblaciones finitas con un nivel de confianza de 95% y un intervalo de confianza del 4.99%. La encuesta fue diseñada con una perspectiva interdisciplinaria, que incluye aspectos económicos, sociales y demográficos. Incluyó un amplio conjunto de preguntas 6 Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. agrupadas de acuerdo con las características socioeconómicas de los agricultores, las características de sus tierras agrícolas y los tipos de cultivos (Kallas, et al., 2010) y su preferencia con respecto a la implementación de acciones de mitigación o adaptación relacionadas con la reducción de los efectos del cambio climático utilizando la técnica AHP. Las actitudes y opiniones de los agricultores que definen su comportamiento hacia el medio ambiente también se determinaron utilizando la escala del nuevo paradigma ecológico (NEP). 1.3 Enfoque metodológico y estructura de la tesis El enfoque metodológico planteado en esta tesis parte del análisis y aplicación de distintos métodos para identificar el nivel de riesgo declarado, las preferencias de los agricultores hacia actividades de adaptación y mitigación del cambio climático y la eficiencia técnica de un grupo de agricultores de una región agrícola del norte del estado de Sinaloa México. El nivel de riesgo es una variable clave de las decisiones bajo incertidumbre. Cada individuo o tomador de decisiones involucrado tiene una actitud diferente hacia el riesgo; por lo tanto, el grado de aversión al riesgo debe cuantificarse para identificar diferencias y similitudes. Existen numerosos métodos que se utilizan con éxito en la gestión económica y agrícola para medir las actitudes declaradas en relación con el riesgo en base a las encuestas de las personas involucradas en actividades económicas. En este caso de estudio, el método seleccionado, como enfoque alternativo para medir las actitudes de riesgo declaradas de los agricultores fue el Método de Listas de Precios Múltiples, propuesto por Holt y Laury (Holt y Laury, 2002), específicamente la variante desarrollada por Brick, Visser and Burns (Brick, et al., 2012). Esta decisión se tomó debido a las características de la población objetivo del estudio, que no tiene un alto nivel de educación, pero está familiarizada con el concepto de loterías. El método seleccionado también se caracteriza por la simplicidad y un tiempo relativamente corto para la recopilación de datos. Además, su 7 Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. aplicación no requiere un conocimiento previo de los conceptos de incertidumbre y riesgo, ni un análisis exhaustivo de varias declaraciones. El método de MPL se ha utilizado en muchos estudios para medir las actitudes de riesgo de los agricultores, entre las que se encuentran aquellas creadas por Olbrich et al., (2014) y Brick et al., (2012). El método MPL “loterías”, consiste en un formato de lista de precios múltiples, por medio del cual a cada uno de los entrevistados se les presenta un conjunto de distintos pares de loterías, dentro de los cuales debe elegir una de las opciones de lotería para cada par (Andersen, et al., 2008). (Relaciona lo niveles de aversión al riesgo con un premio o ganancia). Dentro de la definición del modelo se generó una lista de 8 preguntas (escenarios) con un par de loterías hipotéticas denominadas opción A y opción B, similar al modelo teórico utilizado por Brick, et al., (2012). En dicho modelo al igual que en este se mantienen constantes las probabilidades de cada una de las opciones para mantener el experimento lo más simple posible. En la opción A la probabilidad de obtener la cantidad presentada se fijó a un 100% (opción segura) y dentro de la opción B (opción riesgosa) se fijó a un 50% la probabilidad de obtener la cantidad de ($100) y la con misma probabilidad de 50% no obtener nada ($0) (lanzamiento de una moneda a Cara o Cruz) en todos los escenarios; mientras que la cantidad segura presentada en la opción A, en cada una de las 8 preguntas (escenarios) se va modificando de forma decreciente de acuerdo a las siguientes cantidades ($100, $75, $60, $50, $40, $30, $20 y $10). Otro método utilizado fue el método del Proceso de Análisis Jerárquico (AHP), el cual está clasificado como parte de las técnicas basadas en la teoría de decisión multicriterio TDMC; es un método usado para medir intangibles usando el juicio humano, permite que se realice la priorización de un grupo de alternativas de acuerdo con determinados criterios que facilitan la selección de las soluciones óptimas posibles. Lo que caracteriza a la toma de decisiones multicriterio es la existencia de diversos criterios dentro del proceso de asignación de 8 Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. prioridades para cada una de las distintas alternativas. Este método se utilizó para identificar las preferencias de los agricultores y estimar la importancia relativa (es decir, las prioridades) de diferentes acciones de adaptación y mitigación. El método AHP involucra 3 etapas que forman la estructura metodológica: 1) modelado, 2) evaluación y 3) priorización y síntesis. En la etapa de modelado se realiza la definición del problema y la estructuración de un modelo de decisión en forma de jerarquía. En la etapa de evaluación se realizan las comparaciones pareadas de todos los elementos de cada nivel de grupo usando la escala verbal de comparaciones pareadas propuesta por Saaty, posteriormente, se estima la importancia relativa de las acciones alternativas. En la última etapa correspondiente a la priorización y síntesis, se realiza la identificación de los criterios más preferidos, priorizando de forma conjunta todos los subcriterios propuestos en el modelo; hasta este punto, todas las comparaciones deben trazarse entre los elementos de cada grupo para cada agricultor (matrices Saaty), a través de las cuales se obtienen los pesos locales de los elementos identificados de acuerdo con las preferencias de cada agricultor mediante el uso del Método de la media geométrica [Kallas y Gil, 2012]. En función de las ponderaciones obtenidas para cada individuo se calcula el peso global que corresponderá a cada uno de los atributos, esto, mediante el producto del peso local de cada atributo por el peso local del nodo padre, generándose un vector con los pesos globales de cada una de las alternativas definidas en el modelo. Como parte de la etapa de verificación es importante destacar que con cada matriz generada se realizó el cálculo del nivel de coherencia de las respuestas emitidas por los productores agrícolas. También utilizamos una forma adaptada de la escala del nuevo paradigma ecológico (NEP) que se validó mediante análisis factorial (PCA) para identificar las dimensiones ambientales latentes predominantes. Adicionalmente, se realizó un análisis de heterogeneidad para relacionar las preferencias de los productores agrícolas con las acciones contra los efectos del cambio 9 Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. climático en función de sus actitudes ambientales y de riesgo declaradas hacia sus actividades agrícolas. Otra de las metodologías utilizadas fue el método de frontera estocástica que nos permitió estimar el nivel de eficiencia técnica (ET) de los productores agrícolas de la región de estudio. Este enfoque fue propuesto simultáneamente por Aigner, et al., (1977) y Meeusen y Van Den Broeck (1977). Los niveles de eficiencia técnica se calcularon como una medida orientada a la producción y se definieron como la relación entre la producción observada y la frontera de producción estocástica (FPE) correspondiente, con valores en el rango de cero a uno (Guesmi, et al., 2012). Según los valores estimados de eficiencia técnica, los agricultores se agruparon en tres grupos, teniendo en cuenta la desviación estándar media y una unidad hacia arriba y hacia abajo para establecer a los agricultores con eficiencia baja, media y alta. En el capítulo dos de esta tesis se presentarán los resultados generales de la investigación y posteriormente las conclusiones resultantes y, finalmente en el capítulo tres se presentarán las tres publicaciones que abordan las metodologías descritas. 1.4 Bibliografía  Aigner, D.; Lovell, C.K.; Schmidt, P. (1977). Formulation and estimation of stochastic frontier production function models. J. Econom. 6, 21–37.  Alfaro, Salvador Orlando. (2017). “Riesgos Ambientales Y Desarrollo En La Zona Rural.” Revista Realidad, 355–73.  Altieri, M. A. & Nicholls, C. I. (2008). Los impactos del cambio climático sobre comunidades campesinas y de agricultores tradicionales y sus respuestas adaptativas. Agroecología. 3, 7-28.  Andersen, S. Harrison, G. Lau, M. Rutstrom, E. (2008). Eliciting Risk and Time Preferences. Econometrica. 76, 583-618 10 Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México.  Bermúdez, López. (2000). “Redalyc.Impactos Regionales Del Cambio Climático. Valoración de La Vulnerabilidad.”  Bonatti, M. (2011). Cambios Climáticos, Percepciones Humanas y Desarrollo Rural. Master’s Thesis, Universidad de Buenos Aires, Buenos Aires, Argentina  Brick, K., Visser, M. & Burns, J. (2012). Risk Aversion: Experimental Evidence From South African Fishing Communities. American Journal of Agricultural Economics. 94 (July), 133–152.  Cline, W. R. (2007). Global warming and agriculture: impact estimates by country. Center for Global development, Washington DC.  Galindo, L.; Samaniego, L; Alatorre, J.; Ferrer, J. & Reyes, O., (2014). Cambio climático, agricultura y pobreza en América Latina: Una aproximación empírica. Estudios del cambio climático en américa latina.  Galindo, L.M. (2013). La Economía del Cambio Climático en Mexico. Semarnat.53, 1689– 1699.  Guesmi, B.; Serra, T.; Kallas, Z.; Gil Roig, J.M. (2012). The productive efficiency of organic farming: The case of grape sector in Catalonia. Span. J. Agric. Res.3, 552–566.  Holt, Ch.A., & Laury S. K. (2002). Risk Aversion and Incentive Effects, American Economic Review.92(5), pp1644-1655.  Honty, Gerardo. (2007). “AMERICA LATINA ANTE EL CAMBIO CLIMÁTICO.” Observatorio de La Globalización, 1–12. http://www.flacsoandes.edu.ec/sites/default/files/agora/files/1269363645.honty_america_ latina_frente_al_cambio_climatico_0.pdf.  IPCC. (2014). “CAMBIO CLIMÁTICO 2014.” 11 Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México.  Kallas, Z. & Gil, J., (2012). Combining contingent valuation with the analytical hierarchy process to decompose the value of rabbit meat. Food Quality and Preference. 24, 251–259. https://doi.org/10.1016/j.foodqual.2011.11.006  Kallas, Z.; Serra, T.; Gil, J. (2010). Farmers’ objectives as determinants of organic farming adoption: The case of Catalonian vineyard production. Agric. Econ.41, 409–423.  Makuvaro, V.; Walker, S.; Masere, T.P.; Dimes, J. (2018). Smallholder farmer perceived effects of climate change on agricultural productivity and adaptation strategies. J. Arid Environ. 152, 75–82.  Meeusen, W.; Van den Broeck, J. (1977). Technical efficiency and dimension of the firm: Some results on the use of frontier production functions. Empir. Econ. 2, 109–122.  Ministerio de Agricultura, Pesca y Alimentación–Ministerio para la Transición Ecológica. Qué es el Cambio Climático. (2018). Available online: http://www.mapama.gob.es/es/cambio-climatico/temas/cumbrecambio-limaticocop21/elcambio-climatico/ (accessed on 6 July 2018).  Ocampo, Olga. (2011). “El Cambio Climático Y Su Impacto En El Agro Climate Change and Its Impact on the Agriculture.” Revista de Ingenieria, 115–23.  Olbrich, R., Quaas, M. & Baumgärtner, S. (2014). Risk preferences under multiple risk conditions – survey evidence from semi-arid rangelands I. Introduction. (February 2014), pp.1–15.  Ortiz, R., (2012). El cambio climático y la producción agrícola. Banco Interamericano de Desarrollo.  Rivera, I. & Di Paola, M., (2013). Cambio Climático: Impacto e incidencias de las políticas públicas en el sector agropecuario. Available online: URL http://www.farn.org.ar/wpcontent/uploads/2014/07/informe2013-1.268-291.pdf (accessed on: 06-07-2018). 12 Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México.  Soubry, B., Sherren, K., & Thornton, T. F. (2020). ¿Are we taking farmers seriously? A review of the literature on farmer perceptions and climate change, 2007–2018. Journal of Rural Studies, 74, 210-222.  Valladolid, F., (2017). El impacto de la agricultura en el ecosistema y su efecto en la población rural. Machala.  Vargas, A.R. (2007). Cambio Climático, Agua y Agricultura; Desde la Dirección de Liderazgo Técnico y Gestión del Conocimiento-IICA, 2007, 13.  Yue, Q.; Xu, X.; Hillier, J.; Cheng, K.; Pan, G. (2017) Mitigating greenhouse gas emissions in agriculture: From farm production to food consumption. J. Clean. Prod. 149, 1011–1019.  Zamora, M. (2015). Cambio climático. Revista mexicana de ciencias forestales. 6(31), 0407.  Zhai, S.Y.; Song, G.X.; Qin, Y.C.; Ye, X.Y.; Leipnik, M. (2018). Climate change and Chinese farmers: Perceptions and determinants of adaptive strategies. J. Integr. Agric. 17, 949–963. 13 Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. CAPÍTULO RESULTADOS GENERALES Y CONCLUSIONES Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. La consideración principal de este capítulo es la aportación de la discusión de los resultados de forma breve, de tal manera, que pueda obviarse en la duplicidad de datos con respecto a lo expuesto en el capítulo tercero, en donde se esbozará ampliamente los resultados generales de la investigación, así como las conclusiones de esta. 2.1 Resultados globales En primer lugar, uno de los resultados que reveló la información de la investigación, con respecto al nivel de las actitudes de riesgo de los agricultores, define globalmente a la población estudiada como ligeramente adversa al riesgo, al mostrar una puntuación de 0,32, según la escala de Holt y Laury. De acuerdo con la escala de Holt y Laury (2002), los resultados de MPL con respecto a las actitudes de riesgo indicadas muestran que 51.35% de los agricultores son reacios al riesgo, 7.57% son neutrales y 41.08% son tolerantes al riesgo. Estos resultados son similares a los de Trujillo, et al., (2009), Galarza (2009) y Brick, et al., (2011), quienes encontraron que los agricultores exhibían, en general, una actitud aversa al riesgo. Con respecto al análisis de heterogeneidad del riesgo, los resultados indican que las variables relacionadas con el nivel de aversión al riesgo son: El propósito de los subsidios recibidos (valor de p = 0,002), el género del productor agrícola (valor de p = 0,046) y la edad (p-valor = 0,000). Cabe resaltar que las mujeres resultaron ser más tolerantes al riesgo que sus contrapartes (el porcentaje de hombres que toleran el riesgo = 39%, mientras que el porcentaje de mujeres que toleran el riesgo es del 61%). También, es importante tener en cuenta el nivel promedio de riesgo estimado para las mujeres que fue de -0.035, y el nivel de riesgo estimado para los hombres fue de 0.361, especialmente porque las mujeres son responsables del 60% al 80% de los alimentos del mundo y son mejores administradores del medio ambiente (Doss, et al., 2018). Además, las mujeres son más propensas a participar en actividades extensas relacionadas con la agricultura y están más comprometidas con las actividades agrícolas. Sin 15 Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. embargo, las decisiones relacionadas con el desarrollo del predio agrícola y la adopción de mejoras son tomadas por los hombres, excluyendo a las mujeres en el proceso de toma de decisiones de un nuevo sistema agrícola (Fisher y Carr, 2015). El uso principal de los subsidios y apoyos económicos está altamente relacionado con el nivel de riesgo de los agricultores. Los agricultores que reciben apoyo económico y lo utilizan en inversiones estructurales a nivel de finca son más tolerantes al riesgo, mientras que aquellos que no tienen subsidio tienen un perfil adverso al riesgo (Takeshima y Yamauchi, 2012). La edad también se asoció con el nivel de riesgo, los agricultores "mayores de 60 años" muestran que la experiencia adquirida a lo largo del tiempo les permite tener una menor aversión al riesgo en promedio, en comparación con los agricultores en el rango de 41 a 60 años, que son más conservadores, mostrando un mayor nivel de aversión al riesgo. De acuerdo con el análisis de componentes principales realizado, la primera componente identificada caracteriza la percepción de los agricultores respecto a los patrones climáticos negativos, y la segunda está relacionada con el impacto en su productividad agrícola. La mayoría de los agricultores tolerantes al riesgo, se ubicaron a la derecha de la primera dimensión, mostrando una mayor percepción de los patrones climáticos relacionados con el cambio climático, en comparación con los agricultores reacios al riesgo. Los agricultores altamente tolerantes al riesgo son aquellos que son propensos a usar subsidios en inversiones estructurales a nivel de finca. Los resultados también mostraron que los agricultores con riesgo neutral no tienen percepciones bien definidas sobre el cambio climático, ni los efectos que el cambio climático podría tener en su productividad. Los resultados sobre las preferencias de los agricultores en relación con la implementación de acciones de adaptación y mitigación identificadas de acuerdo con el método AHP utilizado, reflejan la priorización de los agricultores de diferentes formas de enfrentar los impactos del cambio climático en sus actividades. Los pesos promedio estimados muestran que las acciones 16 Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. de mitigación se consideraron las opciones más importantes con una relevancia relativa más alta del 58.18%; siendo el uso de maquinaria menos contaminante la acción más preferida (30.29%). La segunda acción más preferida fue la inversión en la mejora de la infraestructura de riego (17.57%). El cambio de cultivos se consideró la tercera acción más preferida, representando (17.30%) de las respuestas de los agricultores. El manejo de cero labranzas fue la cuarta acción más preferida (16.22%), de todas las acciones el uso de energía renovable fue la opción menos preferida por los agricultores (5.95%). De acuerdo con la relación entre las actitudes ambientales definidas mediante la escala NEP y las preferencias de los agricultores para la adaptación al cambio climático y las acciones de mitigación antes identificadas, aplicando el método de componentes principales identificamos dos comportamientos relevantes principales: actitudes ambientales ecocéntricas y antropocéntricas, que conforman cuatro cuadrantes. Los resultados indican que la mayoría de los agricultores (39%) exhibieron claramente una actitud ecocéntrica positiva (+ eco, - antro), estos agricultores creen que la naturaleza debe protegerse porque es vulnerable a las acciones de los humanos y que los humanos deben limitar su uso y realizar acciones que apoyen la naturaleza. El 27% de los agricultores exhibieron una clara actitud antropocéntrica (- echo, + antro) y un interés en proteger la naturaleza solo para un claro beneficio económico, estos agricultores consideran que los humanos están por encima de la naturaleza y que, por lo tanto, no hay límite para el uso de los recursos naturales. El resto de los agricultores exhibieron opiniones menos claras sobre el medio ambiente, un 15% mostró opiniones negativas hacia las actitudes ecocéntricas y antropocéntricas (- eco, - antro), destacando su desacuerdo con las opiniones que colocan a la naturaleza por encima de los humanos y con aquellos que colocan a los humanos por encima de la naturaleza; y el 19% mostró opiniones positivas hacia las actitudes ecocéntricas y antropocéntricas (+ eco, + antro), 17 Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. son agricultores que están de acuerdo con ambas actitudes a favor de la naturaleza y a favor de las prioridades de los humanos en el uso de los recursos naturales. Estos resultados muestran que las dimensiones ecocéntricas y antropocéntricas están estrechamente relacionadas con las preferencias del agricultor. La acción de mitigación y adaptación al cambio climático más preferida por los agricultores, correspondiente al uso de maquinaria menos contaminante y energéticamente eficiente (M4), fue seleccionada principalmente por los agricultores con una actitud positiva hacia el medio ambiente (+ eco, -anthro). Las acciones de mitigación y adaptación restantes también fueron importantes para los agricultores que exhiben más puntos de vista ecocéntricos (+ eco, -anthro). Como excepción, la acción correspondiente a introducir semillas mejoradas y resistentes (A3) fue preferida por los agricultores que no muestran una actitud clara hacia el medio ambiente (+ eco, + anthro). De igual manera los agricultores con las actitudes más ecocéntricas (+ eco, anthro) exhibieron mayor preferencia por la acción de mitigación correspondiente al uso de energía renovable (M3). El análisis de heterogeneidad entre las actitudes de riesgo establecidas y las preferencias de los agricultores hacia las acciones de adaptación y mitigación no muestra suficiente evidencia de que exista relación alguna. A través del análisis realizado, no se encontró una relación significativa entre las preferencias para las acciones de adaptación y mitigación y el nivel de riesgo establecido, aunque está claramente relacionado con otras variables socioeconómicas y de gestión para los agricultores. De acuerdo con análisis de las características socioeconómicas, se observó que el 89% son hombres y solo el 11% son mujeres, el 52.16% de los agricultores encuestados se encuentran dentro del rango de edad de 41 a 60 años, el 28.38% corresponde a los agricultores mayores de 60 años y 19.46% son menores de 41 años. El número promedio de miembros de la familia se 18 Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. registró en 3.78. El 76% de los ingresos de los participantes se generan a partir de actividades agrícolas. El 68% de los productores reciben un subsidio para realizar su actividad agrícola; dentro de este grupo de productores que reciben subsidio, el 60% lo utilizan para cubrir los costos operativos, mientras que solo el 12.3% de los agricultores lo utilizan para invertir en equipos y tecnología agrícola. La mayoría de los agricultores (63%) no suelen utilizar ningún tipo de seguro agrícola. La mayoría de los agricultores participantes son propietarios del predio agrícola (79%), destacando en su producción el cultivo de trigo en un 29%, de alfalfa con un 24% y el cultivo de soja en un 9.73%. Características socioeconómicas como la edad y sexo del agricultor, además de contar con un contrato de seguro agrícola, un crédito agrícola, pertenecer a una asociación agrícola o el tipo de selección de cultivos se encuentran relacionadas con las acciones de mitigación y adaptación preferidas por los productores agrícolas. En relación con lo anterior, se observó que los agricultores menores de 40 años prefieren la "inversión en la mejora de la infraestructura de riego", los agricultores de 40 a 60 años prefieren el enfoque de "cambio en el cultivo", y los agricultores mayores de 60 años prefieren la "gestión de labranza cero". Los agricultores sin seguro de cosechas prefieren el "cambio en los cultivos", mientras que aquellos con seguro prefieren "el uso de maquinaria menos contaminante y energéticamente eficiente" para reducir los impactos del cambio climático. Por otro lado, los productores con seguro sobre sus cosechas tienen menos preocupaciones con respecto a los impactos del cambio climático y, por lo tanto, muestran preferencia por otras acciones que reducen principalmente los efectos negativos sobre el medio ambiente. De igual manera se observó que los agricultores que cuentan con un crédito agrícola, seguros agrícolas y que pertenecen a una asociación agrícola prefieren "el uso de maquinaria menos contaminante y energéticamente eficiente" y entre sus cultivos seleccionaron cebollas, chiles, maíz, soja, sorgo y triticale. Mientras que los agricultores sin crédito agrícola y bajo un régimen de tenencia de la tierra en propiedad privada que cultivan patatas prefieren 19 Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. aumentar la inversión en la mejora de la infraestructura de riego, y los agricultores sin crédito agrícola, que no pertenecen a una asociación agrícola y cultivan sandía y cártamo principalmente se inclinan por la acción de mitigación identificada como “gestión de labranza cero”. En lo que respecta a la eficiencia técnica (ET), los resultados muestran que la estimación promedio fue de 0.57, lo que indica que los agricultores alcanzan el 57% de su producción potencial máxima, y la mayoría de ellos tienen puntajes de eficiencia promedio (72% de puntaje de TE moderadamente eficiente, 13% de puntaje bajo y 15% de puntaje de TE alto), es decir, existe una gran oportunidad para mejorar la ET en un 43%, mediante un uso más racional y menos arbitrario de los insumos que reduciría los costos de producción y contribuiría al sustento de la granja. Se considera que la baja eficiencia puede estar asociada con un mal uso de la tecnología existente, por lo que la optimización tiene que ver con, entre otras cosas, la optimización de la infraestructura de riego, enfocada en reducir sustancialmente el consumo de agua y energía (Galindo, 2013), dado que, en general, el consumo de energía puede considerarse uno de los gastos más importantes asociados con la producción agrícola. Además, reducir el consumo de energía generaría ahorros económicos y reduciría las emisiones contaminantes en el medio ambiente, lo que daría como resultado una forma de agricultura más sostenible. La eficiencia técnica se relacionó con las variables socioeconómicas: edad, el régimen de propiedad y el porcentaje de ingresos provenientes de la agricultura, Los jóvenes agricultores que son propietarios de sus tierras agrícolas con más del 50% de sus ingresos provenientes de actividades agrícolas son más eficientes técnicamente. Estos resultados están en línea con el hallazgo en Guesmi, et al., (2010) y Perdomo y Mendieta (2007) que destacan la variable edad e ingresos como factores determinantes de ET. 20 Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. En relación con las preferencias de los agricultores, el riesgo y las actitudes ambientales y las percepciones del cambio climático, los resultados muestran que los agricultores más eficientes prefirieron la acción de adaptación “cambio de cultivos”, destacando la importancia de adoptar variedades nuevas, de baja demanda de agua y resistentes a las enfermedades para enfrentar el cambio climático. Estos agricultores exhibieron una actitud antropocéntrica hacia el medio ambiente, destacando relativamente menos sensibilidad a los problemas del cambio climático. En particular, se encontró una percepción más baja con respecto a la variación de fenómenos ambientales como las sequías y los episodios de congelación. Los agricultores con menor eficiencia técnica prefirieron la inversión en instalaciones de riego como una acción de adaptación y exhibieron una actitud ecocéntrica. Este bajo nivel de ET podría estar relacionado con su extenso sistema de producción y con el tipo de gestión agrícola. Estos incluyen la adopción de una estrategia de baja degradación del suelo mediante la aplicación de agricultura de labranza cero, bajo uso de fitosanitarios y siguiendo una rotación natural de cultivos. Los agricultores menos eficientes están aprovechando los recursos naturales disponibles con un bajo nivel de adopción de innovaciones tecnológicas. Este resultado es similar al hallazgo de Álvarez y Del Corral (2010) quienes declararon que los agricultores que adoptan tecnología agrícola intensiva son más productivos y técnicamente más eficientes que los extensivos. En este contexto, la toma de decisiones de los agricultores ecocéntricos está impulsada no solo por sus objetivos económicos sino también por los ambientales. Tienden a preservar el medio ambiente como un aporte de sus actividades que aseguran la generación de un ingreso satisfactorio (Kallas y Gil, 2010). Los agricultores ecocéntricos podrían adoptar tecnologías agrícolas que tengan un impacto positivo en su productividad y sean amigables con el medio ambiente (Parks y Brekken, 2018), como la adopción de un sistema de riego eficiente. Desde el punto de vista de la agricultura sostenible, se requiere un equilibrio entre sus 21 Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. componentes económicos, sociales y ambientales. De lo contrario, cualquier otra posición a favor de solo uno de estos factores supone el detrimento del resto. Su percepción del cambio climático no estaba claramente definida, con una ligera tendencia hacia bajos niveles de percepción. Según estos resultados, podemos deducir que el principal problema que enfrentan los agricultores menos eficientes es la disponibilidad de agua, o específicamente la necesidad de optimizar su uso, dado el alto costo del agua en la producción de cultivos. Los sistemas de riego son un elemento clave para lograr una alta eficiencia técnica en la producción agrícola (Ortiz, 2012). La posición poco clara con respecto al cambio climático no les permite identificar que el costo derivado del consumo de agua se debe al mayor uso del agua debido a la variación en el clima (mayor evapotranspiración debido al aumento de la temperatura, menor humedad en el suelo como resultado de la sequía ) Los efectos del cambio climático, identificados por los agricultores principalmente como el aumento de la temperatura y la mayor incidencia de enfermedades y plagas y problemas de malezas, fueron percibidos en mayor medida por los agricultores moderadamente eficientes. 2.2 Conclusiones Hacer frente de manera efectiva a los impactos del cambio climático en la agricultura implica la implementación de acciones de mitigación y adaptación de acuerdo con las percepciones, actitudes y preferencias de los agricultores. Los responsables de la elaboración de las políticas relacionados con la producción agrícola deben basarse en lo anterior a fin de mejorar la efectividad de las políticas aplicadas, mediante la implementación de acciones con mayor aceptación que mejoren el bienestar social y económico de los agricultores, y de esta manera redirigir el apoyo que se brinda actualmente, al priorizar medidas que promuevan el desarrollo de actividades agrícolas más sostenibles a nivel regional y nacional. 22 Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. En la región estudiada, en términos generales, los agricultores exhibieron una actitud adversa al riesgo, reconociendo la importancia de mejorar la comercialización de sus cultivos y la efectividad de los tratamientos contra enfermedades y plagas. Características como el sexo y la edad de los agricultores se relacionaron con su nivel de riesgo declarado. Los agricultores mayores de 60 años resultaron ser más tolerantes al riesgo que los agricultores más jóvenes. Es importante destacar es que las mujeres agricultoras de la región resultaron ser más tolerantes al riesgo que los hombres y más propensas a invertir a nivel de predio agrícola el dinero recibido en forma de apoyo público o subsidio. Sin embargo, están marginadas como tomadoras de decisiones a nivel de políticas y, por lo tanto, están aún menos involucradas en el diseño y la implementación de políticas públicas en el sector agrícola, principalmente debido a limitaciones culturales. Los agricultores tolerantes al riesgo demostraron ser muy conscientes de los efectos del cambio climático en la producción y demostraron estar de acuerdo con las declaraciones de desarrollo sostenible relacionadas con el medio ambiente, lo que puede generar una mayor resiliencia en la región. Lo anterior refuerza la importancia de que sean involucrados en el proceso de generación de políticas públicas agrícolas. Además, las políticas públicas deben dirigirse de manera efectiva a los agricultores con actitudes adversas al riesgo para construir relaciones seguras con ellos, lo que puede mejorar sus percepciones y capacidades de adaptación en relación con el cambio climático. Las percepciones del cambio climático de los agricultores se destacaron por los efectos adversos sobre la producción, a través de la percepción de una mayor incidencia de plagas y enfermedades en sus cultivos, aumentos en el desarrollo de malezas, aumentos y variaciones de la temperatura y cambios en la temporalidad de los períodos de lluvia. 23 Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. A través del Proceso de Jerarquía Analítica, se descubrió que los agricultores priorizan acciones que implícitamente brindan beneficios económicos a corto plazo. El uso de maquinaria eficiente y menos contaminante se identificó como una de las mejores alternativas no solo por sus impactos positivos en el medio ambiente sino también por sus beneficios económicos en términos de reducción de costos de energía a nivel de granja. Los agricultores con actitudes ecocéntricas exhibieron una mayor disposición a adoptar medidas contra el cambio climático, mientras que aquellos con puntos de vista antropocéntricos exhibieron principalmente preferencias más fuertes por actividades relacionadas con mejoras en su productividad. Los resultados muestran que las preferencias de los agricultores por las acciones de mitigación y adaptación están estrechamente relacionadas con los tipos de cultivos. La inversión en mejorar la infraestructura de riego como una actividad de adaptación fue ampliamente aceptada por los agricultores con problemas de disponibilidad de agua que cultivan batatas. Esta acción de adaptación ayuda a los agricultores a optimizar su uso del agua y abordar los problemas de disponibilidad de agua en la región al aumentar su productividad y limitar el desperdicio de agua. Adoptar un cambio en los cultivos como una acción de adaptación también fue preferido por los agricultores que cultivan sorgo, y la preferencia por el enfoque de mitigación de labranza cero está relacionada con los cultivos de sandía y cártamo. El diseño de políticas públicas agrícolas debe considerar las preferencias de los agricultores hacia las acciones de mitigación y adaptación al diseñar e implementar medidas que aseguren una agricultura sostenible. Las herramientas políticas y las intervenciones deben ser inclusivas y desarrolladas a nivel micro en función de las tipologías agrícolas, y se debe fomentar la diversidad de cultivos. Las áreas rurales que son vulnerables a los impactos del cambio climático, donde la agricultura desempeña un papel económico relevante, muestran la importancia de implementar estrategias 24 Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. de adaptación y mitigación específicas para mejorar el nivel de eficiencia técnica a nivel de granja. La adopción de estas acciones no implica una mayor tecnificación del campo, sino un mejor uso de los recursos e insumos disponibles actualmente mediante la introducción de sistemas alternativos de gestión agrícola, como el uso de la agricultura orgánica y la labranza cero, la adaptación del calendario de siembra y el cambio de cultivos. Los efectos del cambio climático en los sistemas agrícolas dependen del tipo de sistema de producción agrícola implementado. Por lo tanto, es necesario adaptar estos sistemas de acuerdo con los requisitos cambiantes del entorno natural causados por la variación climática, para aumentar la capacidad de producción mediante el uso óptimo de los recursos disponibles, lo que resulta en un mayor nivel de eficiencia técnica. Los resultados sugieren la necesidad de aprovechar los recursos disponibles para enfrentar los efectos adversos del cambio climático. Adoptar el uso de semillas mejoradas y cambiar los cultivos se encuentran entre las acciones más importantes según las preferencias de los agricultores. Los formuladores de políticas agrícolas deben generar incentivos para que los agricultores de la región adopten este tipo de acciones de adaptación y difundir información técnica sobre las mejores prácticas para alentarlos a poner en práctica estas acciones. En relación con los agricultores con menos eficiencia técnica, cuya actitud ambiental era más ecocéntrica, su eficiencia ecológica también debería explicarse mediante un uso más sostenible de los recursos hídricos, ya que este es uno de los principales factores que afecta negativamente el rendimiento agrícola. En el sector agrícola, las políticas económicas que se implementan deben estar dirigidas a aumentar los ingresos y mejorar los medios de vida de los hogares rurales para mejorar sus capacidades y activos productivos mediante el uso sostenible de los recursos. Nuestros resultados muestran que las autoridades competentes deben incluir varias acciones que podrían 25 Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. representar una hoja de ruta en la región estudiada. Primero, es necesario realizar una investigación sobre los impactos del cambio climático en la agricultura en la región desde una perspectiva multidisciplinaria, para identificar el impacto de la implementación de acciones de adaptación y mitigación en los patrones de productividad agrícola. En segundo lugar, esta investigación destaca la importancia de identificar variedades de cultivos que se adapten mejor a las nuevas condiciones climáticas de la región. La adopción de esta medida debe ir acompañada de campañas de difusión para compartir experiencias de otros agricultores y ofrecer información sobre los beneficios económicos, sociales y ambientales de la adopción de estas prácticas. Tercero, las asociaciones agrícolas deberían generar esquemas de capacitación sobre prácticas agrícolas de adaptación y mitigación para mejorar el uso racional y la conservación de los recursos naturales, a fin de reducir la aversión al riesgo y aumentar la probabilidad de implementar las nuevas prácticas. Finalmente, se deben establecer programas de incentivos económicos que puedan ayudar a los agricultores a introducir nuevas prácticas de gestión sostenible a nivel de granja que aumenten su productividad y respeten el medio ambiente, específicamente acciones de mitigación. 2.3 Bibliografía  Alvarez, A.; Del Corral, J. (2010). Identifying diﬀerent technologies using a latent class model: Extensive versus intensive dairy farms. Eur. Rev. Agric. Econ. 37, 231–250.  Brick, K.; Visser, M.; Burns, J. (2012). Risk Aversion: Experimental Evidence from South African Fishing Communities. Am. J. Agric. Econ. 2012, 94, 133–152.  Doss, C.; Meinzen-Dick, R.; Quisumbing, A.; (2018). Theis, S. 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Medición de las actitudes hacia el riesgo en los pequeños productores de piña de Santander, Colombia. Cuad. Desarro Rural. 9, 239– 255. 28 Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. CAPÍTULO LAS PUBLICACIONES Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. 3.1. Primera publicación: Analysis of Farmers’ Stated Risk Using Lotteries and Their Perceptions of Climate Change in the Northwest of Mexico. Orduño Torres, M. A., Kallas, Z., & Ornelas Herrera, S. I. (2019). Analysis of farmers’ stated risk using lotteries and their perceptions of climate change in the northwest of Mexico. Agronomy, 9(1), 4. (Q1- Factor de impacto 2.603) 30 agronomy Article Analysis of Farmers’ Stated Risk Using Lotteries and Their Perceptions of Climate Change in the Northwest of Mexico Miguel Angel Orduño Torres 1, * , Zein Kallas 2 1 2 3 * and Selene Ivette Ornelas Herrera 3 Institute for Research in Sustainability Science and Technology (IS-UPC), Polytechnic University of Catalonia, 08034 Barcelona, Spain Center for Research in Agrofood Economy and Development (CREDA-UPC-IRTA), Polytechnic University of Catalonia, Institute of Agrifood Research and Technology, 08860 Castelldefels, Spain; zein.kallas@upc.edu Faculty of Mathematics and Statistics (FME-UPC), Polytechnic University of Catalonia, 08028 Barcelona, Spain; selene.ivette.ornelas@upc.edu Correspondence: miguel.angel.orduno@upc.edu; Tel.: +34-651-78-76-31 Received: 30 October 2018; Accepted: 18 December 2018; Published: 21 December 2018 Abstract: Risk attitudes are relevant factors affecting production, management and investment decisions at the farm level. They are key factors related to farmers’ attitudes towards the environment and climate change. Several methodological approaches, which were considered to be preferable for measuring the level of risk of an economic agent, ranging from highly risk-tolerant to highly risk-averse attitudes, are available. The Multiple Price List (MPL) method is one of the stated approaches that is gaining relevance. In this study, we apply the MPL and relate the risk outcomes to farmers’ socio-economic characteristics and their perceptions of the environment and climate change. Data were collected using a face-to-face survey, carried out with a group of 370 farmers of an irrigation district, located in the northwest of Mexico. The results showed a risk level of about 0.32, according to the Constant Relative Risk Aversion (CRRA) coefficient, locating farmers of the region in a risk-averse group. The heterogeneity analysis showed that the socioeconomic factors and the perceptions of climate change are related to the farmers´ stated risk level. Farmers who are young women, with a tendency to use public support for structural investment, were shown to be risk-tolerant. Farmers considered floods, hail, diseases, pests, and weed growth incidences to be the most frequent weather patterns in the region. Keywords: risk attitude; farmers; multiple price list; lotteries and climate change 1. Introduction and Objectives Climate change is one of the greatest challenges of our times. This phenomenon refers to the long-term variation of the climate generated by natural causes or human actions that distort climatic parameters, such as temperature and precipitation [1]. The way in which weather patterns occur represents a risk, especially for agricultural production due to floods, storms, droughts and hail [2]. Climate is the most significant component in the practice of agricultural activities and is continuously affecting farmers’ decisions, mainly by increasing the uncertainty of the production [3]. Farmers’ perceptions of climate change are increasingly playing an important role in agricultural output and farmers’ decisions at the farm level [4–6]. The perceptions are a set of understandings and sensitivities, integrating an objective and subjective vision about the environment, that allows for the conceptualization of beliefs, values and norms and determines if attitudes are positive or negative [7]. These perceptions are multidimensional and are mainly related to farmers’ risk attitudes, opinions and Agronomy 2019, 9, 4; doi:10.3390/agronomy9010004 www.mdpi.com/journal/agronomy Agronomy 2019, 9, 4 2 of 18 their socio-economic characteristics [8,9]. They may have a direct impact on the development of public policy programs regarding the agricultural production sector, as well as on farmers’ actions related to energy consumption (type and quantity of energy used in machinery and equipment) [10–12]. In fact, several determinants can be related to the farmer’s risk attitude, which in turn may affect his/her decision at the farm level. The socioeconomic characteristics of farmers (such as age, sex, percentage of income from agriculture, agricultural experience, etc.) are relevant determinants [13]. The farm descriptors (land tenure regime, agricultural subsidies, size, crop diversity and type, rainfed or irrigation land, harvest type, etc.) may also be associated with the determination of risk attitudes [14,15]. Farmers’ perceptions of weather patterns are also significant factors affecting their farming management, crop rotation and investment decisions [16]. Their opinions and attitudes concerning the environment may also directly or indirectly impact their decisions, with a certain level of uncertainty [17]. In this context, the main objective of this paper is to analyze the farmers’ stated risk attitudes in an agricultural region in Mexico, using the Multiple Price Lists (MPL) method, known as “Lotteries”, as an alternative method that belongs to the expected utility risk elicitation approach. We also seek to analyze the heterogeneity of the risk attitude level with farmers’ opinions concerning the environment and perceptions of climate change. The heterogeneity analysis will also be conducted by analyzing the risk attitudes in relation to the abovementioned farms and farmers’ characteristics, perception, attitudes and opinions. Farmers´ attitudes and sensibility concerning the environment will be also included. Finally, we will identify attitude patterns, which allow for the differentiation of groups of producers whose characteristics aid in understanding the decisions that they make regarding their activities in order to inform policy makers on farmers’ preferences. This paper contributes to the existing literature on farmers’ risk attitudes by contributing new insight, using the MPL method in Mexico. Reviewing the existing literature, there seems to be very scarce or no information regarding the measurement of the risk aversion level of farmers’ stated risk in Mexico using the MPL. This paper contributes to the verification of the effectiveness and reliability of this technical risk analysis, with a specific empirical application, and can be easily understood by farmers, without the need for specific prior knowledge of the interviewees. It also offers the opportunity to compare our results with other agricultural systems with similar or different management practices and crops. 2. Background The specific study site is identified as irrigation district DR076, which is located in the Carrizo Valley in the state of Sinaloa in Northwestern Mexico, and is eight meters above sea level (Figure 1). The extension of the irrigation district DR076 is approximately 70,172 ha and has 24,017 inhabitants, distributed in 27 localities, with a Gross Domestic Production (GDP) of $143,052 ($ = Mexican Pesos (MXN); $100 (MXN) = 4.89 USD) [18]. Its main economic activity is based on agricultural and animal production, with land characteristics that are very appropriate for crop farming. The irrigation district DR076 is considered one of the most important regions for crop production, which are mainly cereals, such as wheat, with 76% of the total Agricultural Area Used (UAA), and corn, with 8% [19]. The main irrigation system is by gravity, where water resources are obtained from the “Rio Fuerte” river and the water dam, called “Josefa Ortiz de Dominguez,” with a water volume capacity of one billion and 227 million m3 . In the northwestern region of Mexico, the increase of the temperature and the decrease of the annual precipitation is a generalized trend [20]. In the particular case of the Irrigation District DR076, the atypical climatic conditions have been very extreme in recent years (in particular, in 2013), where the lack of rain, low temperature and strong droughts have been more frequent, causing considerable output losses [21]. In 2011, frost weather led to a total loss of the corn yield [22]. In fact, the effect of climate change is closely related to a reduction in economic growth, making it harder to reduce poverty and ensure the food security of local and marginal agricultural communities. [23]. Agronomy 2019, 9, 4 3 of 18 Agronomy 2018, 8, x FOR PEER REVIEW 3 of 18 Figure Figure 1. 1. Location of the study area: area: Irrigation District DR076, DR076, Sinaloa Sinaloa Mexico Mexico [24]. [24]. The The risk risk attitude attitude is is an an aspect aspect related related to to the the behavior behavior of of the the human human being, being, especially especially that that affecting affecting their economic decisions. According to the neoclassical economic theory, in general terms, individuals their economic decisions. According to the neoclassical economic theory, in general terms, are risk-averse, a lowexhibit risk tolerance andtolerance tend toward maximization [25,26]. Risk attitude individuals are exhibit risk-averse, a low risk and profit tend toward profit maximization [25,26]. is individual-specific behavior that affects personal and professional decisions, in particular, investment Risk attitude is individual-specific behavior that affects personal and professional decisions, in options, such as the amount and periodicity in any economic activity. particular, investment options, such as the amount and periodicity in any economic activity. To To analyze analyze risk risk attitudes, attitudes, several several methodological methodological approaches approaches are are available available that that are are developed developed and implemented with a great emphasis on those aspects associated with economic activities, such as and implemented with a great emphasis on those aspects associated with economic activities, such agriculture. The stated risk elicitation is one of the most widely used approaches, in which individuals as agriculture. The stated risk elicitation is one of the most widely used approaches, in which are asked in aare survey about riskabout behavior. can relyapproach on the multi-affirmation individuals asked in atheir survey theirThis riskapproach behavior. This can rely on themethod, multiwhich often includes the self-assessment of risk attitude. It can also rely on methods affirmation method, which often includes the self-assessment of risk attitude. It that can are alsobased rely on on empirical dataare analysis on the expected utility theory. Expectedutility Utilitytheory. TheoryThe wasExpected initially methods that based and on empirical data analysis and on The the expected introduced by Daniel Bernoulli in 1738. by According to this theory, theAccording analysis of under Utility Theory was initially introduced Daniel Bernoulli in 1738. todecisions this theory, the uncertainty, individuals choose the option that provides the highest expected utility, corresponding analysis of decisions under uncertainty, individuals choose the option that provides the highest to the sumutility, of thecorresponding products of probability and over allofpossible outcomes. Johnover vonall Neumann expected to the sum of utility the products probability and utility possible and Oskar Morgenstern contributed to the original conception of the theory in 1944 by explaining outcomes. John von Neumann and Oskar Morgenstern contributed to the original conception of the the decision under uncertainty that minimizes the risk of an election, and this was followed in this theory in 1944 by explaining the decision under uncertainty that minimizes the risk of an election, research [27,28]. This conception is based on several conditionsisthat areon related to rational equability, and this was followed in this research [27,28]. This conception based several conditions that are in which agents rationally assess the odds of the different scenarios to be evaluated. In this context, related to rational equability, in which agents rationally assess the odds of the different scenarios to the undercontext, risk are the analyzed among different alternatives, the decisions be decisions evaluated.taken In this decisions taken under risk are assuming analyzed that among different have an orderassuming of preference definedhave basedan onorder a probability distribution for defined a certainbased number alternatives, thatand theare decisions of preference and are onofa affirmations or sentences [29]. probability distribution for a certain number of affirmations or sentences [29]. However, on the the expected expected utility, utility, U(X), However, the the risk risk attitude attitude cannot cannot be be only only measured measured directly directly based based on U(X), because it also depends on the strength of the preference (“not to risk”), represented by V(X) [30–32], because it also depends on the strength of the preference (“not to risk”), represented by V(X) [30–32], where order toto obtain a more accurate measure of where XXcorresponds correspondstotothe theincome income(a(aspecific specificamount), amount),inin order obtain a more accurate measure the risk attitude, which is is known asasa a“True of the risk attitude, which known “TrueEquivalent” Equivalent”measure measure[33]. [33].True TrueEquivalent Equivalentisisaa certain certain amount of money, and a person is indifferent to whether there is a secure payment amount of money, and a person is indifferent to whether there is a secure payment or or uncertain uncertain payments payments of of aa certain certain investment investment [34]. [34]. This This measure measure was was applied applied in in this this study. study. 3. Theoretical Framework of the Stated Risk Analysis 3. Theoretical Framework of the Stated Risk Analysis As previously noted, risk is a key variable of decisions under uncertainty. Each involved As previously noted, risk is a key variable of decisions under uncertainty. Each involved individual or decision-maker has a different attitude towards risk; hence, the degree of risk aversion individual or decision-maker has a different attitude towards risk; hence, the degree of risk aversion needs to be quantified in order to identify differences and similarities. There are numerous methods needs to be quantified in order to identify differences and similarities. There are numerous methods that are used successfully in economics and agricultural management to measure stated attitudes Agronomy 2019, 9, 4 4 of 18 that are used successfully in economics and agricultural management to measure stated attitudes concerning risk based on surveys of the individuals involved in economic activities. These methods could be divided into: (i) Methods based on an attitudinal scale, with multiple affirmations or statements, (ii) methods based on the theory of expected utility, and (iii) methods that involve a combination of the previous ones. The attitudinal scale methods are constructed based on the score assigned to multiple statements using a certain scale of values [35]. Using these methods, the risk attitude is considered a latent construct (a dependent variable) that is not directly observable, but can be estimated through other explanatory variables that are related to the statements proposed by different researchers, depending on the topic of study. The empirical methods based on the economic theory of expected utility [36,37] estimate an indicator of risk aversion as a function of probabilities in a non-parametric framework, since it is not a function of the utility that is supposed to govern the behavior of individuals. The following is a brief description of the main applications measuring the stated risk level in agriculture, based on surveys and interviews: • • • • • Bardhan’s attitudinal scale of five points on dairy farmers in India, which identifies the most relevant sources of risk according to farmers’ perceptions and attitudes [38], based on 31 statements related to the social and psychological attributes of each farmer [39]. Risk attitude measuring instrument RAMI of Fausti and Gillespie, applied to cattle producers in the United States [40]. The instrument was made up of five questions, and the last three were defined according to the certainty equivalence framework associated with the expected utility model. Scale of the measurement of risk attitudes, based on three factors of Allub’s scale concerning the hypothesis that risk aversion and income diversification are the factors that influenced the farmers’ decisions in a case study in Argentina [41]. It considered that risk aversion is determined by three factors: The socio-economic status of the farmer, the degree of involvement or participation in the rural development program, and the farmer’s perception of the agro ecological conditions of the farm. Method of measuring the risk attitude with three components of Bard [16]. This scale was implemented on grain producers in the United States. The method consists of three components: The risk attitude scale, a self-assessment question and a model based on the expected utility. The Multiple Price List “Lotteries” (MPL), of Olbrich, Quaas, and Baumgärtner, based on the Theory of Expected Utility (EUT), was implemented to analyze the risk attitude of livestock producers in Namibia’s pastures using the MPL format [42], proposed by Binswanger [43] and studied by Holt and Laury [28]. In this case study, the method selected to measure the farmers’ risk attitudes was the MLP proposed by Holt and Laury [28], specifically the variant developed by Brick, Visser and Burns [44]. This decision was taken because of the characteristics of the study target population, which does not have a high level of education, but is familiar with the concept of lotteries. The selected method is also characterized by simplicity and a relatively short time for data collection. In addition, its application does not require prior knowledge of the concepts of uncertainty and risk, nor extensive analysis of multiple statements. The MPL method has been used in many studies to measure the risk attitudes of farmers, among which are those made by Olbrich et al. [42] and Brick et al. [44]. 4. Materials and Methods The data collected correspond to a representative sample of a total of 370 farmers from irrigation district DR076, and the sample size was determined based on the formula of finite populations, with a confidence level of 95% and an error level of 4.99% [45]. The data collection was carried out in a stratified way through a semi-structured face-to-face questionnaire, carried out during the Agronomy 2019, 9, 4 5 of 18 Agronomy 2018, 8, x FOR PEER REVIEW 5 of 18 period from October to December 2017, with the purpose of identifying factors affecting farmers’ risk attitudes. These factors were classified into socio-economicvariables variablesthat thatinclude include the the particular attitudes. These factors were classified into socio-economic particular farmer characteristics, the farm descriptors and main crops, as well as the environmental opinions and farmer characteristics, the farm descriptors and main crops, as well as the environmental opinions attitudes of farmers and their perceptions of weather patterns changes (the translated questionnaire is and attitudes of farmers and their perceptions of weather patterns changes (the translated available in supplementary file Q_1). On average, the application of the interview lasted 40 min with questionnaire is available in supplementary file Q_1). On average, the application of the interview each support of athe group of students from the Intercultural lastedfarmer 40 minand withwas eachcarried farmerout andwith was the carried out with support of a group of students from the Autonomous University of Sinaloa, who were instructed prior to the application of the survey. Intercultural Autonomous University of Sinaloa, who were instructed prior to the application of the The following methodological approach is summarized in Figure 2. survey. The following methodological approach is summarized in Figure 2. Factors affecting production decisions Risk theoretical framework Semi-structured questionnaire design Socio-economic variables Attitudes and opinions towards the environment Attitudes towards Perceptions of Stated climate change climate change risk level Heterogeneity Analysis Conclusions Figure Figure 2. 2. Methodological Methodological research research approach. approach. The main variables, describing the 370 farmers, their perceptions, opinions and farm The main variables, describing the 370 farmers, their perceptions, opinions and farm characteristics, are described in Tables 1 and 2. characteristics, are described in Tables 1 and 2. Each farmer was presented with a questionnaire, with open-ended questions divided into Each farmer was presented with a questionnaire, with open-ended questions divided into several blocks according to the type of information, collected and presented in Tables 1 and 2. several blocks according to the type of information, collected and presented in Table 1 and 2. This This questionnaire was tested before its final application on a sample of about 25 farmers. The blocks of questionnaire was tested before its final application on a sample of about 25 farmers. The blocks of variables were: (i) Farmers’ socio-economic variables, farm characteristics (economic and management), variables were: (i) Farmers’ socio-economic variables, farm characteristics (economic and investment and land use, following the classification presented by Kallas et al. [13], (ii) environmental management), investment and land use, following the classification presented by Kallas et al. [13], attitudes and opinions, using the New Ecological Paradigm Scale NEP [46], (iii) attitudes or willingness (ii) environmental attitudes and opinions, using the New Ecological Paradigm Scale NEP [46], (iii) to carry out actions to reduce climate change using an array of statements evaluated by farmers, attitudes or willingness to carry out actions to reduce climate change using an array of statements (iv) perception of climate change constructed from literature, and (v) attitudes toward risk using the evaluated by farmers, (iv) perception of climate change constructed from literature, and (v) attitudes MPL method. The methods used are described below: toward risk using the MPL method. The methods used are described below: Table 1. The description of farmers, their perceptions and opinions. SD: Standard Deviation. Variables Gender Male Female Age Under 40 years From 41 to 60 years old Percentage (%) 88.92% 11.08% 19.46% 52.16% Agronomy 2019, 9, 4 6 of 18 Table 1. The description of farmers, their perceptions and opinions. SD: Standard Deviation. Variables Percentage (%) Gender Male Female 88.92% 11.08% Age Under 40 years From 41 to 60 years old Above 60 years 19.46% 52.16% 28.38% Percentage of income from agriculture (%) 76.00% Number of members of the family Generations of the family dedicated to agriculture Mean 3.79 2.28 SD 1.74 0.83 Units members members Farmers’ understanding about aspects related to global warming (%) Melting of the poles 18.38% Rising temperature and warming of the earth 52.97% Pollution 13.92% Emission of gases into the atmosphere 14.73% Farmers’ opinions on the level of improvement required in his activity on a scale from0 (not required at all) to 10 (absolutely required): Commercialization of crops 8.28 2.69 Fight against diseases or pests 7.25 3.14 Choice of crops 6.24 3.45 Quality of the soil 6.84 3.39 Type of tillage 7.32 3.07 Use of efficient irrigation techniques 7.06 3.48 Farmers’ perceptions on climate change on a scale from 0 (not observed at all) to 10 (highly observed): The temperature has increased 7.46 2.32 The level of precipitation has changed 6.72 2.87 The rain periods have changed their temporality 6.86 2.79 The soil has less fertility 6.24 2.96 The periods of drought have increased 6.56 2.79 The harvest has decreased 6.72 2.82 There have been more episodes of droughts 6.61 2.92 There have been more episodes of frost 5.98 3.20 There have been more episodes of floods 6.85 2.68 There have been more episodes of hail 6.08 3.12 There have been more diseases and pests 8.23 1.31 Weeds have increased 7.92 1.88 Farmers’ level of willingness to perform the following actions on a scale from 0 (not willing) to 10 (completely willing): Perform only nightly irrigation 5.53 3.80 Use low-polluting machinery 8.02 2.62 Carry out agro ecological production 7.89 2.50 Use renewable energy sources 7.91 2.52 Do not burn biomass (stubble) 8.01 3.26 Use non-nitrogenous fertilizers 7.99 2.53 Use the zero tillage method 6.60 3.58 Agronomy 2019, 9, 4 7 of 18 Table 2. The description of the farms and main crop activities. Variable Mean Utilized agricultural area Number of irrigations by crop season Volume of water used in irrigation Permanent employees Temporary employees Variable Percentage Type of crop harvest Manual 17.03% Mechanic 82.97% Land tenure regime Private property 32.97% Ejidal * 67.03% Property management regime Owner 79.19% Owner’s family without salary 5.95% Salaried family 0.27% Tenant or associate 14.59% Water availability problems c No 84.05% Yes 15.95% Experience in water collection b No 83.78% Yes 16.22% Type of irrigation Irrigation by gravity 95.68% Sprinkler irrigation 1.62% Mechanized Irrigation by gravity 0.81% Drip irrigation/localized 1.89% 10.66 7.56 15.46 1.52 3.79 SD Units 9.28 ha 4.61 frequency 10.97 m3/ha 1.73 persons 9.39 persons Variable Percentage Have credit for farming activity a No 54.05% Yes 45.95% Receive some subsidy for agriculture No 31.62% Yes 68.38% Purpose of the subsidies received Covering operating expenses 60.50% For investment in equipment 12.30% For agricultural improvement of land 12.60% Other agricultural expenses 14.60% Agricultural insurance d No 63.24% Yes 36.76% Origin of their agricultural training Agrarian Experience 88.92% Agricultural professional-university training 6.76% Courses, conferences, workshops, etc. 2.43% Other education sources 1.89% Main product grown Wheat 28.92% Alfalfa 24.59% Soy 9.73% Peanut 7.03% Corn 5.41% Chilli 4.32% Others (Onion, Sweet potato, Watermelon, 20.00% Bean, Sorghum and Triticale) * Form of farmers’ organizations in Mexico. a Refers to whether the farmer received a credit loan for his agricultural activity. b Indicates if the farmer has used some method to recollect water for crops, such as ponds or dams. c Refers to the problems related to the water availability in their agricultural land, such as a time-restricted access to water resources and the payment of the water quota. d Refers to whether farmers hired an insurance contract for their crops. 4.1. Measuring Farmers’ Environmental Attitudes and Opinions The NEP scale was used to analyze farmers’ environmental opinions. This scale has sixteen statements (Table 3) that express a positive or negative evaluation of the environment and relies on individual’s beliefs about themselves and nature, according to a nine-point Likert type scale, where 1 means absolutely disagree, and 9 means absolutely agree [47,48]. To understand the behavior that humans have toward the environment, it is necessary to understand how they perceive it, that is, what their pro-environmental attitudes are. By performing a factorial analysis using the NEP scale, the dimensionality that characterizes farmers can be identified to determine if the items are clearly associated with an array of dimensions. The NEP scale has been used to analyze farmers’ beliefs concerning environmental issues [49]. This scale reflects the way in which the human being conceptualizes nature and the way he/she behaves in relation to it [48]. Agronomy 2019, 9, 4 8 of 18 Table 3. Statements of the New Ecological Paradigm Scale. Absolutely Disagree Strongly Disagree Moderately Disagree Slightly Disagree Neutral Slightly Agree Moderately Agree Strongly Agree Absolutely Agree 1 2 3 4 5 6 7 8 9 1. A global ecological crisis is exaggerated. 2. The balance of nature supports the impact of industrialized countries. 3. Humans may be able to control nature. 4. Human ingenuity ensures that the earth is not uninhabitable. 5. Humans were created to dominate the rest of nature. 6. Humans have the right to modify the environment to adapt it to their needs. 7. The interference of the human being in nature has disastrous consequences. 8. Plants and animals have the same right to exist as human beings. 9. The human being seriously abuses the environment. 10. The balance of nature is delicate and easily alterable. 11. If things continue as they have been, we will soon experience a great ecological catastrophe. 12. We are approaching the limit number of people that the earth can hold. 13. The earth has limited resources. 14. Despite our special abilities, human beings are still subject to the laws of nature. 15. The land has abundant resources, we just have to learn how to exploit them. 16. Sustainable development needs a balanced situation that controls industrial growth. 4.2. Measuring Farmers’ Perceptions of Climate Change The perception of climate change involves the analysis, according to each farmer, of whether he/she has observed variability in certain meteorological factors or events related to the climate. To address this issue, an array of statements, collected from a literature review (Table 4) and related to climate change was evaluated, according to the farmer’s own perception, on a 9-point Likert type scale (from 1 to 9), where 1 means absolutely disagree, and 9 means absolutely agree. Table 4. Items included analyzing the farmers’ perceptions of climate change. Absolutely Disagree Strongly Disagree Moderately Disagree Slightly Disagree Neutral Slightly Agree Moderately Agree Strongly Agree 1 2 3 4 5 6 7 8 In the last 10 years you have noticed that the temperature has increased: [50,51] In the last 10 years you have noticed that the level of precipitation has changed: [50,52] In the last 10 years you have noticed that rain periods have changed their temporality: [53–55] In the last 10 years you have noticed that the soil has lost fertility: [3,54] In the last 10 years you have noticed that the periods of drought have increased: [50,52] In the last 10 years you have noticed that the harvest has decreased: [56] In the last 10 years you have noticed that there have been more episodes of droughts: [3,50] In the last 10 years you have noticed that there have been more episodes of frost: [50,53] In the last 10 years you have noticed that there have been more episodes of floods: [52] In the last 10 years you have noticed that there have been more episodes of Hail: [57] In the last 10 years you have noticed that there have been more diseases and pests: [58,59] In the last 10 years you have noticed vegetation changes: [60] Absolutely Agree 9 _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ 4.3. Measuring Farmers’ Risk Attitudes 4.3.1. Model Definition Each respondent was presented with a set of different lottery pairs, and one of the lottery options must be chosen from each pair [61]. (This relates the levels of risk aversion with a prize or profit). In the definition of the model, a list of eight scenarios was generated, with a pair of hypothetical lotteries, called option A and option B, which is similar to the theoretical model used by Brick et al. [44]. In this model, as in Brick´s, the probabilities that each one of the options will keep the experiment as simple as possible remain constant. In option A, the probability of obtaining the amount presented was set at 100% (safe option), and in option B (risky option), the probability was set at 50% for obtaining the amount of ($100) and 50% for not getting anything ($0) (tossing a coin for heads or tails) in all scenarios, while the safe amount presented in option A in each of the eight scenarios is modified in a decreasing manner, according to the following amounts ($100, $75, $60, $50, $40, $30, $20 and $10). Agronomy 2019, 9, 4 9 of 18 The level of risk aversion is based on the number of safe answers (option A) that the interviewee selects. According to the experimental design structure (see question 35 in the questionnaire, available in the supplementary file Q_1), only a risk-tolerant participant would select option B in the first scenario, while an extremely risk-averse participant would be the one who, in the eight scenarios, would select option B, and a risk-neutral participant would be the one who selects option A in the first three scenarios, and in the fourth scenario, changes to option B [62]. 4.3.2. Estimation of the Relative Risk Aversion Coefficient “r” We defined the values of the Constant Relative Risk Aversion (CRRA), based on the function, U(y) = y (1 − r) /(1 − r), where y is the income and r the relative risk aversion coefficient for each of the six stages (Table 5). Then, the coefficient r is estimated by means of the logarithmic maximum likelihood function, conditioned to the expected utility model and the CRRA specification, defined as the difference of the expected utility (∇EU = EUA −EUB ). In this case, the maximum likelihood equation used is: ln LEU (r, z, X ) = ∑((ln(∇EU)/z = 1) + (ln(1 − ∇EU)/z = 0)) (1) i where zi = (0,1), 1 indicates that the respondent chose the auction in scenario i, 0 indicates that he/she chose the safe amount, and X corresponds to a vector of variables of personal, agricultural and environmental characteristics and environmental risk. Once the structure of the experiment is established, according to the specification of the choice of theory of expected utility (EUT) under uncertainty, the parameters of the function of CRRA are estimated, which allow the behavior or risk preferences of participants to be modelled [44,63]. According to the theory of the expected utility (EUT, Expected Utility Theory), proposed by von Neumann-Morgenstern, different utility functions have been generated that shape the behavior of people according to their risk preferences. Among the most common is the CRRA, which allows the implicit risk in decision making to be estimated [37]. The expected utility of each lottery is calculated by means of the function: EU = ∑( pi × U ( Xi )) (2) i where pi is the probability of occurrence of the utility of the prize Xi, and U (Xi ) is the utility of the prize Xi . The CRRA function is defined as a non-negative lottery prize, according to the following equation: X 1−r U (X) = (3) 1−r where X corresponds to the lottery prize, r is the latent risk aversion coefficient, where r = 0 indicates risk neutrality, r > 0 indicates risk aversion, and r < 0 indicates taste or tolerance for risk [63]. Table 5. Level of risk coefficients [28]. Number of Safe Choices “A” 0–1 2 3 4 5 6 7 8 Range of CRRA for U(X) = X (1 − r) /(1 − r) −1.71 −0.95 −0.49 −0.15 0.14 0.41 0.68 0.97 r< >r< >r< >r< >r< >r< >r< >r< −0.95 −0.49 −0.15 0.14 0.41 0.68 0.97 1.37 Classification of Risk Preference highly risk-tolerant very risk-tolerant risk-tolerant risk-neutral slightly risk-averse risk-averse very risk-averse highly risk-averse To determine CRRA values, a scenario and its immediate continuum are related, given that each scenario is independent of the rest, and the model is defined with a decreasing level of risk aversion, so it is not necessary to relate it to the subsequent scenarios, meaning that the level of risk aversion is Agronomy 2019, 9, 4 10 of 18 determined by the first change of option A by option B. (Because of this, any inconsistency subsequent to B’s decision can be eliminated). For instance, the choices of an individual, who chooses option A in the first three scenarios and then changes to option B, correspond to a risk aversion coefficient in the range of −0.15 and 0.14, which would define the individual as risk-neutral, while a person who always chooses Option A (the secure lotteries), the CRRA rank that corresponds to it is 0.97–1.37, which defines the person as extremely risk-averse. The expected values of options A and B in each of the scenarios of the experimental design can be seen in Table 6. This also includes the ranges of relative risk aversion coefficients, implicit in each possible choice, under the assumption of the constant relative risk aversion (CRRA). Table 6. Matrix of payments in the risk aversion experiment. Lottery Option A Option B Expected Value Question (Scenario) A PA * B1 PB1 * B2 PB2 * E(A) E(B) Difference 1 2 3 4 5 6 7 8 100 75 60 50 40 30 20 10 1 1 1 1 1 1 1 1 100 100 100 100 100 100 100 100 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0 0 0 0 0 0 0 0 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 100 75 60 50 40 30 20 10 50 50 50 50 50 50 50 50 50 25 10 0 −10 −20 −30 −40 CRRA* Interval −1.71, −0.95 −0.95, −0.49 −0.49, −0.15 −0.15, 0.14 0.14, 0.41 0.41, 0.68 0.68, 0.97 0.97, 1.37 PA, Probability option A; PB1, probability of winning the amount B1 of option B; PB2, probability of winning the amount B2 of option B; CRRA, Constant relative risk aversion. Once the CRRA values are estimated, the choices made by each individual on each pair of options presented in the eight scenarios are taken, and their level of aversion to individual risk is estimated. The instructions and differences regarding the lotteries presented in each scenario were explained in detail to farmers. Then, they were given the sheet to make their choices. The level of individual risk was determined and the group results were subsequently aggregated. By means of contingency tables and the analysis of variance by means of the ANOVA method, the relationship of the data with the variable response level of aversion to risk, presented by the farmers, is explored. Subsequently, the statistical technique of Principal Component Analysis (PCA) was used to reduce the dimensionality based on the estimation of correlations. The obtained results are interpreted, characterizing the farmers of the studied regions. 5. Results and Discussion 5.1. Results on the Level of Farmers’ Risk Attitudes The results of the estimated risk level using the MPL showed a score of about 0.32, which globally defines the studied population as slightly risk-averse, according to the aforementioned scale of Holt and Laury. The results presented in Figure 3 showed the distribution of the sample of farmers, according to the different estimated scores of the attitudes towards risk levels. According to the scale of Holt and Laury [28], the results showed that almost 34% of farmers are highly risk-averse, 7.57% are risk-neutral, and 22% of farmers are highly risk-tolerant. These results are similar to those of Trujillo et al. (2009) [64], Galarza (2009) [65], Pennings and Garcia (2001) [17] and Brick et al. (2011) [44], who found that farmers exhibited, in general, a risk-averse attitude. according to the different estimated scores of the attitudes towards risk levels. According to the scale of Holt and Laury [28], the results showed that almost 34% of farmers are highly risk-averse, 7.57% are risk-neutral, and 22% of farmers are highly risk-tolerant. These results are similar to those of Trujillo et al. (2009) [64], Galarza (2009) [65], Pennings and Garcia (2001) [17] and Brick et al. (2011) Agronomy 2019, 9, 4 that farmers exhibited, in general, a risk-averse attitude. 11 of 18 [44], who found Risk-tolerant Risk-averse 34.32% n=127 21.89% n=81 9.73% n=36 highly risk very risk tolerant tolerant 9.46% n=35 risk tolerant 7.57% n=28 risk neutral 9.19% n=34 2.43% n=9 5.41% n=20 slightly risk averse very risk highly risk risk averse averse averse Figure 3. Distribution of the farmers according to their risk level. n: numbers. Figure 3. Distribution of the farmers according to their risk level. n: numbers. 5.2. Risk Heterogeneity Analysis 5.2. Risk Heterogeneity Analysis The risk response (risk-CRRA) is a categorical variable and represents the risk level of the farmers The risk response (risk-CRRA) is Laury. a categorical variable represents analysis the risk indicate level ofthat the according to the scale used by Holt and The results of theand heterogeneity farmers according to the scale used by Holt and Laury. The results of the heterogeneity analysis the variables related to the level of risk aversion are: The purpose of the subsidies received (p-value = indicate the variables related to the level of risk=aversion are: the subsidies 0.002), thethat gender of the agricultural producer (p-value 0.046) and theThe age purpose (p-value =of0.000). Results received (p-value = 0.002), the gender of the agricultural producer (p-value = 0.046) and the age (pshowed that women consider themselves a bit more risk-tolerant than their counterparts (percentage value = 0.000). Results showed that women consider themselves a bit more risk-tolerant than their of men who tolerate risk = 39%, while the percentage of women who tolerate risk = 61%). The average counterparts (percentage of men was who− tolerate riskthe = 39%, while the ofwas women who tolerate level of estimated risk for women 0.035, and estimated riskpercentage level of men 0.361. This result risk = 61%). The average level of estimated risk for women was −0.035, and the estimated risk level of should be taken into account, particularly because women are responsible for 60% to 80% of the world’s men was This resultstewards should be into account, particularly because women responsible food, and0.361. they are better of taken the environment [66]. Furthermore, women are are more prone to for 60% to 80% of the world’s food, and they are better stewards of the environment [66]. participating in extensive activities related to agriculture and are more committed to farming activities. Furthermore, women are more prone to participating in extensive activities related to agriculture However, decisions that are related to farm development and improvement adoption are takenand by are more committed to farming activities. However, decisions are related to farm development men, excluding women in the decision-making process of a newthat agricultural system [67]. and improvement adoption are excluding women in the related decision-making process of a The variable collecting the taken main by usemen, of the subsidies was highly to farmers’ risk level. new agricultural system [67]. Farmers who receive economic support and use it in structural investment at the farm level are more The variable usean of adverse the subsidies highly toalso farmers’ risk level. risk-tolerant, whilecollecting those whothe do main not have profilewas to risk [68]. related Age was associated with Farmers who receive economic support and use it in structural investment at the farm level more the risk level, which is similar to the findings of Brick et al., in their experiment on Africanare fishing risk-tolerant,[44]. while whowe dofound not have an adverse profile to risk [68]. show Age was associated communities Inthose our case, that farmers “above 60 years of age” thatalso the experience with the risk level, which is similar to the findings of Brick et al., in their experiment on acquired over time allows them to have a lower risk aversion on average, compared to farmersAfrican in the fishingofcommunities [44]. ourare case, we conservative, found that farmers 60 years of age” show that the range 41 to 60 years’ old,Inwho more with a“above higher level of risk aversion. experience time allows themresearch, to have awhich lowermeasured risk aversion on average, compared to Severalacquired pieces ofover agricultural economic risk attitudes according to the expected utility theory, found that they are related to a set of farm and farmers’ variables. Fahad et al. (2018) mentioned that factors, such as age, farming experience, and land size, may influence the farmers’ decisions to contract crop insurance as a risk management strategy [14]. De Pinto et al. (2013) found that the implications for climate change mitigation projects are related to farmers’ risk level [69]. Dörschner and Musshoff (2013) mentioned that the income variation and the farmers’ risk attitudes can constitute an explanatory approach for the low acceptance of the actions that face the impact of climate change, showing that a better understanding of farmers’ risk attitudes is necessary to make more effective agricultural policies [70]. The variables shown in Table 7, correspond to the attitudinal variables included in the heterogeneity analysis relating the risk level and attitudes associated with, and perception of, the environment and climate change. Agronomy 2019, 9, 4 12 of 18 Table 7. Variables related to the level of risk based on the ANOVA, classified by the type of information. Type of Information Variables Utilized agricultural area Volume of water used in irrigation Income Number of hectares of rain fed crops Volume of water irrigated (m3 /ha) Percentage of income from agriculture Attitudes and Opinions towards the Environment (NEP Statements) A global ecological crisis is exaggerated The balance of nature supports the impact of industrialized countries Humans may be able to control nature Human ingenuity ensures that the earth is not uninhabitable The interference of the human being in nature has disastrous consequences The human being seriously abuses the environment The balance of nature is delicate and easily alterable We are approaching the limit number of people that the earth can hold The earth has limited resources The land has abundant resources, we just have to learn how to exploit them Sustainable development needs a balanced situation that controls industrial growth Attitudes towards Climate Change Level of disposition to perform only nightly irrigation Level of willingness to use low-polluting machinery Level of disposition to carry out agro ecological production Level of disposition to use of renewable energy sources Level of disposition not to burn biomass (stubble) Level of willingness to use non-nitrogenous fertilizers Level of willingness to use zero tillage Perception of Climate Change Level of impact of global warming on their crops Percentage of climate change influence on production costs Temperature increase More episodes of floods More episodes of hail More diseases and pests Weed increase Source: Own elaboration, based on the data provided by the interviewees. Given that farmers perceive that they have a significant interest with respect to climate change, we reduced the dimensionality of this group of variables, seeking a minimum loss of information through a Principal Components Analysis (PCA). The objective is to determine a smaller set of unrelated variables that explain the existing relationships between the observed variables, identify the number of underlying variables, evaluate the individuals in relation to these new variables, and interpret the extracted component. Before starting the analysis, the Kaiser Meyer Olkin test (KMO) was applied to verify the applicability of the set of available variables. The results obtained, of 0.697 and 0.74, indicating that the relation of these is between medium and low. The results of the PCA allowed two components to be identified, which explain 51% of the total variability. The first factor identified is defined by the climatic variables, such as floods, hail, diseases and pests. The second factor is defined by variables related to the impact of climate change on the farming activity, with variables related to weed increase, the impact of the global warming on crops and the influence of climate change on production. These results indicated that the first component characterizes the perception of farmers towards the negative weather patterns, and the second is related to the impact on their agricultural productivity. The distribution of the point´s cloud, which represents the farmers in relation to the two main components and the variables previously described, is shown in Figure 4. Most of the risk-tolerant farmers (red points) are located to the right of the first dimension, exhibiting a higher perception of the weather patterns related to climate change, compared to the risk-averse farmers. This result is similar to the finding of Pomareda (2008) [71] and Brown et al. (2017), who found that effect of natural shocks affects risk attitudes and perceptions [72]. Furthermore, risk-tolerant farmers (red points) are more The distribution of the point´s cloud, which represents the farmers in relation to the two main components and the variables previously described, is shown in Figure 4. Most of the risk-tolerant farmers (red points) are located to the right of the first dimension, exhibiting a higher perception of the weather patterns related to climate change, compared to the risk-averse farmers. This result is similar to the finding of Pomareda (2008) [71] and Brown et al. (2017), who found that effect of natural Agronomy 2019, 9, 4 13 of 18 shocks affects risk attitudes and perceptions [72]. Furthermore, risk-tolerant farmers (red points) are more concentrated in the upper part of the second dimension, showing that they perceive to a greater extent that climate change hasofathe high impact on their production. Inthey this context, concentrated in the upper part second dimension, showing that perceive our to aresults greatershowed extent thatclimate the highly farmers are those that are prone to using subsides in structural that changerisk-tolerant has a high impact on their production. In this context, our results showed that the investment at the farm level. also showed thatsubsides risk-neutral farmers investment do not haveatwellhighly risk-tolerant farmers are The thoseresults that are prone to using in structural the defined perceptions regarding climate nor the effects do thatnot climate could have on their farm level. The results also showed thatchange, risk-neutral farmers have change well-defined perceptions productivity. regarding climate change, nor the effects that climate change could have on their productivity. Figure offarmers farmersaccording accordingtoto their perceptions of climate change. (A) Impact of Figure4.4. Distribution Distribution of their perceptions of climate change. (A) Impact of global global warming on their crops; (B) Percentage of climate change influence on production costs; warming on their crops; (B) Percentage of climate change influence on production costs; (C) (C) Temperature increase; More episodes of floods; More episodes of hail; More diseases Temperature increase; (D)(D) More episodes of floods; (E)(E) More episodes of hail; (F) (F) More diseases and and pests; Changes in weed development. pests; (G)(G) Changes in weed development. The Theresult resultofofPCA, PCA,carried carriedout outon onfarmers’ farmers’opinions opinionsand andattitudes attitudestowards towardsthe theenvironment, environment, using the NEP scale and their relation to risk level, is presented in Figure 5. As can using the NEP scale and their relation to risk level, is presented in Figure 5. As canbe beseen, seen,the thefirst first two components explain an accumulated variance of 47.4%. The first factor was principally defined two components explain an accumulated variance of 47.4%. The first factor was principally defined by bythe thevariables, variables,“The “Theearth earthhas haslimited limitedresources” resources”and and“Sustainable “Sustainabledevelopment developmentneeds needsaabalanced balanced situation”. This factor can be characterized as an ecocentric attitude. The second factor situation”. This factor can be characterized as an ecocentric attitude. The second factorwas wasmainly mainly defined by the variables, “Humans may be able to control nature”, “The balance of nature supports the defined by the variables, “Humans may be able to control nature”, “The balance of nature supports impact of industrialized countries”, and “Human ingenuity ensures that the earth is not uninhabitable,” which are related to an anthropocentric attitude towards the environment [73]. Based on the points dispersion, the results showed that farmers with a risk-tolerant attitude (red points) have a positive perception regarding the aspects related to ecocentric attitudes. The risk-tolerant farmers are those that allow for better differentiation of the aspects related to their positive environmental opinions. They have a clearer vision of the aspects related to their positive environmental opinions. Furthermore, in this context, risk-tolerant farmers are willing to perform positive actions relating to the environment, such as using low-polluting machinery, not burning biomass (stubble) or using non-nitrogenous fertilizers (identified in Table 7), while there is no clear trend in the environment opinions of risk-averse farmers. However, their risk-averse attitude does not preclude their potential adoption of positive actions towards the environment, as stated in Dörshner and Musshoff (2013) [70]. environmental opinions. Furthermore, in this context, risk-tolerant farmers are willing to perform positive actions relating to the environment, such as using low-polluting machinery, not burning biomass (stubble) or using non-nitrogenous fertilizers (identified in Table 7), while there is no clear trend in the environment opinions of risk-averse farmers. However, their risk-averse attitude does not preclude their potential adoption of positive actions towards the environment, as stated in Agronomy 2019, 9, 4 14 of 18 Dörshner and Musshoff (2013) [70]. Figure 5. 5. Distribution according to totheir theirenvironmental environmental opinions. (1)global A Figure Distributionofoffarmers, farmers, interviewed interviewed according opinions. (1) A global ecological crisis exaggerated;(2)(2)The Thebalance balance of nature of industrialized ecological crisis is is exaggerated; naturesupports supportsthe theimpact impact of industrialized countries; (3) (3) Humans may be able to control nature; (4) (4) Human ingenuity ensures thatthat thethe earth is is countries; Humans may be able to control nature; Human ingenuity ensures earth notnot uninhabitable; (5) The interference of the human being in nature has disastrous consequences; uninhabitable; (5) The interference of the human being in nature has consequences; (6) (6) The balance of of nature is delicate andand easily The human human being being seriously seriouslyabuses abusesthe theenvironment; environment;(7)(7)The The balance nature is delicate easily alterable; (8) (8) WeWe are are approaching the the limit number of people thatthat the the earth cancan hold; (9) (9) TheThe earth hashas alterable; approaching limit number of people earth hold; earth limited resources; (10)(10) TheThe land hashas abundant resources; wewe justjust have to learn how to exploit them; limited resources; land abundant resources; have to learn how to exploit them; (11)(11) Sustainable development needs a balanced situation. Sustainable development needs a balanced situation. 6. Conclusions 6. Conclusions There is a wide variety of stated methods for measuring the risk level in the agricultural sector, There is a wide variety of stated methods for measuring the risk level in the agricultural sector, although the MPL method (lotteries) is shown to be a valid approach in the agricultural sector. It is although the MPL method (lotteries) is shown to be a valid approach in the agricultural sector. It is characterized by its simplicity and is easily understood. Furthermore, its application does not require characterized by its simplicity and is easily understood. Furthermore, its application does not require prior knowledge of the concepts of uncertainty and risk, nor extensive analysis of multiple statements. prior knowledge of the concepts of uncertainty and risk, nor extensive analysis of multiple In general, in the studied area, farmers do not have high academic preparation, which makes the MPL statements. In general, in the studied area, farmers do not have high academic preparation, which a suitable method in this case. The MPL is a useful tool for comparing the stated risk attitudes of makes the MPL a suitable method in this case. The MPL is a useful tool for comparing the stated risk individuals who face the same lottery game. attitudes of individuals who face the same lottery game. The farmers in the studied region exhibited, in general terms, a risk-averse attitude, which is The farmers in the studied region exhibited, in general terms, a risk-averse attitude, which is consistent with the main finding of other stated approaches to risk studies in the agriculture consistent with the main finding of other stated approaches to risk studies in the agriculture sector. sector. Farmers recognize the importance of improving the commercialization of their crops and Farmers recognize the importance of improving the commercialization of their crops and the the effectiveness of treatments against diseases and pests. Farmers’ perceptions of climate change were highlighted by the adverse effects on their output through higher disease and pest incidences on their crops, weed development increases, temperature increases and variations, and changes in the temporality of the rain periods. The farmers´ characteristics, such as sex and age, were related to their stated risk level. An important finding to highlight is that women in the region were more risk-tolerant than men and were more prone to using public support to invest at the farm level. However, they are marginalized as decision-makers at the policy level and are therefore still less involved in the design and implementation of public policy in the agricultural sector, mainly due to cultural limitations. Our results also showed that farmers above 60 years old are more risk-tolerant than younger farmers. Agronomy 2019, 9, 4 15 of 18 As part of the findings of this research, risk-tolerant farmers were shown to be highly aware of the effects of climate change on production and were shown to agree with sustainable development statements concerning the environment, which may generate greater resilience in the region. Better involvement of these farmers in the process of generating agricultural public policies is recommended. Additionally, public policies should effectively target farmers with risk-averse attitudes in order to build confident relationships with them, which may improve their perceptions and adaptive capacities in relation to climate change. One of the limitations of this paper is that the comparability of the results on the level of risk depends on the use of the MPL method under the same payment scheme, given that the difference in payments can generate heterogeneous results. Thus, for future research, it should be considered that the applicability and validity test using MPL could be verified by its application in other agricultural areas with similar characteristics. This would also corroborate and validate the results, generating a comparative map of attitudes towards risk and climate change perceptions. Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4395/9/1/4/s1, supplementary file: supplementary file Q_1. Author Contributions: M.A.O.T. conceived and designed the study, investigates, performed data collection, and wrote manuscript; Z.K. conceived and designed the research, made review and editing, participates in writing the manuscript and was the supervisor of all procedures; S.I.O.H. made the formal analysis of the data and participates in writing the manuscript. Funding: This research received no external funding. Acknowledgments: The National Council of Science and Technology (CONACYT) is thanked for the support to carry out this research. Conflicts of Interest: The authors declare no conflicts of interest. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Ministerio de Agricultura, Pesca y Alimentación–Ministerio para la Transición Ecológica. Qué es el Cambio Climático. 2018. Available online: http://www.mapama.gob.es/es/cambio-climatico/temas/cumbrecambio-climaticocop21/el-cambio-climatico/ (accessed on 6 July 2018). Cárdenas, M.J. 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World Dev. 2018, 104, 310–325. [CrossRef] [PubMed] Reyna, C.; Bressán, E.; Mola, D.; Belaus, A.; Ortiz, M. Validating the structure of the New Ecological Paradigm scale among Argentine citizens trough different approaches. Pensam. Psicol. 2018, 16, 107–118. [CrossRef] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). Análisis de preferencias hacia las acciones de adaptación y mitigación al cambio climático: Perspectiva del agricultor en el noroeste de México. 3.2. Segunda Publicación: Farmers’ environmental perceptions and preferences regarding climate change adaptation and mitigation actions – towards a sustainable agricultural system in México Orduño Torres, M. A., Kallas, Z., & Ornelas Herrera, S. I. Farmers’ environmental perceptions and preferences regarding climate change adaptation and mitigation actions – towards a sustainable agricultural system in México. Land Use Policy, august 2020, (Q1- Factor de impacto 4.236). 31 Land Use Policy 99 (2020) 105031 Contents lists available at ScienceDirect Land Use Policy journal homepage: www.elsevier.com/locate/landusepol Farmers’ environmental perceptions and preferences regarding climate change adaptation and mitigation actions; towards a sustainable agricultural system in México T Miguel Angel Orduño Torresa,, Zein Kallasb, Selene Ivette Ornelas Herreraa a Institute for Research in Sustainability Science and Technology (IS-UPC), Polytechnic University of Catalonia, 08034 Barcelona, Spain Center for Research in Agrofood Economy and Development (CREDA-UPC-IRTA), Polytechnic University of Catalonia, Institute of Agrifood Research and Technology, 08860 Castelldefels, Spain b ARTICLE INFO ABSTRACT Keywords: Climate change Adaptation Mitigation Sustainable agriculture Environmental factors Analytical hierarchy process New ecological paradigm scale Climate change compromises sustainable agricultural development. It has deep economic, environmental, and social impacts, particularly on vulnerable rural regions in developing countries where agriculture constitutes the backbone of the economy. This study analyzes farmers’ preferences regarding the potential implementation of several mitigation and adaptation actions addressing climate change. Data were collected on 370 farmers in the “Valle del Carrizo” region of northwestern México. Using the Analytical Hierarchy Process (AHP) methodology, the farmers’ preferred mitigation and adaptation actions were identified and related to their stated attitudes regarding risks using the Multiple Price List (MPL) lotteries approach. Farmers’ environmental beliefs and perceptions as key means of understanding concepts of sustainability were related to their preferences. The use of less polluting machinery and investment in improving irrigation infrastructure were identified as the most preferred actions. Environmental opinions reviewed using the New Ecological Paradigm (NEP) scale allowed for the identification of the participants’ ecocentric and anthropocentric attitudes, highlighting the commitment of most farmers to the sustainable use of natural resources. Agricultural policies should be developed according to farmers’ preferences and behaviors. The design and implementation of measures and policy tools addressing climate change should be inclusive and developed at the micro-level considering farm and farmer typologies. 1. Introduction Climate change is one of the most significant challenges facing human society. The ways in which weather events are developing pose social, economic, and environmental risks and are raising more concern with the appearance of various unexpected phenomena such as floods, storms, droughts, and heat waves. Climate change refers to the varia tion of the earth’s climate generated either by natural causes or human actions that affect the variability of climatic parameters such as tem perature, rainfall, and drought (Gan et al., 2016). Climate change compromises sustainable agricultural development, which is based on three converging levels of environmental, economic, and social impact. Climate change is not only an environmental phe nomenon, but it has also deep economic and social consequences, especially for vulnerable developing countries, posing great challenges to their agricultural development and welfare (Tesfahunegn et al., 2016). The effects of climate change are closely related to a decline in ⁎ economic growth, complicating efforts to reduce poverty and to ensure the food security of marginalized local agricultural communities (López and Hernández, 2016). Agriculture is of great importance to the economic development of developing countries and constitutes the backbone of their economies by providing their populations with food, raw materials, and employ ment opportunities (Ogen, 2007). Socially, agriculture forms the basis for achieving food security, which basically depends on the eradication of extreme poverty and hunger (Von Braun et al., 2005). Agriculture is essential to community livelihoods in rural and marginal areas. In this context, agricultural policies and public intervention in rural commu nities are necessary tools that contribute to the reduction of poverty as part of an economic and social development approach (Croppenstedt et al., 2018). Climatic patterns are the most significant input factor for agri cultural production (Frutos et al., 2018), and their variability is closely related to output productivity. At the same time, the agricultural sector Corresponding author. E-mail addresses: miguel.angel.orduno@upc.edu (M.A. Orduño Torres), zein.kallas@upc.edu (Z. Kallas), selene.ivette.ornelas@upc.edu (S.I. Ornelas Herrera). https://doi.org/10.1016/j.landusepol.2020.105031 Received 21 January 2019; Received in revised form 22 June 2020; Accepted 18 August 2020 0264-8377/ © 2020 Published by Elsevier Ltd. Land Use Policy 99 (2020) 105031 M.A. Orduño Torres, et al. and animal farming in particular constitute an important source of greenhouse gas (GHG) emissions, which are closely related to climate change (Rivera and Di Paola, 2013). Agriculture in regions of Africa and Latin America is most vulnerable to climate change due to its geo graphic positioning and because local economies and populations rely heavily on agriculture activities for subsistence purposes, especially in rural and marginal areas (Ortiz, 2012). In the study region examined in the present work, climatic condi tions are extreme and have in recent years become even more atypical with high levels of precipitation occurring over short periods and with lower temperatures than normal recorded (Lara et al., 2017). Such patterns have affected levels of agricultural production and crop quality and jeopardized food security within the region and country. Ad ditionally, climate change projections associated with global warming establish temperature increases of 0.5 °C–1.0 °C for 2020 and of 2 °C–4 °C for 2080, variations in rainfall of + 10 % to -20 % by 2050, and a decrease in rainfall of 5%–30% by 2080 (Flores et al., 2012). Such patterns will increase vulnerability to flooding and other natural dis asters and lead to changes in water availability mainly affecting the agricultural and livestock sectors (López, 2000). Climate change is also related to societal development. Relationships between society, agriculture and economic development in rural areas are closely linked to the consequences of climate change (Valladolid, 2017; Maia et al., 2018). Currently, the effects of climate change in different regions are heterogeneous due to specific human activities and regional economic, climatic, and social characteristics (Frutos et al., 2018). Therefore, the implementation of strategies to adapt production in agricultural systems or mitigate effects of climate change on outputs must be implemented according to each region, farmers’ characteristics and farming activities (Aguiar and Cruz, 2018; López and Hernández, 2016). Climate change adaptation actions corresponds to initiatives and measures focused on reducing the vulnerability of natural and human systems to effects of actual or expected climate change (IPCC, 2014) or on reducing the likelihood of an object, person or system suffering negative impacts. Not considering the effects of climate change has negative implications for adaptation capacities, resulting in a more vulnerable situation that does not contribute to environmentally sus tainable agriculture (Wheaton and Kulshreshtha, 2017). Vulnerability is generally associated with levels of poverty within a region. Adaptation is intended to limit damage caused by current and projected climate change as much as possible (Aguiar and Cruz, 2018). With respect to climate change adaptation, no industry has more at stake than the agricultural sector (Lee et al., 2014). Traditional agricultural practices can be considered adaptation tools when applying improved, droughttolerant strategies while avoiding monoculture production (Altieri et al., 2015; Galindo et al., 2014). Mitigation actions, according to the FAO, are measures adopted to reduce greenhouse gas emissions and/or encourage the elimination of carbon through sinks. Climate change mitigation can be achieved by limiting or preventing the generation of greenhouse gas (GHG) emis sions and through activities that reduce their concentrations in the at mosphere (IPCC, 2014). To mitigate climate change, it will be necessary to reduce demand for energy and ensure that energy consumption is based on the use of low-carbon fuels. According to the two above de scribed concepts of adaptation and mitigation, it can be generalized that mitigation is responsible for addressing the causes of climate change while adaptation focuses on reducing the effects of climate change. Since farmers depend heavily on their crops, levels of pro duction positively or negatively affect (their income) their sustain ability, reinforcing the need to implement adaptation strategies. Adaptation strategies are key to improving the efficiency and pro ductivity of the agricultural sector (Di Falco et al., 2011) by reducing agricultural vulnerability to climate change. Adaptation activities can range from testing and introducing new more resistant crop varieties to building retaining walls and storm barriers to protect residents and property from flooding (O’Garra and Mourato, 2016). According to Khanal et al. (2018), adaptation actions with the greatest impacts on productivity are those related to soil and water management, which is followed by a change in the sowing ca lendar and in crop variety selection (Khanal et al., 2018). Specifically, a water management adaptation involves investment in the improvement in irrigation infrastructure, which results in more security in the availability of water for irrigation, in turn reducing dependence on rain cycles, allowing for the reduction of evapotranspiration, and thereby achieving more productivity with less water consumption. Similarly, the implementation of crop and variety changes or of changes in the sowing calendar as adaptation strategies ensures a higher level of production (Khanal et al., 2019). Climate change mitigation actions are necessary to ensure that long-term agricultural productivity and food security are not compromised, ensuring the sustainability of agri cultural production (Acquah, 2011). Through the implementation of mitigation strategies such as zero tillage methods, which allow for soil conservation as erosion decreases, it is possible to generate gains in food productivity (Di Falco et al., 2011). According whit the last, sustainable agriculture faces two main challenges: the total exploitation of natural resources and environ mental pollution (Hoang and Rao, 2010). The development of sus tainable agriculture can help address the impacts of climate change. Sustainable agriculture is based on the implementation of actions that help conserve environmental and economic resources such as water and land inputs (Bertoni et al., 2018). Sustainable agriculture involves the production of food and other inputs through farmers' efforts and in stitutional participation in the use of new technologies while preserving the environment and natural resources to meet current societal needs and guarantee a better quality of life without compromising the re sources of future generations (Mubiru et al., 2017). Therefore, understanding farmers’ views and perceptions regarding climate change and the actions that they consider most effective against its impacts is critical. In particular, the analysis of farmers’ preferences for different mitigation and adaptation actions can lead to the devel opment of more sustainable agricultural systems. Such preferences are also related to farmers’ views regarding environmental issues and to their ecocentric or anthropocentric beliefs. Environmental and ecolo gical beliefs and opinions are key factors in understanding sustain ability concept when related to agricultural activities (Reyna et al., 2018). Within this context, the objectives of this research were to identify the relative importance of several climate change adaptation and mi tigation actions related to agriculture activities in a marginal region in México in order to guide policy makers through the prioritized solu tions that contribute to the sustainability of agricultural systems. Furthermore, farmers’ attitudes, opinions, and beliefs towards the en vironment were evaluated in association with their preferences’ pat terns. The relation between farmers’ preference structures with their risk attitudes and their socioeconomic characteristics was also ana lyzed. 2. Materials and methods To reach the abovementioned objectives, several methodological approaches were applied.. The Analytical Hierarchy Process (AHP) was used to identify farmers’ preferences and to estimate the relative im portance (i.e. priorities) of different mitigation and adaptation actions. We also used an adapted form of the New Ecological Paradigm (NEP) Scale that was validated via factorial analysis (PCA) to identify pre dominant latent environmental dimensions. Using the Multiple Price Lists (MPL) method or “lotteries,” an alternative approach to expected utility risk elicitation, the farmers' stated risk attitudes were estimated. Finally, a heterogeneity analysis was carried out to relate framers’ preferences to actions against climate change effects based on their environmental and stated risk attitudes toward their farming activities. 2 Land Use Policy 99 (2020) 105031 M.A. Orduño Torres, et al. Fig. 1. Methodological research approach. Intercultural University of Sinaloa following the ethical principles of the Declaration of Helsinki and according to confidentiality rules and a privacy policy guaranteeing the security of the personal data of each participant. In addition to the above, each participant was informed of the survey’s focus and of how he/she should respond to questions and was asked to sign a consent form to participate in the study. Fig. 1 summarizes the methodological approach applied in this study. In the following section, more information on our theoretical background and empirical application is given. 2.1. The case study and sample of farmers The data was collected through the application of a face to face survey, corresponding to a representative sample of 370 farmers from an agricultural area identified as Irrigation District 076 (DR076) in northwestern Mexico (Fig. 2). The sample size was determined based on the formula of finite populations with a confidence level of 95 % and an error level of 4.99 % (Rojas, 2015). Data collection was carried out in a stratified manner according to farm sizes (large and small), farmers’ ages (young and old) and sex to represent both men and women within the sample using a quota sampling approach. The farmers completed semi-structured, face-to-face questionnaires from October to December 2017. The questionnaire included 108 questions and was divided into several blocks according to types of information collected. These were classified as 1) farmers’ preferences for climate change adaptation and mitigation actions, 2) environmental attitudes and opinions derived from the NEP scale, 3) stated risk attitudes derived from the MPL ap proach, and 4) farmers’ socio-economic features (Kallas et al., 2010) and farm characteristics (Kallas et al., 2012). Each farmer took approximately 40 min to answer the interview questions, and interviews were carried out with the support of students from the Autonomous Intercultural University of Sinaloa who were trained to deliver the survey. Before the interviews, the survey was reviewed and approved by the ethics committee of the Autonomous 2.2. Description of the AHP methodology The AHP method is a multicriteria analysis tool that was developed by Saaty at the end of the 1970s (Saaty, 2001). It allows for the im provement in decision-making processes, in turn generating added value in terms of knowledge (Moreno, 1998). It is important to high light that decision making should be understood as a methodical pro cess by which a person or group of people choose(s) between two or more alternatives with different quantitative or qualitative attributes to achieve an individual or common good that complies with previously conceived expectations (Moody, 1992). The AHP technique has been widely used in agricultural research mainly in analyzing farmers to establish priorities in decision making, resolve agrarian and environ mental problems and analyze marketing issues related to consumers’ preferences (Kallas and Gil, 2012; Ndamani and Watanabe, 2017; Aslam et al., 2018). The AHP method involves 3 main stages: 1) modeling, 2) assess ment, and 3) prioritization and synthesis. These stages form the methodological structure described below. Stage 1. Modeling The activities of this stage, which are described below, include 1) 3 Land Use Policy 99 (2020) 105031 M.A. Orduño Torres, et al. Fig. 2. Location of the study area. problem definition and 2) structuring a decision model in the form of a hierarchy. 2.3.2. A2. Change in crops Niggol and Mendelsohn (2008) noted that in Latin America, farmers use crops change as a way to adapt to climate change, especially where temperature and precipitation affect the selection of crops, crop yields, and incomes (Niggol and Mendelsohn, 2008). Changing cultivation methods is a good measure of adaptation, especially when it comes to reducing dependence on water resources, as is the case when less waterintensive crops are used, for instance (Moniruzzaman, 2015). 2.2.1. Problem identification and definition We found that there was a lack of information on farmers’ pre ferences in northern Mexico regarding climate change mitigation and adaptation as a normative framework in the establishment of public policies related to agricultural production to reduce effects of climate change. Accordingly, several alternative actions were evaluated from a literature review. Actions implemented to strengthen the resilience of food security systems to climate change at multiple levels were defined as measures of adaptation, and actions aimed at reducing greenhouse gas (GHG) emissions from agriculture were defined as mitigation measures while taking into account limitations inherent to the analyzed region (Mussetta et al., 2017). Identified adaptation and mitigation actions (criteria) representing the factors based on which the hierarchical analysis was carried out include: 2.3.3. A3. Introduce improved and resistant seeds Improved seeds can be used by farmers in different regions to adapt to climate change. Improved seeds, among their other characteristics, develop quickly; generate high yields; are drought, plague, and pest resistant; and are more resistant to flooding (Mohamed et al., 2018). 2.3.4. A4. Sowing calendar adaptation As a measure of climate change adaptation, the adaptation of the sowing calendar to changes at the start of the rainy season guarantees optimal growth scenarios and lower risks of drought in significant periods of planting evolution. On the other hand, the use of rainwater has greater utility and increases crop yields (Waha et al., 2013). 2.3. Adaptation measures 2.4. Mitigation measures 2.3.1. A1. Investment in improving irrigation infrastructure A lack of basic irrigation infrastructure restricts agricultural adap tation to climate change. Irrigation infrastructure and to a lesser extent temperature control techniques (greenhouses) facilitate adaptation to climate change by reducing climate dependence (Castells et al., 2018). 2.4.1. M1. Organic agriculture According to Xiaohong et al. (2011), organic farming uses new varieties of efficient and sustainable ecological technology and has created new ways to mitigate agroecosystem emissions through, for 4 Land Use Policy 99 (2020) 105031 M.A. Orduño Torres, et al. example, the use of bio-digesters and those that reduce water con sumption (Xiaohong et al., 2011) each cluster for each farmer k, from which the corresponding Saaty matrices are obtained (Âk), through which local weights of the identi ˆ fied elements are obtained Wí K according to the preferences of each farmer using the Row Geometric Mean Method (RGMM) (Kallas and Gil, 2012). 2.4.2. M2. Zero tillage management Zero tillage methods effectively mitigate climate change by enhan cing and/or maintaining organic matter in the soil, which lowers greenhouse gas emissions (Mangalassery et al., 2015) ˆ The estimation of priorities (Wí K ) was carried out using Super Decisions software [Super decision, 2018] designed for the im plementation of the AHP methodology. An example of results of pair wise comparison called judgments (âijk) for farmer k in cluster 2 re ferring to adaptation measures is shown in Table 3. All judgments (âijk) obtained from the pairwise comparison lead to the construction of a Saaty matrix for farmer k (Âk) with dimensions (n x n = 4 × 4) as follows: For the example shown in Table 3, the Saaty matrix is: 2.4.3. M3. Renewable energy use The agricultural sector can actively mitigate climate change by using manure as an alternative to fertilizers and by converting agri cultural crops and waste into energy to reduce reliance on non-re newable sources (e.g., through biomass production) (Liu et al., 2017). 2.4.4. M4. Use of less polluting and energy efficient machinery While greenhouse gas emissions are generally attributed to the en ergy sector due to the use of fossil fuels via agricultural machinery such as tractors, irrigation pumps, etc., the use of less polluting agricultural machinery can help mitigate impacts of climate change (Yue et al., 2017). a 1.1k a 2.1k a 3.1k a 4.1k Âk = a 1.2k a 2.2k a 3.2k a 4.2k a 1.3k a 2.3k a 3.3k a 4.3k a 1.4k a 2.4k a 3.4k a 4.4k Based on the Saaty matrix, the relative importance (i.e., the weights ˆ ˆ ˆ ˆ or priorities) of different actions W nk = W1k, ... Wik , ... Wnk are estimated using the RGMM: 2.4.5. Structuring a decision model as a hierarchy Our hierarchical scheme (Fig. 3) prioritizes main criteria (adapta tion and mitigation) and sub-criteria (actions) based on what is most accepted according to farmers’ preferences. Stage 2. Assessment This stage corresponds to the third phase in the empirical applica tion of the AHP: 3) model evaluation through paired comparisons of all elements of each cluster level (Fig. 3) using the verbal scale of paired comparisons proposed by Saaty (Table 1), from which the relative im portance of alternative actions is then estimated. For the upper cluster level, only one pairwise comparison is applied [n⋅(n-1)/2 = 2⋅(2−1)/2 = 1] on adaptation and mitigation actions. For each of the lower level clusters according to dimension n = 4 (4 al ternatives actions), 6 pairwise comparisons are used [n⋅(n-1)/2 = 4⋅ (4−1)/2 = 6], where each alternative of the hierarchy is compared to the remaining alternatives within its cluster at the same hierarchical level depending on the satisfaction it provides to the respondent (farmers). Pairwise comparisons were collected using the scheme out lined below (Table 2): Stage 3. Prioritization and synthesis This phase involves 4) synthesis to identify the best alternative and 5) the examination and verification of a decision that corresponds to the last two activities of the hierarchical analysis process from which priorities (i.e., the relative importance) are estimated. ˆ i=n Wik = n i=n i=1 ˆaijk The previously estimated weights are normalized to the unit. ˆw = 1 ik i=1 (1) (2) 2.4.7. Examination and verification of the decision As part of the verification stage, it is important to note that for each generated matrix, the Consistency Ratio (CR) of farmers’ answers was calculated according to corresponding mathematical expressions: CR=CI/RI; (3) where CI is the Consistence Index obtained as: CI = n max n (4) 1 where n = is the number of alternatives and max is the maximum value of components of the eigenvector obtained as: max = 2.4.6. Synthesis to identify the most preferred criteria For this activity, the joint prioritization of all sub-criteria proposed in the model to select the one that addresses a given problem is carried out; to this point, all comparisons must be drawn between elements of ˆ âijk wik i j (5) RI is the Random Index, which is obtained by multiple random extractions of the Saaty matrix of size n x n (Table 4). A value of CR lower than 10 % indicates satisfactory consistency for the pairwise comparisons (Siraj et al., 2015). The AHP is also Fig. 3. Decision hierarchy model and identification of clusters that form the decision hierarchy model. 5 Land Use Policy 99 (2020) 105031 M.A. Orduño Torres, et al. Table 1 Verbal scale used for paired comparisons (Saaty, 1997). Degree of importance Scale definition 1 3 5 7 9 2, 4, 6, 8 Both criteria are of the same importance. The two compared elements contribute equally to the fulfillment of the parent node. The preferred criterion is slightly more important than the other. The preferred criterion is moderately more important than the other. The preferred criterion is much more important than the other. The preferred criterion is significantly more important than the other. Judgments are made to define the relative importance of compared elements. considered a valid technique for the analysis of group decisions (Easley et al., 2000). Thus, to obtain an averaged aggregated of different mi tigation and adaptation measures for the sample, corresponding in ˆ The first identified component is referred to as anthropocentrism and was measured with affirmations focused on the supremacy of hu mans over nature. The second component, the ecocentric dimension, was measured with statements focused on the unbalanced state humans have created in nature. The third component reflects consciousness regarding the existence of a limit on nature related to resources of the biosphere. The fourth component measures confidence in human to manage natural resources correctly. The last component reflects per ceptions of infinite natural resources and thus humans’ indifference to their consumption given the presence of abundant natural resources. dividual weights (Wí k ) were aggregated across farmers to obtain a ˆ synthesis of weights for each set of criteria (Wi ). The aggregation was carried out using the Geometric Mean (GM) procedure, which is con sidered the most suitable method for aggregating individual priorities in a social collective decision-making context (Forman and Peniwati, 1998): wi = K k=K k=1 wik i (6) 2.6. Stated risk attitude: the lotteries approach The stated risk attitude level is related to human behavior, which is specific to each individual decision maker. Individuals prefer options that ensure more utility based on their risk preferences (Mejía, 2015; Brick et al., 2012; Galarza, 2009). Several methodological approaches have been developed to measure individuals’ stated risk attitudes and their relations to actions under a certain degree of uncertainty. The Multiple Price List (MPL) or “lotteries” have recently been used in agriculture based on the theory of the expected utility u (x) and strength of risk preferences v (x) with the “True Equivalent” used to measure attitudes toward risk (Pennings and Garcia, 2001; Jianjun et al., 2015; Orduño et al., 2019). The MPL method allows one to identify levels of risk tolerance or aversion through a set of questions posed to decision makers and in our case to farmers. The method ex amines 8 scenarios with different lottery pairs where one lottery option (option A or option B) is chosen (Drichoutis and Lusk, 2012; Brick et al., 2012). The level of risk aversion is based on the number of safe answers (option A) the interviewed farmer selects. A farmer who is risk tolerant selects a risky option (option B) for the first scenario. A farmer who is risk neutral selects option A for the first 3 scenarios and selects option B for the remaining scenarios from (4–8 scenarios) while an extremely risk averse farmer selects option A for all 8 scenarios (March et al., 2014). In the model, the safe option (option A) corresponds to a 100 % probability of succeeding, and the risky option (option B) corresponds to a 50 % probability of obtaining $100 and a 50 % probability of obtaining $0 (based on a coin toss) in all scenarios. Amounts provided by option A are progressively decreased across all 8 scenarios to the following amounts: $00, $75, $60, $ 50, $40, $30, $20, and $10. The experimental design structure of the risk elicitation question is illu strated in the questionnaire available in the supplementary file Q_1v2 (Question 35). 2.5. New ecological paradigm (NEP) scale According to Hawcroft and Milfont, environmental attitudes can be observed through psychological tendencies expressing positive or ne gative evaluations of the natural environment and that cannot be ob served directly and thus it must be inferred. Numerous tools allow one to measure environmental attitudes, among which three psychometric tools are highlighted: The Ecology Scale, The Scale of Environmental Concern and The New Ecological Paradigm. The first two scales refer to very specific environmental issues, while the NEP scale, which is the most widely used, allows one to measure general beliefs based on re lationships between humans and their environments (Hawcroft and Milfont, 2010). According to some studies, farmers’ beliefs regarding environmental issues can be measured using the NEP scale. This scale analyzes re lationships between subjects’ beliefs about themselves and nature. The scale reflects the ways in which humans conceptualize nature and in teract with it (Vozmediano and San Juan, 2005; Dunlap et al., 2000; Lezak and Thibodeau, 2016). In this study, farmers’ preferences regarding climate change adap tation and mitigation actions were analyzed in relation to their en vironmental beliefs measured through the NEP scale. Predominant la tent environmental dimensions of farmers could then be identified. The NEP scale was presented to farmers with an array of statements using a 9-point Likert type scale (Table 5). Individuals’ views of the environment can be revealed from their perceptions and attitudes. Using the NEP scale, an exploratory factorial analysis (Principal Component Analysis, PCA) was performed to iden tify the dimensionality that characterizes farmers by associating the scale’s items with several independent dimensions. The identified di mensions allowed us to define latent factors that are present in the participants’ environmental attitudes (Gomera et al., 2013). An ex ploratory factor analysis (PCA) was carried out with Varimax rotation and using the Statistical Package for the Social Sciences (SPSS, version 23.0). Before carrying out the factorial analysis, the Kaiser-Meyer-Olkin sample adaptation measure (KMOS) was applied (Table 6). Theoretically, according to Gomera et al. (2013) and Vozmediano and Guillen (2005), the application of factorial analysis should reveal five dimensions 1) a component related to anthropocentrism, 2) an ecocentric component, 3) limited consciousness, 4) a component re lated to human confidence in nature and 5) a last component related to perceptions of infinite natural resources. 2.7. Hypotheses analyzed Based on the above literature, the below hypotheses are tested: 1 H1: Farmers’ estimated preferences regarding climate change adaptation and mitigation (AHP) are related to their attitudes and opinions regarding the environment (NEP scale). 2 H2: Farmers’ preferences regarding climate change adaptation and mitigation (AHP) are related to their stated risk attitudes (MPL lotteries). 6 8 7 6 5 M1. Organic agriculture 9 8 7 M1. Organic agriculture 9 8 7 M1. Organic agriculture 9 8 7 M2. Zero tillage management 9 8 7 M2. Zero tillage management 9 8 7 M3. Use of renewable energy 9 8 7 5 5 5 5 5 5 6 6 6 6 6 6 M. Comparison of mitigation actions (cluster 3) A1. Investment in the improvement in irrigation infrastructure 9 8 7 6 5 4 A1. Investment in the improvement in irrigation infrastructure 9 8 7 6 5 4 A1. Investment in the improvement in irrigation infrastructure 9 8 7 6 5 4 A2. Change in crops 9 8 7 6 5 4 A2. Change in crops 9 8 7 6 5 4 A3. Introduce improved and resistant seeds 9 8 7 6 5 4 A. Comparison of adaptation actions (cluster 2) 9 A. Adaptation Measures Comparison of measures (cluster 1) Table 2 Paired comparisons included in the questionnaire. 7 4 4 4 4 4 4 4 2 2 2 2 3 3 3 3 3 3 3 3 3 2 3 3 2 3 3 2 2 2 2 2 2 1 1 1 1 1 1 2 2 3 4 1 1 1 1 1 1 5 5 5 5 5 5 5 M2. Zero tillage management 2 3 4 5 M3. Use of renewable energy 2 3 4 5 M4. Use of less polluting and energetically efficient machinery 2 3 4 5 M3. Use of renewable energy 2 3 4 5 M4. Use of less polluting and energetically efficient machinery 2 3 4 5 M4. Use of less polluting and energetically efficient machinery 2 3 4 5 A2. Change in crops 2 3 4 A3. Introduce improved and resistant seeds 2 3 4 A4. Adaptation of the sowing calendar 2 3 4 A3. Introduce improved and resistant seeds 2 3 4 A4. Adaptation of the sowing calendar 2 3 4 A4. Adaptation of the sowing calendar 2 3 4 1 M. Mitigation Measures 6 6 6 6 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 8 8 8 8 8 8 9 9 9 9 9 9 9 9 9 9 9 9 9 M.A. Orduño Torres, et al. Land Use Policy 99 (2020) 105031 Land Use Policy 99 (2020) 105031 M.A. Orduño Torres, et al. Table 3 Example of the calculation of weights based on paired comparisons corresponding to cluster 2, adaptation (A) attributes for individual k = 1. Functions A1 A2* A1* A3* A1* A4* A2* A3* A2* A4* A3* A4* Judgment (âij) 9 â12 = 9 â21 = 1/9 9 â13 = 9 â31 = 1/9 9 â14 = 9 â41 = 1/9 2 â23 = 2 â32 = 1/2 2 â24 = 2 â42 = 1/2 â34 = 1/2 2 â43 = 2 A1. Investment in the improvement in irrigation infrastructure. A2. Change in crops. A3. Introducing improved and resistant seeds. A4. Adaptation of the sowing calendar. action was investment in the improvement in irrigation infrastructure (17.57 %). The changing of crops was deemed the third most preferred action, accounting for (17.30 %) of the farmers´ answers. Zero tillage management was the fourth most preferred action (16.22 %). The use of renewable energy was the least preferred option and was selected by 5.95 % of the farmers. Table 4 Values of the random consistency index (RI) based on the size (n) of the matrix (Saaty, 1994). n 1 2 3 4 5 6 7 8 9 10 RI 0.00 0.00 0.58 0.90 1.12 1.24 1.32 1.41 1.45 1.49 3.2. H1: Relations between environmental attitudes and farmers’ preferences for climate change adaptation and mitigation actions 3 H3: Farmers’ preferences regarding climate change adaptation and mitigation (AHP) are related to socioeconomic and farm character istics. According to the results of our first PCA applied to items of the NEP scale, with a KMOS of 0.747 indicating that the reduction in di mensionality is relevant, the variability explained by the model with 5 components is 67.11 %. For this PCA, the first component included items 10, 11, 12, 13, 14 and 16 on ecocentric attitudes. The second component grouped items 2, 3, 4, 5, 6 and 8 related to an anthropo centric attitude, among which item 8 is negatively related. The last three components correspond to one or no significant item with rela tively low percentages of explained variance. Furthermore, items 1 and 7 do not contribute significantly to any component. Another PCA was then carried out on the 12 items related to the anthropocentric and ecocentric dimensions. In this case, the KMOS test generated a result of 0.754 and the variability explained by the factorial analysis of the two 2 components was measured as 52.98 %. This reduction in the NEP scale allowed for a better definition of components by clearly differentiating the regrouping of item 8 with attitudes related to an ecocentric attitude. All the above hypotheses were tested through an analysis of var iance using the ANOVA method. Preferences regarding climate change adaptation and mitigation were related to the two main latent factors (ecocentric and anthropocentric) defined from the NEP via factorial analysis (PCA). 3. Results 3.1. Farmers’ preferences for adaptation and mitigation actions The estimated average weighting of adaptation and mitigation ac tions based on the AHP is presented in Fig. 4. The results reflect farmers’ prioritization of different ways to face the impacts of climate change on their activities. Weights (i.e., relative importance) were es timated at the local (i.e., for each cluster from local weights) and global levels (i.e., for the hierarchy level from global weights). The estimated average weights show that mitigation actions were deemed the most important options with a higher relative relevance of 58.18 %. For each farmer we then estimated actions deemed the most preferred (Fig. 5). According to the farmers´ preferences, which were identified from the global weight of each individual farmer, the use of less polluting machinery was the most preferred action. The second most preferred 4. Ecocentric and anthropocentric environmental attitudes The farmers’ distribution according to the reduced NEP scale can be observed in Fig. 6. Two main relevant behaviors are identified: ecocentric and anthropocentric environmental attitudes. Accordingly, each farmer is positioned within two principal axes representing the main factors. Four potential positions are specified in four quadrants: quadrant (+ eco, +anthro) corresponds to farmers agreeing with both attitudes Table 5 . Statements of the New Ecological Paradigm Scale. Fully disagree 1 Strongly disagree 2 Moderately disagree 3 Slightly disagree 4 Neutral 5 1 The global ecological crisis has been exaggerated 2 The balance of nature supports the impacts of industrialized countries 3 Humans may be able to control nature 4 Human ingenuity will ensure that the earth will not become uninhabitable 5 Humans were created to dominate nature 6 Humans have the right to modify the environment and adapt it to their needs 7 Human interference in nature will have disastrous consequences 8 Plants and animals have the same rights to exist as human beings 9 Humans have seriously damaged the environment 10 The balance of nature is delicate and easily alterable 11 If things continue as they have, we will soon experience a significant ecological catastrophe 12 We are approaching the earth’s limit in terms of sustaining the global human population 13 The earth has limited resources 14 Despite our special abilities, human beings are still subject to the laws of nature 15 The land has abundant resources, and we just need to learn to exploit them 16 Sustainable development must apply a balanced approach that controls industrial growth 8 Slightly agree 6 Moderately agree 7 Strongly agree 8 Fully agree 9 Land Use Policy 99 (2020) 105031 M.A. Orduño Torres, et al. Table 6 Grouped reduced NEP scale according to each item’s contribution to the new components. New ecological paradigm scale items Factor 1 Ecocentric Factor 2 AnthropoCentric 11. If things continue as they have, we will soon experience a significant ecological catastrophe 10. The balance of nature is delicate and easily alterable 14. Despite our special abilities, human beings are still subject to the laws of nature 12. We are approaching the earth’s limit in terms of sustaining the human population 16. Sustainable development must apply a balanced approach that controls industrial growth 8. Plants and animals have the same rights to exist as human beings 13. The earth has limited resources 3. Humans may be able to control nature 4. Human ingenuity will ensure that the earth will not become uninhabitable 5. Humans were created to dominate nature 6. Humans have the right to modify the environment to adapt it to their needs 2. The balance of nature supports the impact of industrialized countries Extraction method: PCA. Rotation method: Varimax standardization with Kaiser. Total explained variance 0.81 0.78 0.69 0.63 0.63 0.59 0.52 0.00 0.06 0.04 0.04 0.16 −0.08 0.06 0.16 0.13 0.26 −0.18 0.46 0.80 0.77 0.75 0.71 0.70 52.98 % Fig. 4. Average relative relevance weights determined by AHP analysis according to farmers’ opinions (WA: local weight of adaptation measures group, WM: local weight of mitigation measures group, WLA: local weight of a specific (n) adaptation measure, WLM: local weight of a specific (n) mitigation measure, WGA: global weight of a specific (n) adaptation measure and WGM: global weight of a specific (n) mitigation measure). in favor of nature and in favor of humans’ priorities in using natural resources. This space may represent inconsistencies between farmers regarding their attitudes towards the environment. For this same context, quadrant (-eco, -anthro) may also reflect farmers’ inconsistencies regarding their stated opinions towards the environment, highlighting their disagreement with views that place nature above humans and with those that place humans above nature. Quadrant (- echo, + anthro) refers to farmers who agreed with anthropocentric attitudes but disagreed with ecocentric views, thus representing farmers who believe that humans are above nature and that there is therefore no limit to the use of natural resources. The protection of nature in this case should only be aim at enhancing the quality of human life. Finally, quadrant (+eco, - anthro) groups farmers who agreed with ecocentric attitudes and showed disagreement with anthropocentric behaviors. These farmers believe that nature should be protected be cause it is vulnerable to the actions of humans and that humans must limit its use and perform actions that support nature. The farmers’ distribution on the abovementioned four quadrants shows that the majority (39 %) exhibited a clearly positive ecocentric attitude (+ eco, - anthro), highlighting positive views of the environ ment in the studied region. However, 27 % of the farmers exhibited a 9 Land Use Policy 99 (2020) 105031 M.A. Orduño Torres, et al. Fig. 5. Farmers’ preferences for climate change adaptation and mitigation actions. Fig. 6. Farmers’ distributions on the reduced NEP scale, ecocentric and anthropocentric dimensions, and relations to farmers´ preferences for climate change adaptation and mitigation actions. +eco denotes that farmers agree with ecocentric attitudes, -eco denotes that farmers disagree with ecocentric attitudes, +anthro denotes that farmers agree with anthropocentric attitudes, and -anthro denotes that farmers disagree with anthropocentric attitudes. clear anthropocentric attitude (- echo, + anthro) and an interest in protecting nature only if for a clear economic benefit. The remaining farmers exhibited less clearly defined opinions regarding the environ ment where 15 % exhibited negative views toward ecocentric and anthropocentric attitudes (- eco, - anthro) while 19 % exhibited positive views toward ecocentric and anthropocentric attitudes (+ eco, + an thro). The two abovementioned factors are related to farmers’ preferences 10 Land Use Policy 99 (2020) 105031 M.A. Orduño Torres, et al. towards mitigation and adaptation actions obtained from the AHP. The results (Fig. 7) show that the ecocentric and anthropocentric dimen sions are closely related to the farmer’s preferences. The mitigation and adaptation actions presented in Fig. 7 are ordered according to their relative importance as discussed in Fig. 6. An interpretation of the re sults shown in Fig. 7 must be carried out horizontally by comparing the relative importance (%) of each action across the four quadrants. The most preferred climate change adaptation and mitigation action (the use of less polluting and energetically efficient machinery, M4) was principally selected by farmers who exhibited a positive view of the environment (+eco, -anthro). The remaining mitigation and adaptation actions were also more important for farmers exhibiting more eco centric views of the environment (+eco, -anthro). As an exception, one action (to introduce improved and resistant seeds, A3) was preferred more by farmers that do not exhibit a clear attitude toward the environment (+eco, +anthro). The results listed vertically in Fig. 7 show that farmers with the most ecocentric attitudes (+ eco, -anthro) exhibited the strongest pre ferences for the use of renewable energy (M3). Approximately, 68 % of the producers had received a subsidy mainly used (60 %) to cover operating costs while 12.3 % of farmers had ap plied it to invest in agricultural equipment and technology. Most of the farmers (63 %) do not usually use any type of agricultural insurance. Most of the participants owned their agricultural land (79 %), and the main products grown included wheat (29 %), alfalfa (24 %) and soy beans (9.73 %). Socioeconomic characteristics measured related to preferred miti gation and adaptation actions included the following: adopting con tracted agricultural insurance, having credit for a farming land tenure regime, belonging to an agricultural association, selection of crops, and farmer’s age and sex. The results for these variables show that farmers without crop in surance prefer the “change in crops” measure, while those with in surance prefer “the use of less polluting and energetically efficient machinery” to reduce the impacts of climate change. On the other hand, framers with crop insurance have less concerns regarding the impacts of climate change and thus exhibit a preference towards other actions that principally reduce negative effects on the environment. Farmers with credit for farming activities and agricultural insurance and belonging to an agricultural association prefer “the use of less polluting and energetically efficient machinery” and grow onions, chili peppers, corn, soybeans, sorghum, and triticale. Furthermore, farmers without credit for farming activity and with private property under a land tenure regime who grow sweet potatoes prefer to increase in vestment in the improvement in irrigation infrastructure. Mitigation action “zero tillage management” was preferred by farmers without credit for farming activity, who do not belong to an agricultural association and principally grow watermelon and cartamo. Finally, farmers under 40 years of age prefer “investment in the improvement in irrigation infrastructure,” farmers 40–60 years of age prefer the “change in crop” approach, and farmers over 60 years of age prefer “zero tillage management.” 4.1. H2: stated risk attitudes and farmers’ preferences for climate change adaptation and mitigation actions The MPL results regarding stated risk attitudes show that 51.35 % of the farmers are risk averse, 7.57 % are neutral, and 41.08 % are risk tolerant. The heterogeneity analysis shows that the stated risk attitudes and farmers´ preferences for adaptation and mitigation actions are not clearly related. Through the analysis conducted, no significant re lationship was found between preferences for adaptation and mitiga tion actions and the stated risk level, though it is clearly related to other socioeconomic and management variables for farmers. 4.2. H3: Farmers’ preferences for climate change adaptation and mitigation actions and their socioeconomic characteristics 5. Discussion Regarding the socioeconomic characteristics of the sample, most of the farmers surveyed were between 41 and 60 years of age (52 %), followed by farmers over 60 years of age (28.38 %) and those under 41 years of age. Only 11 % of the agricultural producers were women, and the average number of family members was recorded as 3.78. Our analysis of socioeconomic characteristics also shows that 76 % of the participants’ incomes are generated from agricultural activities. 5.1. Farmers’ preferences for adaptation and mitigation actions Overall, the above results show that farmers in the study region prefer to implement mitigation actions to address climate change. These results are in agreement with those obtained by Bragado (2016), who found that mitigation actions are prioritized within the Fig. 7. Farmers’ distribution by preferences according to a combination of their positive or negative views of ecocentric and anthropocentric attitudes (4 quadrants). 11 Land Use Policy 99 (2020) 105031 M.A. Orduño Torres, et al. agricultural sector in addressing climate change effects. The most preferred action among the studied farmers involves the “use of less polluting machinery,” which indicates that public policy decisions should focus on promoting the use of less polluting and highly efficient agricultural machinery. This outcome was also proposed by Xu and Lin, who recommend that local governments encourage the use of energy efficient, less polluting agricultural machinery to support en vironmentally friendly production (Xu and Lin, 2017). Due to water scarcity, which it is becoming more frequent in the studied region, water management agencies have been forced to fre quently restrict volumes and periods of water use for irrigation, sub jecting crops to water stress (Ojeda et al., 2012) and causing farmers to prefer investment in improving irrigation infrastructure. Investment in irrigation infrastructure increases water use efficiency (Nelson et al., 2009a, 2009b) and may lead to a high degree of water loss. It is worth mentioning that in the presence of poor irrigation infrastructure, more than 55 % of water used is wasted (Sifuentes et al., 2015). Crop change (polyculture) methods exhibit more stability with less loss of productivity during drought seasons because they allow crops to reach acceptable levels of productivity even under unusual climatic conditions and environmental stress. Crop change can ensure a certain level of productivity in the midst of climate change. The approach can also address future social and economic needs as Altieri and Nicholls indicate (Altieri and Nicholls, 2009), corroborating our finding that farmers favor such actions third in terms of their preferences. Alternative zero tillage management was identified as the fourth most preferred mitigation strategy among farmers in the study region. Lau, Jarvis and Ramírez (2011) and Nichols and Altieri (2013) have also advocated for zero tillage as a feasible mitigation action (Lau et al., 2011; Altieri and Nicholls, 2013). All the above actions are closely related to economic benefits at the farm level. The adoption of less polluting and efficient machinery re duces fuel oil consumption and thus reduces production costs. Investment in irrigation infrastructure increases the productivity and quality of crops, optimizes the use of water, and decreases water waste (Nelson et al., 2009a, 2009b and Khanal et al., 2019). Crop changes increase productivity and decreases costs due to a lesser use of fertili zers and agrochemicals, which positively affects farm productivity (Moniruzzaman, 2015 and Khanal et al., 2018). The adoption of zero tillage management reduces production costs, as it lowers tilling labor costs and may reduce the use of chemicals and phytosanitary methods. Zero tillage methods are usually related to organic agriculture, which may also increase the price of products (Kallas et al., 2010). The use of renewable energy was preferred least by the farmers corroborating studies showing the need for strong investment to encourage the use of renewable energy facilities that may mitigate climate change (Kung and McCarl, 2018). In general terms, farmers prefer options that minimize the impacts of climate change while at the same time providing them a perceived benefit in the short run at the farm level. nature should be protected showed the strongest preference for the use of renewable energy and mitigation actions to face climate change. This group clearly exhibited the strongest concerns regarding the environ ment and a clear tendency toward using more environmentally friendly technology (Hajjar and Kozak, 2015). 5.3. H2: stated risk attitudes and farmers’ preferences for climate change adaptation and mitigation actions Our risk level results show that most of the studied farmers were risk averse. This is at first unexpected, as most of the studied farmers do not use agricultural insurance. However, our findings are in line with those of Jianjun et al. (2015), who used MPL and found an unclear relation between risk attitudes and preferences for climate change adaptation and mitigation (Jianjun et al., 2015). According to Palm (1998), most risk-averse individuals tend to take preventive and protective actions against potential damages (López and De Paz, 2007). Farmers in our study region were found to be mostly risk averse, which would imply that they have a strong willingness to carry out actions in favor of reducing the effects of climate change through adaptation or mitigation actions. The non-significant relationship found between preferences for adaptation and mitigation actions and the stated risk level could be explained by the fact that all actions were identified by farmers as protective measures against potentially negative impacts of climate change. Preferences for adaptation and mitigation measures among farmers in the study region are also related to other variables con cerning farmers’ and farm characteristics and farmers’ decisions made in relation to their activities (Orduño et al., 2019). 5.4. H3: Farmers’ preferences for climate change adaptation and mitigation actions and their socioeconomic characteristics Our results show that farmers without crop insurance preferred the “change in crops” adaptation strategy, while those with insurance preferred “the use of less polluting and energetically efficient ma chinery.” This result may be attributed to the fact that a change in crops increases productivity and thus insures farmers’ incomes against im pacts of climate change. This preference affords farmers confidence in terms of having enough income to support their planting commitments (Altieri and Nicholls, 2009). Our findings show that farmers who do not need credit for their agricultural activities and who grow potatoes prefer “investment in im proving irrigation infrastructure,” which may be related to the fact that potato crops are very sensitive to a lack of water (FAO, 2008). These preference patterns show that farmers are more concerned with using water solution technologies to reduce the impacts of climate change in the region. This same outcome was found for farmers under 40 years of age, showing that young individuals are more sensitive to water use and waste (Rodríguez and Jiménez, 2014). Farmers aged 40 to 60 years in stead prefer the “change in crop” approach, which may be linked to an interest in ensuring economic benefits. Finally, farmers over 60 years of age prefer “zero tillage management,” which could be associated with farmers’ experience. The “zero tillage management” approach is also preferred by farmers who grow watermelon and cartamo and who do not have credit for their farming activities. This outcome could be related to the fact that watermelon and cartamo do not require an extensive land preparation, thus rendering zero tillage methods a viable mitigation option (Moreno et al., 2013; Valdez et al., 2012). 5.2. H1: Relations between environmental attitudes and farmers’ preferences for climate change adaptation and mitigation actions Regarding farmers’ environmental attitudes, which are described by Gomera et al. (2013) and Reyna et al. (2018) as ecocentric and anthro pocentric environmental attitudes, and regarding farmers’ preferences to mitigate or adapt to climate change, the most preferred action, “the use of less polluting and energetically efficient machinery,” was selected by farmers with positive attitudes toward the environment. As Hajjar and Kozak (2015) argue, ecocentrics might be interested in using more environmentally sustainable technologies, while farmers without clear views on the environment prefer “introducing improved and resistant seeds.” For this adaptation measure, farmers may seek to enhance their economic benefits through the implementation of a simple mitigation or adaptation action without considering positive or negative effects on the environment. Ecocentric farmers believing that 6. Conclusions This study contributes to the literature by furthering available knowledge that can inform policy makers regarding support and sub sidies related to agricultural production that better meet framers’ needs and preferences. This may enhance the effectiveness of policy measures 12 Land Use Policy 99 (2020) 105031 M.A. Orduño Torres, et al. by stimulating preferred actions that improve farmers’ social and eco nomic welfare. It may also guide current public support to prioritize measures that promote the development of more sustainable agriculture activities at regional and national levels. At the methodological level, this paper contributes to the few studies jointly using the AHP in re lation to farmers’ preferences with the NEP scale and MPL risk ap proach, particularly in reference to México. To effectively face the impacts of climate change on agriculture implies the implementation of mitigation and adaptation actions ac cording to farmers’ interests and preferences. In general terms, farmers tend to prefer adaptation actions or mitigation actions because the former are perceived to offer benefits sooner when adopted. Farmers with ecocentric attitudes exhibited a greater willingness to adopt measures against climate change, while those with anthropocentric views principally exhibited stronger preferences for activities related to improvements in their productivity. Through the Analytical Hierarchy Process, farmers were found to prioritize actions that implicitly provide economic benefits over the short run. The use of efficient, less polluting machinery was identified as one of the best alternative options not only due to its positive impacts on the environment but also due to its economic benefits in terms of reducing energy costs at the farm level. Our results show that farmers’ preferences for mitigation and adap tation actions are closely related to the types of crops cultivated. Investment in improving irrigation infrastructure as an adaptation activity was widely accepted by farmers with water availability issues who grow sweet potatoes. This adaptation action helps farmers optimize their water use and address water availability issues in the region by increasing their productivity and limiting the water waste. Adopting a change in crops grown as an adaption action was also preferred by farmers who grow sorghum. Also, a preference for the zero tillage mitigation approach was found to be related to watermelon and cartamo cultures. Agricultural public policy decisions must consider farmers’ pre ferences towards mitigation and adaptation actions when designing and implementing measures that ensure sustainable agriculture. Policy tools and interventions must be inclusive and developed at the micro-level based on farm typologies, and crop diversity must be encouraged. Declaration of Competing Interest The authors report no declarations of interest. Acknowledgments The National Council of Science and Technology (CONACYT) is acknowledged for supporting this research. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.landusepol. 2020.105031. References Acquah, H.D., 2011. Farmers perception and adaptation to climate change: a willingness to pay analysis. J. Sustainable Dev. Africa 13 (5), 150–161. 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Author statement I am writing to return the 3th revised version of the Manuscript identified as LUO_2019_108 entitled: “Farmers’ environmental percep tions and preferences regarding climate change adaptation and miti gation actions – towards a sustainable agricultural system in México”, for further consideration in Land Use Policy Journal. We have made all changes according to the reviewers’ suggestions and comments. In the revised manuscript we kept the track control activated, so you can see all changes made on last sent version. Attached, you can see the revised manuscript and a set of responses to the reviewer’s comments. We appreciate your willingness to consider this new revision. While we hope these changes have alleviated the referee concerns, we stand ready to make additional changes if needed. Funding This research received no external funding. 13 Land Use Policy 99 (2020) 105031 M.A. Orduño Torres, et al. environmental beliefs from the NEP scale. 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Sustainability, 11(12), 3291(Q2- Factor de impacto 2.592) 32 sustainability Article Is Technical Efficiency Affected by Farmers’ Preference for Mitigation and Adaptation Actions against Climate Change? A Case Study in Northwest Mexico Miguel Angel Orduño Torres 1, * , Zein Kallas 1, * , Selene Ivette Ornelas Herrera 1 Bouali Guesmi 2,3 1 2 3 * and Institute for Research in Sustainability Science and Technology (IS-UPC), Polytechnic University of Catalonia, 08034 Barcelona, Spain; selene.ivette.ornelas@upc.edu Center for Research in Agrofood Economy and Development (CREDA-UPC-IRTA), Polytechnic University of Catalonia, Institute of Agrifood Research and Technology, 08860 Castelldefels, Spain; bouali.guesmi@upc.edu University of Carthage, Mograne Higher School of Agriculture, LR03AGR02 SPADD, Zaghouan 1121, Tunisie Correspondence: miguel.angel.orduno@upc.edu (M.A.O.T.); zein.kallas@upc.edu (Z.K.) Received: 25 April 2019; Accepted: 10 June 2019; Published: 14 June 2019 Abstract: Climate change has adverse effects on agriculture, decreasing crop quality and productivity. This makes it necessary to implement adaptation and mitigation strategies that contribute to the maintenance of technical efficiency (TE). This study analyzed the relationship of TE with farmers’ mitigation and adaptation action preferences, their risk and environmental attitudes, and their perception of climate change. Through the stochastic frontier method, TE levels were estimated for 370 farmers in Northwest Mexico. The results showed the average efficiency levels (57%) for three identified groups of farmers: High TE (15% of farmers), average TE (72%), and low TE (13%). Our results showed a relationship between two of the preferred adaptation actions against climate change estimated using the analytical hierarchy process (AHP) method. The most efficient farmers preferred “change crops,” while less efficient farmers preferred “invest in irrigation infrastructure.” The anthropocentric environmental attitude inferred from the New Ecological Paradigm (NEP) scale was related to the level of TE. Efficient farmers were those with an anthropocentric environmental attitude, compared to less efficient farmers, who exhibited an ecocentric attitude. The climate change issues were more perceived by moderately efficient farmers. These findings set out a roadmap for policy-makers to face climate change at the regional level. Keywords: technical efficiency; adaptation and mitigation preferences; climate change perception; environmental attitudes; farmers’ risk attitude 1. Introduction and Objectives For a growing number of researchers and policy-makers, the sustainable development of present and future societies, depends on paying immediate attention to the environmental problems related to climate change [1,2]. The uncertainty of changes generated by this phenomenon exposes the environment to high vulnerability [3]. Article 1 of the United Nations Framework Convention on Climate Change (UNFCCC) states that climate change is “attributed directly or indirectly to human activity that alters the composition of the global atmosphere and that adds to the natural variability of the climate observed during comparable periods of time” [4]. There are different strategies to address the adverse effects and impacts of climate change, and these can be realized by adopting two Sustainability 2019, 11, 3291; doi:10.3390/su11123291 www.mdpi.com/journal/sustainability Sustainability 2019, 11, 3291 2 of 15 approaches: Adaptation and mitigation actions. Agriculture is one of the most sensitive sectors to climate change. Any variation in climate systems has a significant potential impact on productivity and on the processes related to farming activities [5]. Therefore, adaptation and mitigation actions can be implemented as differentiation strategies to reduce the risk of food insecurity [6] and production variability [7]. Adaptation is the ability to adjust to the impacts of climate change in the short term of natural or human systems [8]. Mitigation refers to actions aimed at reducing greenhouse gas emissions, with agriculture being an important source of the generation of these emissions [9]. Agriculture is of vital importance for the development of countries, especially developing nations, where the economy and family subsistence are based on food production and agricultural activities [10], given that in countries with lower incomes, 63% of the economically active population are employed in agriculture, while in developed countries only 3% are engaged in the agricultural sector [11]. Therefore, identifying the most preferred adaptation and mitigation actions turns out to be highly valuable. These preferences not only depend on farming activities, regions, and farmers’ characteristics [12], but also could be related to technical efficiency (TE) at the farm level [13]. Agricultural TE implies obtaining the maximum production or output by using the minimum resources or input [14]. TE can be positively or negatively affected [15,16] by farmers’ decisions and preferences when adopting adaptation and mitigation actions. The agricultural TE level is related to the type of irrigation, sowing calendar, quality of seeds, water infrastructure, technology and machinery, quality of the land, and fertilizer use. It can also be related to farmers’ risk and environmental attitudes, preferences for investment decisions, and socioeconomic characteristics, among other variables [16,17]. In this context, it is also assumed that TE at the farm level can be related to farmers’ perception of climate change [18] and socioeconomic characteristics, among other variables. The aim of this study is fourfold: First, to measure the level of technical efficiency using the parametric stochastic production frontier (SPF) method in order to identify its relationship to farmers’ stated preferences for several climate change adaptation and mitigation actions, using the analytical hierarchy process (AHP). We seek to identify the direction and magnitude of the relationship between TE and farmers’ preferences [13,19]. The second objective is to analyze the relationship between the level of TE and farmers’ environmental attitude defined in two main dimensions, anthropocentric and ecocentric, using the New Ecological Paradigm (NEP) scale. The aim is to identify whether agricultural producers at a higher level of efficiency have an anthropocentric attitude [20]. The third objective is to establish whether there is a relationship between the level of TE and the stated risk attitude estimated using the multiple price list (MPL) method, also known as the lottery approach. The aim is to identify whether the most efficient farmers are risk-tolerant [21]. The fourth objective is to analyze the relationship between TE and the farmers’ perception of climate change. The aim is to explore whether farmers who have a greater perception of the variation of climate change are more technically efficient [22]. The case study of the irrigation district DR076 belongs to the state of Sinaloa, the leading nationwide in grains, vegetables, and fruit production that supply several national and international markets. According to the FAO (2011), Sinaloa was considered the granary of Mexico, after its crops production accounted for 75% of the country’s production. Given that in the study region there is no literature about TE at the farm level and its relationship with farmers’ preferences when it comes to mitigation and adaptation actions, as well as with their environmental attitudes and risk behavior, this research aims to provide new knowledge that may help policy-makers to identify more effective strategies, involving stakeholders in creating agricultural and rural development policies. Climate change is negatively affecting current agricultural systems, especially the rural areas in the developing countries due to their economic vulnerability and dependency on farming activities. The studied region shows similar features and characteristics with other agricultural regions in Mexico and agricultural systems worldwide. These similarities can be characterized in terms of the related adverse effects of climate change as well as farmers’ characteristics and crops production systems. The results of this study provide evidence that support the idea that farmers’ preferences for the Sustainability 2019, 11, x FOR PEER REVIEW 3 of 15 related adverse effects Sustainability 2019, 11, 3291of climate change as well as farmers’ characteristics and crops production 3 of 15 systems. The results of this study provide evidence that support the idea that farmers’ preferences for the adaptation and mitigation actions against climate change is related to their technical adaptation andpreferences’ mitigation actions against climate change is related to their These efficiency. These patterns can provide the decision makers withtechnical valuableefficiency. information preferences’ patterns can provide the decision makers with valuable information that allow them to that allow them to identify effective policy measures replicable in other similar farming systems. identify effective policy measures replicable in other similar farming systems. Description of the Study Area Description of the Study Area Irrigation District DR076, Valle del Carrizo, is located in the state of Sinaloa, Mexico, bordered Irrigation District DR076, Valle del Carrizo, is located in the state of Sinaloa, Mexico, bordered on the north by the state of Sonora, on the south by the Sierra Madre Occidental, and on the west by on the north by the state of Sonora, on the south by the Sierra Madre Occidental, and on the west the Gulf of California. Its geographic location is at latitude 26°05’ north and longitude 108°53’ west, by the Gulf of California. Its geographic location is at latitude 26◦ 050 north and longitude 108◦ 530 including part of the municipalities of Ahome and El Fuerte [23]. Its main economic activity is crop west, including part of the municipalities of Ahome and El Fuerte [23]. Its main economic activity is and livestock production; cereals such as wheat and corn are the most important crops, at 76% and crop and livestock production; cereals such as wheat and corn are the most important crops, at 76% 8% of total cultivated area, respectively [24]. The DR076 irrigation district is characterized by an and 8% of total cultivated area, respectively [24]. The DR076 irrigation district is characterized by an extreme climate, in which atypical conditions have been highlighted in recent years, with high levels extreme climate, in which atypical conditions have been highlighted in recent years, with high levels of precipitation in short periods of time as well as low temperatures [25]. This has repercussions on of precipitation in short periods of time as well as low temperatures [25]. This has repercussions on the the technical efficiency of crop production and represents a threat to the food security of the region technical efficiency of crop production and represents a threat to the food security of the region and the and the country [26]. country [26]. 2. Materials andand Methods 2. Materials Methods 2.1.2.1. Survey Data Survey Data This study reports on on thethe findings of aofsurvey carried outout with 370370 agricultural producers from This study reports findings a survey carried with agricultural producers from DR076, whose sample size was determined based on the formula of finite populations, with a DR076, whose sample size was determined based on the formula of finite populations, with a confidence confidence level of 95% and a confidence interval of 4.99% [27]. The survey was designed with an level of 95% and a confidence interval of 4.99% [27]. The survey was designed with an interdisciplinary interdisciplinary perspective, including social, and demographic aspects. a perspective, including economic, social,economic, and demographic aspects. It included a wideItsetincluded of questions wide set of questions grouped according to the farmers’ socioeconomic characteristics, grouped according to the farmers’ socioeconomic characteristics, characteristics of their agricultural characteristics theirofagricultural and preference the types ofregarding crops [28], their preference regarding or land and theoftypes crops [28], land and their theand implementation of mitigation theadaptation implementation of mitigation or adaptation actions related to reducing the effects of actions related to reducing the effects of climate change using the AHP technique.climate Farmers’ change using the AHP technique. Farmers’ attitudes and opinions that define their behavior toward attitudes and opinions that define their behavior toward the environment were also determined using thethe environment also determined using the NEP scale. Furthermore, their attitude about NEP scale. were Furthermore, their stated attitude about risk and perception ofstated climate change–related riskimpacts and perception of climate impacts were identified (see Figure 1). The students survey was were identified (see change–related Figure 1). The survey was administered by undergraduate of the administered by undergraduate students of the Intercultural Autonomous University of Sinaloa and Intercultural Autonomous University of Sinaloa and was conducted from October to December 2017. was conducted fromwas October to December 2017. The questionnaire was approved by theUniversity ethics The questionnaire approved by the ethics committee of the Intercultural Autonomous committee ofand the was Intercultural of Sinaloa and was conducted according to of Sinaloa conductedAutonomous according toUniversity ethical principles in social science, with specific care taken ethical principles in social science, with specific taken to protect personal information according to protect personal information according to care national regulations. Before the survey was conducted, to farmers nationalsigned regulations. Before the survey was conducted, farmers signed a consent form and a consent form and received an explanation of the questionnaire. received an explanation of the questionnaire. Figure 1. Methodological approach. Sustainability 2019, 11, 3291 4 of 15 2.2. Technical Efficiency The parametric stochastic production frontier (SPF) was used to estimate the technical efficiency (TE) scores. This approach was simultaneously proposed by Aigner et al. (1977) and Meeusen and Van den Broeck (1977) [29,30]. The SPF model can be specified as Yi = f (Xi ; β) exp(ei ); ei = vi − ui , i = 1, 2, . . . , N (1) The dependent variable (Yi ) is expressed in currency units, Mexican pesos (Mex$), and represents the total farm income. Among the inputs considered as explanatory variables is the total land devoted to crop production (X1 ), measured in hectares. Total labor inputs (X2 ) is composed of family and hired labor, representing the number of employees on the farm. (X3 ) defines expenditure on seeds, expressed in Mex$. Chemical inputs (X4 ) aggregates the value of fertilizers and crop-protection products used in the production process. Farming overhead (X5 ) includes irrigation, energy, fuel, and other expenses and is also measured in monetary units. Using these variables, TE scores were computed as an output-oriented measure and defined as the ratio of observed output to the corresponding SFP, with values in the range of zero to one [31]. According to the estimated TE values, farmers were grouped into three groups, taking account of the mean and one unit upward and downward standard deviation to establish farmers with low, medium, and high efficiency. 2.3. Farmers’ Preferences for Climate Change Mitigation and Adaptation Actions Taking a multicriteria approach to decision-making problems, farmers’ preferences for different climate change adaptation and mitigation actions were identified using the analytical hierarchy process (AHP). This technique is widely used in the resolution of problems of an agrarian and environmental nature [32]. The AHP was developed by Saaty in the 1970s [33]. This tool has a mathematical approach that breaks down any problem into parts or clusters and analyzes them in a hierarchical structure. It allows the assessment of quantitative and qualitative criteria using a common scale [34]. The hierarchical modeling carried out in AHP allows the conversion of subjective evaluations according to the relative importance between different attributes or criteria into a set of weights or global weights to facilitate decision-making. According to the AHP, the criteria (in our case mitigation and adaptation actions) are compared using a pairwise approach, in which the researcher estimates the relative importance of each criterion in order to identify the best alternative as a solution to the problem proposed. By applying the AHP, it was possible to perform an analysis of each option in relation to the others through paired comparisons, facilitating an estimation of farmers’ preferences. The methodological structure of this tool is given by three stages: Modeling, evaluation, and prioritization. During the modeling stage, the problem is defined (i.e., identification of farmers’ preferences for mitigation and adaptation actions). In this case, the appropriate criteria were identified from a literature review [12]. The identified criteria are shown in Table 1. Table 1. Criteria of climate change impact reduction. A Adaptation Measures [35] M Mitigation Measures [36] A1 Invest in improved irrigation facilities [37] A2 Change crops [39] A3 Introduce improved and resistant seeds [41] A4 Adapt the sowing calendar [43] M1 Use organic agriculture [38] M2 Use zero tillage management [40] M3 Use renewable energy [42] M4 Use low-polluting emission and energy-efficient machinery [44] Accordingly, the decision model was structured in the form of a hierarchy, grouping the clusters into different levels (Figure 2), which were used to carry out the paired comparisons of the elements of each cluster based on the verbal scale. Sustainability Sustainability2019, 2019,11, 11,3291 x FOR PEER REVIEW Cluster 1 55 ofof1515 Actions against climate change A. Adaptation M. Mitigation Cluster 2 A1 Cluster 3 A2 A3 M1 A4 M2 M3 M4 Figure 2. Identification of clusters that make up the decision hierarchy model [8]. Figure 2. Identification of clusters that make up the decision hierarchy model [8]. The comparison structure has a pair of criteria (A and M) and a scale in both directions from one The comparison structure has a pair of criteria (A and M) and a scale in both directions from to nine; this allows establishing which of the two criteria was preferred and a measure of the relative one to nine; this allows establishing which of the two criteria was preferred and a measure of the importance of the preferred criterion (see Table 2). relative importance of the preferred criterion (see Table 2). Table 2. Structure of comparison scale between criteria (cluster 1). Table 2. Structure of comparison scale between criteria (cluster 1). A. Adaptation Measures. M. Mitigation Measures 9 9 8 8 A. Adaptation Measures. 7 6 5 4 3 7 6 5 4 3 2 2 1 1 2 2 M. Mitigation Measures 3 4 5 6 7 3 4 5 6 7 8 8 9 9 Scale: One means that both criteria have the same importance, two means that the selected criterion has slightly Scale: One means that both criteria have the same importance, two means that the selected criterion higher importance than the other, up to nine, which means the selected criterion has absolute importance with has slightly higher respect to the other [45].importance than the other, up to nine, which means the selected criterion has absolute importance with respect to the other [45]. Once the hierarchy is structured, the actions corresponding to the assessment stage are carried out. Once the hierarchy is structured, the actions corresponding to the assessment stage are carried This stage corresponds to administering the questionnaire, when the interviewees are asked for the out. This stage corresponds to administering the questionnaire, when the interviewees are asked for degree of importance they assign to their preference in each pair of criteria in each cluster, identified the degree of importance they assign to their preference in each pair of criteria in each cluster, as mitigation or adaptation actions. (The complete experimental design of the paired comparisons identified as mitigation or adaptation actions. (The complete experimental design of the paired question can be seen in the questionnaire, available in the Supplementary Materials, question 36.) comparisons question can be seen in the questionnaire, available in the Supplementary file The third stage is prioritization and synthesis. At this stage, the farmers’ expressed preferences in Farmer_interview.pdf, question 36.) the paired comparisons âik are used to construct the Saaty matrix (Âk ) of each cluster, with dimensions The third stage is prioritization and synthesis. At this stage, the farmers’ expressed preferences n × n = 4 × 4 (where n corresponds to the number of actions within the cluster) and the vector of aˆ ik are in the paired comparisons usedcorresponding to construct the Saaty matrix (Âk) of each cluster, eigenvalues with the normalized weights to the cluster in question, which servewith to dimensions n × nimportance, = 4 × 4 (where corresponds to the number actions the cluster) and the obtain the relative alsonknown as the priority or local of weight ŵikwithin of the attribute, according of eigenvalues with the normalized weights corresponding toestimated the cluster question, tovector the weighting of judgments issued by the individual (k), which can be byinmeans of thewhich row geometric mean method (RGMM), which will multifunctionality to be carried wˆ ik ofout. serve to obtain the relative importance, alsoallow known as the priorityassessment or local weight the The estimation of relative importance (ŵik carried issued out using Decisions [46]. attribute, according to the weighting of) was judgments by the theSuper individual (k), software which can be The verification of farmers’ consistency using the consistency ratio [47] showed an average value less estimated by means of the row geometric mean method (RGMM), which will allow than 10%, which is acceptable according to the AHP literature [48]. Finally, in order to obtain the ˆ ik ) was multifunctionality assessment to be carried out. The estimation of relative importance ( w general results of the weight of each attribute, the process was repeated for each individual who was carried out using(kthe Decisions software [46]. The verification of farmers’ consistency using part of the sample = 1Super to 370). the consistency ratio [47] showed an average value less than 10%, which is acceptable according to theFarmers’ AHP literature [48]. Finally, in order to obtain general results of the weight of each attribute, 2.4. Environmental Attitudes and Opinions bythe NEP Scale Method the process was repeated for each individual who was part of the sample (k = 1 to 370). Given current environmental concerns, the study of human attitudes and behaviors toward the environment has become increasingly relevant. These environmental attitudes or opinions can be 2.4. Farmers’ Environmental Attitudes and Opinions by NEP Scale Method approximated and expressed by positive or negative evaluations of the relationships between humans Given current environmental study of human behaviorsindividuals’ toward the and the natural environment [49,50].concerns, There arethe several methods andattitudes scales to and approximate environment has increasingly relevant. These environmental attitudes opinions can be attitudes toward thebecome environment. The Environmental Concerns alternative is a scaleorthat pays specific approximated and expressed by positive or negative evaluations of the relationships between attention to issues related to transportation, environmental pollution, biodiversity protection, and humans and theuse, natural environment [49,50]. There are several methods and scalesEnvironmental to approximate natural resource as well as consumption behavior [51]. The Proactive—Reactive individuals’ scale attitudes toward the environment. Environmental Concerns alternativepractices, is a scale Management is another alternative that allowsThe the evaluation of different environmental that pays specific attention to issues related to transportation, environmental pollution, biodiversity protection, and natural resource use, as well as consumption behavior [51]. The Proactive—Reactive Sustainability 2019, 11, 3291 6 of 15 in which the proactive actions are aimed at modifying the processes that negatively affect the environment and the reactive ones are aimed at repairing current negative effects [52]. Among the array of alternative methods to measure environmental attitude, the New Ecological Paradigm (NEP) scale is one alternative that can be adapted to identify the psychological tendency reflecting the relationship that humans have with the environment [53,54] in an economic context, such as agricultural production. Individuals’ attitudes can be identified as latent environmental dimensions principally reflecting an ecocentric or anthropocentric perspective. An ecocentric attitude is related to statements in which someone expresses a vision in favor of nature, considering that humans cause disequilibrium by their use of the environment, while an anthropocentric attitude considers that the environment can support the intensive use of natural resources, expressed through statements in which humans are said to have supremacy [55]. In this study, we used an NEP scale composed of 16 statements in order to analyze farmers’ environmental attitudes. The agreement level for each statement was measured according to a 9-point Likert-type scale, where one represents total disagreement with the statement, five corresponds to a neutral opinion, and nine represents total agreement with the statement. The statements used in the experimental design to identify farmers’ latent environmental dimensions can be seen in the questionnaire, available in the Supplementary Materials, question 27. 2.5. Farmers’ Risk Attitude Using the MPL Lottery Method Decision-making under uncertainty depends to a large extent on the risk attitude of economic agents. It is related to each person’s behavior and influenced by socioeconomic factors and experiences [56]. In the agricultural field, the decisions made by farmers are affected by their level of aversion to or tolerance of risk that is related to their farm management and crop cultivation strategy and periodicity. They are also related to farmers’ attitudes toward the environment and their perception of climate change [12]. The multiple price list (MPL), also known as a “lottery,” is one of the tools most recently used to identify farmers’ stated attitudes about risk under uncertainty (i.e., the level of risk tolerance or aversion). It relates levels of risk with reward or loss in a lottery. According to this method, an array of eight questions is proposed as lottery scenarios, with a pair of hypothetical alternatives from which to select, option A and option B, both with constant probabilities in each scenario. Option A (the safe option) determines the level of risk aversion, and depending on the number of scenarios in which the interviewee selects this option, once he/she decides to change to option B (the risky option), the interview must be stopped to avoid inconsistencies. If a farmer selects the risky option (option B) in the first scenario, it indicates that he/she is tolerant of risk; if the farmer selects option A in the first three scenarios and it then changes to option B in scenario four, it indicates that he/she has a neutral attitude toward risk; and a risk-averse farmer will choose the safe option (option A) in all scenarios [57]. According to the Holt and Laury scale, a risk-tolerant person corresponds to a risk coefficient from −1.75 to less than −0.15, a risk—neutral person corresponds to a risk coefficient from −0.15 to less than 0.14, and a risk—averse person corresponds to a risk coefficient of 0.14 or greater [58]. The experimental design used to identify the declared level of risk can be seen in the Supplementary Materials, question 35. 2.6. Farmers’ Perceptions of Climate Change The perception of climate change in farming activities is based on an analysis of the variability of phenomena such as increased temperature; varied levels and timing of rainfall; decreased soil fertility; increased periods of drought; decreased yields; increased drought episodes, frosts, floods, hailstorms, and plagues; and changes in vegetation [59]. Farmers’ perceptions of climate change are also relevant as they are related to their farming decisions and practice [60]. Statements related to these phenomena identified from the literature [61–69] were used to evaluate farmers’ agreement or disagreement using a Likert-type scale ranging from one to nine, where one corresponds to absolute disagreement and nine Sustainability 2019, 11, 3291 7 of 15 corresponds to absolute agreement with the statement. The proposed statements allow an assessment of farmers’ opinions on issues such as the instability of meteorological factors and events related to climatic conditions. 3. Results and Discussion 3.1. Technical Efficiency Results show that the average TE estimate is 0.57, indicating that farmers reach 57% of their maximum potential output, and the majority of them have average efficiency scores (72% moderately efficient, 13% low TE score, and 15% high TE score). Moreover, results suggest that farmers could increase their output by 43% on average if they used available resources more effectively with the same production technology. Thus, there is a large opportunity to improve TE by a more rational and less arbitrary use of inputs that would reduce production costs and contribute to the farm’s livelihood. It is considered that low efficiency may be associated with a misuse of existing technology, so optimization has to do with, among other things, optimizing the irrigation infrastructure, focused on substantially reducing water and energy consumption [70], given that, generally, energy consumption can be considered one of the most significant expenses associated with agricultural production. Additionally, reducing energy consumption would bring economic savings and reduce polluting emissions in the environment, resulting in a more sustainable form of agriculture. Results also show that farmers who use family labor to a greater extent instead of hiring labor dedicated exclusively to agricultural activities have less technical efficiency, which indicates a need for greater professionalization and specialization of the people in charge of tasks in the field. This result differs from that obtained in a study [71], in which the presence of family labor greatly improved technical efficiency [72]. This difference could be attributed to the size of the farm, which in our case study was about 10.60 ha on average. When the size of the farm increases, more specialized labor is required and family labor may not be able to satisfy the need. In any case, it is important to note that Marquez et al. (2013) also indicate that the significance of this relationship can also be related to the methodological framework used to estimate TE. Another action that we believe contributes to an improvement of technical efficiency is changing crops, given that the type of crop defines the amount of resources required, such as water, fertilizers, labor, machinery, etc. [73]. In addition, implementing more ecological techniques such as zero tillage contributes to increased technical efficiency due to a decreased use of energy resources and labor on the farm [74] (unlike conventional agriculture, which uses intensive tillage, significantly increasing costs for energy consumption, causing additional soil erosion, runoff, and pollution by sediment, with fertilizers and pesticides that will impact the subsoil and aquifers). 3.2. Adaptation and Mitigation Preferences The results of the analysis of preferences estimated by the AHP method (Table 3) reveal that mitigation actions (M = 58.19%) were relatively more preferred compared to adaptation ones (A = 41.81%). The highest in relative importance compared to other actions is the mitigation alternative “use low-polluting emission and energy-efficient machinery” (M4 = 17.94%). These results, in relation to climate change impact reduction, show that mitigation actions must be a priority, as also affirmed by Bragado (2016) [75]. The second most popular option was the alternative “use zero tillage management” (M2 = 14%), and the third was “use renewable energy” (M3 = 13.71%). Sustainability 2019, 11, x FOR PEER REVIEW 8 of 15 Sustainability 2019, 11, 3291 8 of 15 Sustainability 11, x FORprioritization PEER REVIEW of different actions (relative relevance) faced by the impact of climate 8 of 15 Table2019, 3. Farmers’ change on their activities. Table Farmers’ prioritizationofofdifferent differentactions actions (relative (relative relevance) of of climate Table 3. 3. Farmers’ prioritization relevance)faced facedby bythe theimpact impact climate Criteria. Local Weight Attributes Local Weight Global Weight change on their activities. change on their activities. (Cluster 1) (Cluster 2) Cluster 1 × Cluster 2 Criteria. Local Weight Attributes Local Weight Global Weight A1 Invest in improving irrigation facilities 22.74% 9.27% (Cluster 1) (Cluster 2) Cluster 1 × Cluster 2 A2 Change crops 25.99% 10.60% Adaptation 41.81 A1 Invest in improving irrigation facilities 22.74% 9.27% A3 Introduce improved and resistant seeds 28.03% 11.43% A2 Change crops 25.99% 10.60% Adaptation 41.81 A4 Adapt sowing calendar 23.25% 9.48% A3 Introduce improved and resistant seeds 28.03% 11.43% Local Total 100.00% – A4 Adapt sowing calendar 23.25% 9.48% (Cluster 3) Cluster 1 × Cluster 3 Local Total 100.00% – M1 Use organic agriculture 22.77% 13.48% (Cluster 3) Cluster 1 × Cluster 3 M2 Use organic zero tillage management 23.79% M1 Use agriculture 22.77% 13.48%14.09% Mitigation 58.19 M3 Usetillage renewable energy 23.15% M2 Use zero management 23.79% 14.09%13.71% Use low-polluting emission and Mitigation 58.19 M3 Use renewable energy 23.15% 13.71% M4 30.29% 17.94% energy-efficient machinery Use low-polluting emission and M4 30.29% 17.94% Localmachinery Total 100.00% – energy-efficient Global total Local Total 100.00% – 100.00% Global total 100.00% According to the relative relevance assigned to adaptation and mitigation options (see Figure According to how the relative relevance assigned to adaptation and mitigation options (see Figure 3),According we identified manyrelevance farmers in the sample assigned the global weight to each option. to the relative assigned to adaptation andhighest mitigation options (see Figure 3), 3), we identified how many farmers in the sample assigned the highest global weight to each option. action how mostmany preferred byinfarmers to reduce thethe effects of global climateweight change “Use weThe identified farmers the sample assigned highest to was each M4, option. The action most preferred by farmers to reduce the effects of climate change was M4, “Use low-polluting emission and energy-efficient machinery,” a mitigation action; the second and third The action most preferred by farmers to reduce the effects of climate change was M4, “Use low-polluting low-polluting emission and energy-efficient machinery,” a mitigation action; the second and third preferred were A1,machinery,” “Invest in improved irrigation and A2, “Change crops,” emission andactions energy-efficient a mitigation action;infrastructure,” the second and third preferred actions preferred actions were A1, “Invest in improved irrigation infrastructure,” and A2, “Change crops,” both adaption were A1, “Invest options. inoptions. improved irrigation infrastructure,” and A2, “Change crops,” both adaption options. both adaption M4. Use of low M4. Use of low polluting polluting emission and emission and energetically energetically efficient efficient machinery machinery 18.65% (69) 18.65% (69) M3. Use of M3. Use of renewable energy renewable energy 5.95% (22) 5.95% (22) A1. inin A1.Investment Investment improvement of improvement of irrigation irrigation infrastructure infrastructure 17.57% 17.57%(65) (65) A2. of of A2.Change Change crops crops 17.30% (64) 17.30% (64) A4. Adaptation of A4. Adaptation of sowing calendar sowing calendar 6.75% 6.75% (25) (25) M1. Organic M1. Organic Agriculture Agriculture 7.56%7.56% (28) (28) A3. Introduce A3. Introduce improved and and improved resistant seedsseeds resistant 10.00% (37) (37) 10.00% M2. M2. ZeroZero tillage tillage managment managment 16.22% (60) 16.22% (60) Figure Distributionofoffarmers’ farmers’preferences preferences for for climate change adaptation and actions. Figure 3. 3. Distribution climate change adaptation andmitigation mitigation actions. Figure 3. Distribution of farmers’ preferences for climate change adaptation and mitigation actions. EnvironmentalAttitudes Attitudes 3.3.3.3. Environmental 3.3. Environmental Attitudes Accordingtotothe theanalysis analysisofofenvironmental environmental attitudes, attitudes, the two-thirds of of According theNEP NEPscale scaleshowed showedthat that two-thirds According to the analysis of environmental attitudes, the NEP scale showed that two-thirds of farmers had a positive position on the environment, emphasizing their ecocentric attitude (see farmers had a positive position on the environment, emphasizing their ecocentric attitude (see Figure 4), farmers4),had a positive position the environment, emphasizing their ecocentric attitude Figure means that most on farmers nature to be good thing that should be (see which means which that most farmers consider natureconsider to be a good thing thata should be protected regardless Figure 4), which means that most farmers consider nature to be a good thing that should be whether it generates directasbenefits not, such as through responsible of protected whether itregardless generatesofdirect benefits or not, such throughorresponsible consumption and use of protected regardless of whether it generates direct benefits or not, such as through responsible consumption and use of clean energy. Furthermore, expressed a higher preference for clean energy. Furthermore, farmers expressed a higherfarmers preference for maintaining environmental consumptionenvironmental and use of clean energy. Furthermore, farmers expressed a higher preference for maintaining resources and the regeneration of natural processes. The remaining resources and the regeneration of natural processes. The remaining farmers had an undefined position maintaining resourcesonand regenerationThey of natural processes. The remaining farmers had environmental an undefined position thetheenvironment. expressed agreement with on the environment. They expressed agreement with affirmations where the importance of nature farmers had an the undefined position on isthe environment. They expressed affirmations where importance of nature highlighted and with affirmations thatagreement defend the with is highlighted and with affirmations that defend the human right to use environmental resources. human right towhere use environmental resources. However, in other and cases,with bothaffirmations affirmations that received a the affirmations the importance of nature is highlighted defend However, in other cases, both affirmations received a high level of disagreement, showing an unclear human right to use environmental resources. However, in other cases, both affirmations received a relationship. There is no conclusive opinion on the environment, that is, they generally do not have a Sustainability 2019, 11, x FOR PEER REVIEW 9 of 15 Sustainability 2019, 11, 3291 9 of 15 high level of disagreement, showing an unclear relationship. There is no conclusive opinion on the environment, that is, they generally do not have a clear environmental opinion; they agree with clear environmental opinion; agree with some ecocentric attitudes and anthropocentric attitudes, some ecocentric attitudes andthey anthropocentric attitudes, or they absolutely disagree with both [76]. or they absolutely disagree with both [76]. Distribution of farmer attitudes toward the environment 39% (145) 27% (99) 19% (72) 15% (54) (+eco,+antro) (-eco,+antro) Inconsistent Anthropocentric (-eco,-antro) (+eco,-antro) Inconsistent Ecocentric Figure 4. Farmers’ environmental attitudes. 3.4. Risk Risk Attitudes Attitudes 3.4. Through the the analysis analysis of ofrisk riskattitudes attitudesusing usingthe theMPL MPLmethod, method,it it was observed that according Through was observed that according to to the scale proposed Holt andLaury Laury[58], [58],approximately approximately51% 51%ofoffarmers farmershad hadan an attitude attitude of of risk risk the scale proposed byby Holt and aversion, while risk-tolerant andand 8% had a neutral attitude about risk. The estimated average aversion, while41% 41%were were risk-tolerant 8% had a neutral attitude about risk. The estimated of the farmers’ risk level had a value of 0.32, placing the farmers studied as slightly averse to risk average of the farmers’ risk level had a value of 0.32, placing the farmers studied as slightly averse to according to the scale. This result is consistent with results on the level of risk aversion obtained in risk according to the scale. This result is consistent with results on the level of risk aversion obtained other studies, such as as Pennings and Garcia (2001) andand Brick et al. (2012) [57,77]. in other studies, such Pennings and Garcia (2001) Brick et al. (2012) [57,77]. 3.5. Climate Change Perceptions 3.5. Climate Change Perceptions The results of the analysis of climate change perceptions show that farmers perceived a high degree The results of the analysis of climate change perceptions show that farmers perceived a high of variation in all climate phenomena presented in the experimental design, according to the answers degree of variation in all climate phenomena presented in the experimental design, according to the given during the interview using a 9-point Likert-type scale, where one is absolute disagreement and answers given during the interview using a 9-point Likert-type scale, where one is absolute nine absolute agreement with the statement. Most important was the increasing incidence of diseases disagreement and nine absolute agreement with the statement. Most important was the increasing and pests (8.23), followed by increased weed problems (7.92), increased temperature (7.46), and the incidence of diseases and pests (8.23), followed by increased weed problems (7.92), increased presence of frost episodes (5.98). temperature (7.46), and the presence of frost episodes (5.98). 3.6. Socio-Economic Characteristicvs 3.6. Socio-Economic Characteristicvs Finally, the TE efficiency was related to the socio-economic variables (age, education level, income Finally, the TE efficiency was related to the socio-economic variables (age, education level, and percentage of income coming from agricultural activities, origin of their agricultural training income and percentage of income coming from agricultural activities, origin of their agricultural education, number of generations devoted to agriculture and property management regime). Results training education, number of generations devoted to agriculture and property management showed that age, property regime and the percentage of income coming from agriculture were regime). Results showed that age, property regime and the percentage of income coming from significantly related to TE. Young farmers that are owner of their agricultural land with more than 50% agriculture were significantly related to TE. Young farmers that are owner of their agricultural land of their income coming from agriculture activities are more technically efficient. These results are in with more than 50% of their income coming from agriculture activities are more technically efficient. line to the finding in Guesmi et al., (2010) and Perdomo and Mendieta (2007) highlighting the age and These results are in line to the finding in Guesmi et al., (2010) and Perdomo and Mendieta (2007) income variable as determinant factors of TE [31,78]. highlighting the age and income variable as determinant factors of TE [31,78]. 3.7. Technical Efficiency with Regard to Farmers’ Preferences, Risk, and Environmental Attitudes and Climate 3.7. Technical Efficiency with Regard to Farmers’ Preferences, Risk, and Environmental Attitudes and Climate Change Perceptions Change Perceptions As commented above, technical efficiency scores were related to farmers’ preferences for adaptation commented above, technical efficiency were related to farmers’ preferences for and As mitigation actions, environmental attitudes,scores risk attitude, and climate change perceptions. adaptation mitigation actions, environmental attitudes, risk attitude, and climate An analysis and of heterogeneity was made by ANOVA and PCA to statistically contrast whetherchange any of perceptions. An analysis offarmers’ heterogeneity was made by ANOVA and PCA these results characterizing attitudes were related to their level of TE.to statistically contrast whether any of these results characterizing farmers’ attitudes were related to their level of TE. Sustainability Sustainability 2019, 2019, 11, 11, 3291 x FOR PEER REVIEW 10 of of 15 10 15 Results showed two clearly independent dimensions (see Figure 5) that explain 52.28% of the Results showed two clearly independent dimensions (see Figure 5) that explain 52.28% of the total variance. In Figure 5, the horizontal axis represents climate change perception. Farmers on the total variance. In Figure 5, the horizontal axis represents climate change perception. Farmers on right side are more sensitive and farmers on the left side are less sensitive to climate change the right side are more sensitive and farmers on the left side are less sensitive to climate change problems. The vertical axis represents TE, environmental attitudes, and preferences for significant problems. The vertical axis represents TE, environmental attitudes, and preferences for significant actions against climate change. Farmers on the upper part are more efficient compared to the lower actions against climate change. Farmers on the upper part are more efficient compared to the lower part. part. The same description also holds for anthropocentric and ecocentric attitudes. Finally, the The same description also holds for anthropocentric and ecocentric attitudes. Finally, the preference preference for crop changes is located on the opposed side of the preference for improving irrigation for crop changes is located on the opposed side of the preference for improving irrigation systems as systems as adaptation actions. adaptation actions. Figure Technical efficiency Figure 5. 5. Technical efficiency as as related related to to farmers’ farmers’ preferences, preferences, environmental environmental attitudes, attitudes, and and climate climate change perception. change perception. Results show that the most efficient farmers (green dots) preferred the “change crops” Results show that the most efficient farmers (green dots) preferred the “change crops” adaptation action, highlighting the importance of adopting new, productive, low-water-demand adaptation action, highlighting the importance of adopting new, productive, low-water-demand and disease-resistant varieties to face climate change. According to Estrada et al. (2006), crops must and disease-resistant varieties to face climate change. According to Estrada et al. (2006), crops must be diversified to maximize yields, yet generally farmers do not diversify but specialize in a specific be diversified to maximize yields, yet generally farmers do not diversify but specialize in a specific crop, citing economic factors for this decision [79]. These farmers exhibited an anthropocentric attitude crop, citing economic factors for this decision [79]. These farmers exhibited an anthropocentric toward the environment, highlighting relatively less sensitivity to climate change issues. In particular, attitude toward the environment, highlighting relatively less sensitivity to climate change issues. In lower perception was found regarding the variation of environmental phenomena such as droughts and particular, lower perception was found regarding the variation of environmental phenomena such freezing episodes. Derived from previous results, as also commented by Ovares (2016), climate change as droughts and freezing episodes. Derived from previous results, as also commented by Ovares perception is related to anthropocentric attitudes [80], and we can argue that a lack of environmental (2016), climate change perception is related to anthropocentric attitudes [80], and we can argue that a interest or ignorance of the effects of production activities on the environment is related to unawareness lack of environmental interest or ignorance of the effects of production activities on the environment of climate variation. In this context, it is necessary to identify whether technical efficiency has been is related to unawareness of climate variation. In this context, it is necessary to identify whether achieved to the detriment of the environment. technical efficiency has been achieved to the detriment of the environment. Farmers with lower technical efficiency (black dots), preferred the investment in irrigation facilities Farmers with lower technical efficiency (black dots), preferred the investment in irrigation as an adaptation action and exhibited ecocentric attitude. This low level of TE could be related to their facilities as an adaptation action and exhibited ecocentric attitude. This low level of TE could be extensive production system and to the type of farming managements. These include the adoption of related to their extensive production system and to the type of farming managements. These include low soil degradation strategy by applying zero or low tillage agriculture, low use of phytosanitary and the adoption of low soil degradation strategy by applying zero or low tillage agriculture, low use of following a natural crop rotation. The less efficient farmers are taking advantage of the available natural phytosanitary and following a natural crop rotation. The less efficient farmers are taking advantage resources with low adopting level of technological innovations. This result is similar to the finding of the available natural resources with low adopting level of technological innovations. This result is similar to the finding of Alvarez and Del Corral (2010) who stated that farmers adopting intensive Sustainability 2019, 11, 3291 11 of 15 of Alvarez and Del Corral (2010) who stated that farmers adopting intensive agriculture technology are more productive and more technically efficient than the extensive ones [81]. In this context, the decision-making of ecocentric farmers is driven not only by their economic objectives but also by the environmental one. They tend to preserve the environment as an input of their activities that ensure the generation of a satisfactory income [28]. The ecocentric farmers could adopt agricultural technologies that positively impact their productivity and are environmental friendly [82], such as the adoption of efficient irrigation system. From a sustainable agriculture point of view, a balance is required between their economic, social and environmental components. Otherwise, any other position in favour of only one of these factors it supposes the detriment of the rest. Their perception of climate change was not clearly defined, with a slight tendency toward low perception levels. According to these results, we can deduce that the main problem faced by less efficient farmers is the availability of water, or specifically the need to optimize its use, given the high cost of water in the production of crops. Irrigation systems are a key element in achieving high technical efficiency in agricultural production [83]. The unclear position regarding climate change does not allow them to identify that the cost derived from water consumption is due to the increased use of water because of variation in the climate (greater evapotranspiration due to increased temperature, lower humidity in soil as a result of drought). The effects of climate change, identified by farmers mainly as increasing temperature and increased incidence of diseases and pests and weed problems, were perceived to a greater extent by moderately efficient farmers. Finally, according to the results of this study, non significant evidence was found for a relationship between the stated attitude about risk and technical efficiency. This could be related to the nature of the data, with relatively homogeneous outcomes, with most producers in the region being risk averse and moderately efficient. 4. Conclusions Rural areas that are vulnerable to the impacts of climate change, where agriculture plays a relevant economic role, show the importance of implementing region-specific adaptation and mitigation strategies to improve the level of technical efficiency at the farm level. Adopting these actions does not imply greater technification of the field, but better use of currently available resources and inputs by introducing alternative farm management systems such as using organic agriculture and zero tillage, adapting the sowing calendar, and changing crops. The effects of climate change on agro systems depend on the type of agricultural production system implemented. Thus, it is necessary to adapt these systems according to the changing requirements of the natural environment caused by climatic variation, in order to increase production capacity through the optimal use of available resources, resulting in a higher level of technical efficiency. Our results suggest a need to take advantage of available resources in order to face the adverse effects of climate change. Adopting the use of improved seeds and changing crops are among the most important actions according to farmers’ preferences. Agricultural policy-makers must generate incentives for farmers in the region to adopt these types of adaptation actions, and disseminate technical information on best practices to encourage them to put these actions into practice. In relation to farmers with less technical efficiency, whose environmental attitude was more ecocentric, their ecological efficiency should also be accounted for through more sustainable use of water resources, as this is one of the main factors that negatively affects agricultural performance. In the agricultural sector, the economic policies that are implemented must be aimed at increasing the income and improving the livelihoods of rural households in order to improve their capacities and productive assets through the sustainable use of resources. Our results show that competent authorities need to include several actions that could represent a roadmap in the region studied. First, there is a need to conduct research regarding the impacts of climate change on agriculture in the region from a multidisciplinary perspective, in order to identify the impact of implementing adaptation and mitigation actions on agricultural productivity patterns. Second, this research highlights the Sustainability 2019, 11, 3291 12 of 15 importance of identifying crop varieties that are better adapted to the new climatic conditions in the region. The adoption of this measure should be accompanied by dissemination campaigns to share experiences from other farmers and offer information on the economic, social, and environmental benefits of adopting these practices. Third, training schemes on agricultural practices of adaptation and mitigation should be generated by agricultural associations to improve the rational use and conservation of natural resources, in order to reduce the risk aversion and increase the likeliness of implementing the new practices. Finally, economic incentive programs should be established that could help farmers to introduce new sustainable management practices at the farm level that increase their productivity and respect the environment, specifically mitigation actions. In this study, one of the limitations was related to the data collection process in terms of the veracity of the economic information gathered from farmers. Some producers were not able to provide all the needed information and others refrained from responding reliably to the questions, due to either a lack of understanding or lack of interest. For further research, it could be interesting to include variables accounting for social and environmental externalities in the calculation of efficiency to better highlight a more sustainable indicator. Supplementary Materials: The following are available online at http://www.mdpi.com/2071-1050/11/12/3291/s1. Author Contributions: M.A.O.T. conceived and designed the study, investigated, performed data collection, and wrote the manuscript; Z.K. conceived and designed the research, reviewed and edited, participated in writing the manuscript and was the supervisor of all procedures; S.I.O.H. made the formal analysis of the data and participated in writing the manuscript; B.G. participated in analysis of the data. Funding: This research received no external funding. 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https://openalex.org/W2095146796	https://europepmc.org/articles/pmc3753306?pdf=render	English	null	Medical and Household Characteristics Associated with Methicillin Resistant Staphylococcus aureus Nasal Carriage among Patients Admitted to a Rural Tertiary Care Hospital	PloS one	2,013	cc-by	7,815	Abstract This is an open-access article distributed under the terms of the Creative Commons Attribut unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Center for Research Resources (grant number UL1RR025747), the NC Occupational Safety and Health Education Research Center (5-52665), and the H. Michael and Barbara Arrighi Endowed Scholarship Fund. The content is the sole responsibility of the authors and does not represent the official views of the National Center for Research Resources, the National Institutes of Health, or the National Institutes for Occupational Safety and Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Keith Ramsey has provided consultancy for BD GeneOhm, is on the Speakers Bureaus of Cubist Pharmaceuticals and Sanofi-Pasteur, and has served as an expert witness in legal proceedings related to healthcare-associated infections, and received fees for his services. Pia D. M. MacDonald is employed by Social & Scientific Systems, Inc. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors. * E-mail: schinasi@email.unc.edu complex and distinctions between CA and HA strains are increasingly difficult to disentangle. Leah Schinasi1, Steve Wing1, Pia D. M. MacDonald1,2, David B. Richardson1, Jill R. Stewart3, Kerri L.Augustino4,5, Delores L. Nobles4,5, Keith M. Ramsey4,5 1 Department of Epidemiology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America, 2 Social & Scientific Systems, Inc., Durham, North Carolina, United States of America, 3 Department of Environmental Sciences and Engineering, The University of NC at Chapel Hill, Chapel Hill, North Carolina, United States of America, 4 Department of Infection Control, Vidant Medical Center, Greenville, North Carolina, United States of America, 5 Division of Infectious Diseases, The Brody School of Medicine at East Carolina University, Greenville, North Carolina, United States of America Abstract Background: Methicillin resistant Staphylococcus aureus (MRSA) poses a threat to patient safety and public health. Understanding how MRSA is acquired is important for prevention efforts. This study investigates risk factors for MRSA nasal carriage among patients at an eastern North Carolina hospital in 2011. Methods: Using a case-control design, hospitalized patients ages 18 – 65 years were enrolled between July 25, 2011 and December 15, 2011 at Vidant Medical Center, a tertiary care hospital that screens all admitted patients for nasal MRSA carriage. Cases, defined as MRSA nasal carriers, were age and gender matched to controls, non-MRSA carriers. In-hospital interviews were conducted, and medical records were reviewed to obtain information on medical and household exposures. Multivariable conditional logistic regression was used to derive odds ratio (OR) estimates of association between MRSA carriage and medical and household exposures. Results: In total, 117 cases and 119 controls were recruited to participate. Risk factors for MRSA carriage included having household members who took antibiotics or were hospitalized (OR: 3.27; 95% Confidence Interval (CI): 1.24–8.57) and prior hospitalization with a positive MRSA screen (OR: 3.21; 95% CI: 1.12–9.23). A lower proportion of cases than controls were previously hospitalized without a past positive MRSA screen (OR: 0.40; 95% CI: 0.19–0.87). Conclusion: These findings suggest that household exposures are important determinants of MRSA nasal carriage in hospitalized patients screened at admission. Citation: Schinasi L, Wing S, MacDonald PDM, Richardson DB, Stewart JR, et al. (2013) Medical and Household Characteristics Associated with Methicillin Resistant Staphylococcus aureus Nasal Carriage among Patients Admitted to a Rural Tertiary Care Hospital. PLoS ONE 8(8): e73595. doi:10.1371/journal.pone.0073595 Citation: Schinasi L, Wing S, MacDonald PDM, Richardson DB, Stewart JR, et al. (2013) Medical and Household Characteristics Associate Staphylococcus aureus Nasal Carriage among Patients Admitted to a Rural Tertiary Care Hospital. PLoS ONE 8(8): e73595. doi:10.137 Editor: Tara C. Smith, University of Iowa, United States of America Editor: Tara C. Smith, University of Iowa, United States of America Received December 7, 2012; Accepted July 26, 2013; Published August 26, 2013 Copyright: 2013 Schinasi 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: 2013 Schinasi et al. Medical and Household Characteristics Associated with Methicillin Resistant Staphylococcus aureus Nasal Carriage among Patients Admitted to a Rural Tertiary Care Hospital Leah Schinasi1, Steve Wing1, Pia D. M. MacDonald1,2, David B. Richardson1, Jill R. Stewart3, Kerri L.Augustino4,5, Delores L. Nobles4,5, Keith M. Ramsey4,5 Introduction When it was first identified in the 1960s, Methicillin resistant Staphylococcus aureus (MRSA) was a nosocomial pathogen [1]. In the 1990s, the epidemiology of this virulent pathogen changed; it was identified in people without recent medical contact [2]. Healthcare associated (HA) and community associated (CA) MRSA strains are genetically distinct [3]. However, CA strains have been detected in hospital outbreaks [4], and people without recent hospitalizations may carry HA strains [5]. Indeed, the epidemiology of MRSA is In addition to medical related exposures [6], household and environmental exposures are gaining recognition as potentially important determinants of transmission and acquisition [7,8]. For example, MRSA carriage or infection has been associated with living with household members with a history of infections [8,9] and with presence of MRSA in the household environment [8]. There is evidence that MRSA may be transmitted between humans and animals [9,10]. There have been reports of MRSA August 2013 \| Volume 8 \| Issue 8 \| e73595 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 1 August 2013 \| Volume 8 \| Issue 8 \| e73595 MRSA and Medical Household Exposures infections and outbreaks among athletics participants [11] and associations between smoking and MRSA infection [12]. Geno- typic, clinical and demographic differences in the epidemiology of MRSA have been observed among regions and sub-communities within the same area [13,14]. Therefore, region specific investi- gations of the sources of acquisition are important for informing infection prevention. Case identification Cases were defined as inpatients who screened positive for MRSA nasal carriage upon admission to the hospital. They were identified by reviewing daily reports from the electronic medical record, which captured MRSA screening results and demographic data on all admitted patients to the hospital. p Although there is a growing body of literature on risk factors for MRSA carriage in urban communities [8,15,16], data on predictors of carriage in rural areas remains limited. The eastern section of North Carolina is largely rural [17], and compared to the statewide population, a higher proportion of eastern North Carolina residents are Black and live in poverty–approximately 28% and 20% of eastern North Carolinians, respectively, versus 21% and 17% of North Carolina’s total population [18]. Vidant Medical Center (VMC) is an 861-bed teaching hospital of the Brody School of Medicine at East Carolina University and the tertiary care center for 29 eastern North Carolina counties. Since February of 2007, VMC has screened all patients for MRSA within 24 hours of admission using duplicate swabs of the anterior nares. Since the initiation of this program, all MRSA carriers have been placed on contact isolation, prescribed 5-day courses of mupirocin for application to their nares, and bathed with chlorhexidine soap [19]. Ethics statement The Institutional Review Boards at the University of North Carolina at Chapel Hill and East Carolina University approved this work. Participants provided written informed consent and signed Health Information Portability and Accountability Act authorization forms. Control identification Controls were defined as inpatients who screened negative for MRSA upon admission to the hospital. For every case, an eligible control was identified by reviewing the same daily reports of the electronic medical record that were used to identify cases. When more than one patient was eligible to serve as a control, a random number generator was used to select the control. If that potential control declined to participate or was discharged from the hospital before the interviewer approached them, another control was identified. Because of the above-stated objective of the parent study to investigate the relationship of MRSA carriage with occupational exposures, and because gender and age are strong potential determinants of occupational exposure and case status that could be controlled by design rather than analysis only, controls were matched to cases based on age (65 years) and gender. VMC’s screening program provided the opportunity to investigate risk factors for MRSA carriage among hospitalized patients in eastern North Carolina. The objective of this study was to investigate household exposures, hospitalization and/or MRSA screening history, demographic characteristics, and smoking history as predictors of MRSA nasal carriage in patients who were recently admitted and screened at VMC. To investigate differences in the epidemiology of the genetic subtypes of MRSA, a repetitive sequence-based polymerase chain reaction (rep-PCR) [20] was used to characterize isolates from study participants as CA or HA, and associations between exposures and CA or HA MRSA carriage were investigated. Interviews and medical record review Participant enrollment and structured interviews were conduct- ed with cases and controls from July – December 2011. The interviewer approached eligible cases and controls in their hospital rooms, introduced the study, and invited them to participate in a brief interview. The structured interview included questions about medical and antibiotic use history in the past 12 months for participants and their household member(s), indoor pets, sports participation or gym use in the past 2 weeks, smoking history in the past 12 months, and demographics. One author (L.S.) conducted all interviews. The following information was abstracted from medical records: prior MRSA screening results, surgery, hospitalizations, and antibiotic prescriptions within the past 12 months; and diagnoses listed in the discharge summary for the current hospitalization. If participants reported not taking antibiotics but their medical records showed antibiotic prescriptions, including mupirocin for previous MRSA carriage, the data were coded to reflect the record. If participants reported antibiotic use but their medical records showed no antibiotic prescriptions, participants’ reports were used. Because of the way the question about antibiotic use was asked during the interview, it was not possible to tell if participants self-reported mupirocin use as an antibiotic exposure; therefore, mupirocin prescriptions recorded in the medical chart were included with other antibiotic exposures. One author (L.S) abstracted all medical record data. Participant eligibility Eligible participants were admitted patients at VMC, screened for MRSA within 24 hours of their admission and English or Spanish speakers. The same eligibility criteria were applied to cases and controls. These data arise from a parent study, the objectives of which were to investigate relationships between occupational exposures to livestock and MRSA nasal carriage. To increase the probability of employment, participants were restricted to ages 18–65. To increase the probability of exposure to livestock for the parent study, participants were restricted to residents of swine-producing zip codes in which the permitted number of swine was equal to or greater than the median number listed with the North Carolina Division of Water Quality (North Carolina zip codes with 1,032,750 or more permitted swine). Of 1,083 zip codes in North Carolina [21], 176 were eligible. Statistical analysis Conditional logistic regression models, which adjusted for matching by age and gender, were used to estimate odds ratios (OR) and 95% confidence intervals (CIs). The following variables were examined in a multivariable model: education; self-reported race/ethnicity; visiting a gym or playing sports; tobacco smoking history; indoor cats or dogs; household member presence, hospitalization or antibiotic use; and participant hospitalization and MRSA screening history. These variables were selected a priori, based on the hypothesis that they might be associated with MRSA nasal carriage. Variables were coded to provide the best predication with the fewest degrees of freedom, as indicated by Akaike Information Criterion (AIC) statistics. The variable representing the presence, hospitalization and antibiotic use history of household members was coded as a 3-level categorical variable (0 = participant lived alone, 1 = participant lived with 1 or more household members, but none used antibiotics in the past 4 weeks or were hospitalized in the past 12 months, 2 = participant lived with 1 or more household members who used antibiotics in the past 4 weeks or were hospitalized in the past 12 months). Similarly, history of participant hospitalization and MRSA screening was coded as a 3-level categorical variable (0 = not hospitalized in the past 12 months, 1 = hospitalized but never screened positive for MRSA in the past 12 months, 2 = hospital- ized and screened positive for MRSA at least once in the past 12 months). These variables were coded to create mutually exclusive categories that could be included in the same multivariable model. All the other variables included in the multivariable model were coded dichotomously. Five cases had concomitant MRSA clinical infections— abscesses (n = 2), cellulitis/abscess, pneumonia, and bacteremia (Table S1). Thirteen (11.1%) cases and 3 (3.4%) controls had cellulitis or soft tissue infection diagnoses. Seven (6.0%) cases and 3 (2.5%) controls had diarrhea. Thirty-eight (31.9%) cases and 31 (26.5%) controls had diabetes. Odds ratios were derived from a conditional, multivariable model (Table 2). Cases had a higher odds of having less than a high school degree (OR: 2.04, 95% CI: 0.75–5.50), being a non- white race, having indoor cats or dogs (OR: 1.79; 95% CI: 0.88– 3.63), smoking cigarettes in the past 12 months (OR: 1.60; 95% CI: 0.80–3.19), and visiting a gym or playing sports in the past 2 weeks (OR: 2.64; 95% CI: 0.64–10.88). MRSA and Medical Household Exposures MRSA and Medical Household Exposures DNA was extracted using an UltraCleanTM Microbial DNA Isolation Kit (Mo Bio Laboratories, Solana Beach, CA). The NanoDropH ND-1000 Spectrophotometer (Isogen, Ijssel stein, The Netherlands) was used to estimate the genomic DNA concentrations. Extracts were diluted to give a final DNA concentration of 35 ng/ml. DNA was extracted using an UltraCleanTM Microbial DNA Isolation Kit (Mo Bio Laboratories, Solana Beach, CA). The NanoDropH ND-1000 Spectrophotometer (Isogen, Ijssel stein, The Netherlands) was used to estimate the genomic DNA concentrations. Extracts were diluted to give a final DNA concentration of 35 ng/ml. Because patients were not always available to be interviewed due to rapid turnover or medical treatments and surgery, and because not all eligible patients agreed to participate, at the end of data collection some participants were left without a matching case/control. To avoid double loss of information, in analysis, case and control matched sets were pooled. Pooled sets were created by identifying the appropriate gender/age match who was admitted within the shortest amount of time of the unmatched case/control. The use of the same control for more than one case has been described previously as a valid method that should not bias measures of association [24]. The DiversiLabH Staphylococcus kit for DNA fingerprinting (bioMerieux, Boxtel, Netherlands), a repetitive sequence-based PCR (rep-PCR), was used to amplify regions between repetitive, noncoding sequences in DNA samples. The DiversilabH system is a commercially available strain typing kit that is popular in clinical settings because it is less time consuming compared to pulsed-field gel electrophoresis and, unlike sequence based typing methods such as Staphylococcus Protein A (spa) or multi locus sequence typing, does not require access to DNA sequencing facilities [20]. The protocol was run according to manufacturer specifications. Software from the DiversiLabH system (version v.r.3.3.40) was used for genotyping [20]. DiversiLabH produces a dendogram, a graph of fluorescence intensity that corresponds to banding patterns, and a similarity matrix. The rep-PCR profiles were compared to the DiversiLabH MRSA library, which contains 70 samples of 14 representative USA pulsed field gel electrophoresis types [3]. Fingerprints with no banding differences were consid- ered matches and assigned the library type. Isolates without a match in the library were considered non-typeable, or no match. MRSA isolates were subsequently classified as CA, HA or non- matches. All statistical analyses were performed using Statistical Analysis Software version 9.3. Results Of 164 cases and 190 controls invited, 121 (73.8%) and 122 (64.2%) consented to participate, respectively. Subsequently, 4 cases and 3 controls were excluded based on having addresses outside the eligible zip codes, leaving 117 cases and 119 controls for analysis. One hundred (89.3%) matched sets had 1 case per control. Because cases and controls were pooled to avoid loss of information, 7 sets (6.3%) had 2 controls per case and 5 (4.5%) had 2 cases per control. All participants lived in 22 counties in eastern North Carolina or in the eastern-most portion of central NC. Sixty-eight (57.1%) controls and 67 (57.3%) cases were female (Table 1). Fifty-eight controls (48.7%) and 54 (46.2%) cases identified as non-Hispanic white. In the past 12 months, 94 cases (80.3%) and 93 controls (78.2%) used or were prescribed antibiotics. Cases and controls had similar numbers of household members. MRSA detection and typing VMC’s clinical microbiology laboratory used the BD Gen- OhmH MRSA polymerase chain reaction (PCR) to test nasal swabs for MRSA [22]. The BD GeneOhmH real-time PCR identifies MRSA by targeting the junction of the SCCmec right extremity (SRE) with a section of the orfX gene, which is specific to S. aureus [23]. Swabs were stored at 4uC for 24 – 48 hours, then transferred to the infection control laboratory and streaked onto a CHROMagarH MRSA plate (CHROM agar Microbiology, Paris, France). The CHROMagarH plate was incubated for 24 – 48 hours at 37uC. Mauve colonies were identified as MRSA. August 2013 \| Volume 8 \| Issue 8 \| e73595 PLOS ONE \| www.plosone.org 2 Statistical analysis Cases had a higher odds of past hospitalization with a positive MRSA screen (OR: 3.21; 95% CI: 1.12–9.23) but a lower odds of hospitalization without a positive screen in the past 12 months (OR: 0.40; 95% CI: 0.19– 0.87). Additionally, cases had 3.27 times the odds of living with someone who had used antibiotics in the past 4 weeks and/or was hospitalized in the past 12 months (95% CI: 1.24–8.57). Of 117 cases, 108 duplicate swabs were available for culturing and 49 (45.4%) grew MRSA colonies. Using the multivariable model, the 49 culture positive cases were compared to 52 matched controls (Table S2). Because of the small numbers of participants in these analyses, the resulting measures of association were imprecise. Being hospitalized but never screening positive for MRSA was negatively associated with MRSA carriage (OR: 0.29; 95% CI: 0.06–1.39). Living with someone who used antibiotics in the past 4 weeks and/or was hospitalized in the past year (OR: 4.76; 95% CI: 0.88–25.72), having less than a high school degree (OR: 2.65, 95% CI: 0.53–13.24); and having indoor pets (OR: 1.26; 95% CI: 0.40–3.99) was associated with a higher odds of MRSA carriage. Similar proportions of cultured cases and controls had prior hospitalization in addition to prior MRSA carriage (OR: In addition to the analysis of the full data set, cases whose isolates grew MRSA colonies were compared to their matched controls. Also, the relationship between HA or CA MRSA carriage and the explanatory variables was examined. In the analysis of CA and HA carriers, samples sizes were small; therefore, each explanatory variable was investigated in a separate model. PLOS ONE \| www.plosone.org August 2013 \| Volume 8 \| Issue 8 \| e73595 August 2013 \| Volume 8 \| Issue 8 \| e73595 3 MRSA and Medical Household Exposures Table 1. Characteristics of methicillin resistant Staphylococcus aureus nasal carriers and their gender and age matched controls. No. (%) Controls Cases (n = 119) (n = 117) Female 68 57.1 67 57.3 Age, y 18–29 29 24.4 24 20.5 30–39 15 12.6 16 13.7 40–49 17 14.3 22 18.8 50–59 40 33.6 37 31.6 60–65 18 15.1 18 15.4 Race or ethnicitya Non-Hispanic White 58 48.7 54 46.2 Non-Hispanic Black 55 46.2 53 45.3 Hispanic or Latino 3 2.5 4 3.4 Other 3 2.5 6 5.1 No. Discussion From the inception of the universal MRSA screening program at VMC in February of 2007 until December of 2011, approximately 7.9% of nearly 225,000 screened patients, exclud- ing the neonatal population, were identified as MRSA nasal carriers by PCR. We compared participants who were hospitalized and screened positive for MRSA on a previous occasion within the past year to participants who were not hospitalized in the past year; MRSA carriage identified by PCR predicted nasal carriage at later hospital admission. VMC prescribes mupirocin to MRSA carriers to decolonize their nares; patients complete the treatment either while they are hospitalized or as outpatients. Of 36 participants who screened positive for MRSA carriage on a previous visit to VMC within the past year, all but 1 were prescribed mupirocin after their last positive MRSA screen. This suggests that MRSA carriers who retested positive were re- colonized in the community. The finding that past MRSA carriage predicted a positive MRSA screen upon readmission could be partially explained by carriage of mupirocin resistant MRSA strains. Based on point prevalence estimates, low- and high-level mupirocin resistance was detected in 3.5% and 6.0% of isolates collected from VMC patients in 2011, respectively (unpublished data). Therefore, it is unlikely that mupirocin resistance fully explains these results. 1.03; 95% CI: 0.25–4.24). Also, lower proportions of culture positive cases than controls were non-Hispanic white (OR: 0.89; 95% CI: 0.26–3.09), smoked cigarettes in the past year (OR: 0.61; 95% CI: 0.14–2.62), and lived with someone who did not use antibiotics and was not hospitalized (OR: 0.60; 95% CI: 0.15– 2.46). Because of sparse data, the effect of gym use or sports participation in the past 2 weeks was not estimable. 1.03; 95% CI: 0.25–4.24). Also, lower proportions of culture positive cases than controls were non-Hispanic white (OR: 0.89; 95% CI: 0.26–3.09), smoked cigarettes in the past year (OR: 0.61; 95% CI: 0.14–2.62), and lived with someone who did not use antibiotics and was not hospitalized (OR: 0.60; 95% CI: 0.15– 2.46). Because of sparse data, the effect of gym use or sports participation in the past 2 weeks was not estimable. Participants who were hospitalized in the past 12 months without a prior positive MRSA screen had lower odds of MRSA carriage than participants who were not hospitalized in the past year. Previous research suggests that past hospitalization is associated with MRSA carriage [15,26,27]. Statistical analysis household members Mean and standard error 3.2 5.5 3.1 1.9 Min and max 0 .10 0 .10 Antibiotic use, past 12 mo.b 93 78.2 94 80.3 Surgery, past 12 mo. 40 33.6 46 39.3 Hospitalized, past 12 mo. 70 58.8 71 60.7 Abbreviations: y, year; mo, month; min, minimum; max, maximum. aRace/ethnicity was self-reported by participants during the interview. The other category includes participants identifying their race or ethnicity as Asian, American Indian, or other. bAny participant who was previously prescribed mupirocin for MRSA nasal carriage within the past year was coded as having taken antimicrobials. doi:10.1371/journal.pone.0073595.t001 CI: 0.14–2.51); were a non-white race (OR: 1.49; 95% CI: 0.32– 6.91; OR: 0.60; 95% CI: 0.14–2.51); and had household members who did not use antibiotics in the past 4 weeks and were not hospitalized in the past year (OR: 1.19; 95% CI: 0.19–7.53 and OR: 0.89; 95% CI:0.18–4.48). Compared to controls, higher proportions of CA and HA carriers had less than a high school degree (OR: 2.56; 95% CI: 0.46–14.29; OR: 1.50; 95% CI: 0.25– 8.98) and smoked cigarettes in the past year (OR: 2.36; 95% CI: 0.44–12.56; OR: 1.67; 95% CI: 0.40–6.97). Higher proportions of CA carriers had household members who used antibiotics in the past 4 weeks and/or were hospitalized in the past 12 months (OR: 3.64; 95% CI: 0.34–39.22). Due to sparse data, this effect was not estimable for HA carriers, and the effect of gym use/sports participation was not estimable for CA or HA carriers. CI: 0.14–2.51); were a non-white race (OR: 1.49; 95% CI: 0.32– 6.91; OR: 0.60; 95% CI: 0.14–2.51); and had household members who did not use antibiotics in the past 4 weeks and were not hospitalized in the past year (OR: 1.19; 95% CI: 0.19–7.53 and OR: 0.89; 95% CI:0.18–4.48). Compared to controls, higher proportions of CA and HA carriers had less than a high school degree (OR: 2.56; 95% CI: 0.46–14.29; OR: 1.50; 95% CI: 0.25– 8.98) and smoked cigarettes in the past year (OR: 2.36; 95% CI: 0.44–12.56; OR: 1.67; 95% CI: 0.40–6.97). Higher proportions of CA carriers had household members who used antibiotics in the past 4 weeks and/or were hospitalized in the past 12 months (OR: 3.64; 95% CI: 0.34–39.22). Due to sparse data, this effect was not estimable for HA carriers, and the effect of gym use/sports participation was not estimable for CA or HA carriers. Discussion Patients previously hospitalized with a negative MRSA PCR on admission may be less susceptible or less exposed in the community. August 2013 \| Volume 8 \| Issue 8 \| e73595 Molecular typing Patterns of MRSA carriage are commonly classified as persistent, intermittent, and absent. Previous research has shown that a high percentage of patients who initially screen negative for MRSA return to the hospital with negative screens on future visits, whereas high proportions of those who screen positive on an initial visit show patterns of persistent or intermittent carriage [28]. Although results should be interpreted tentatively since most of the measures of association were imprecise due to sparse data, results from our study contribute to evidence that the household environment may affect MRSA acquisition [7,8,29,30] and could play a role in intermittent or persistent carriage. Compared to living alone, living with household members who either used antibiotics or were hospitalized was positively associated with MRSA carriage. It is possible that household members acquired MRSA through past medical related exposures and then exposed MRSA carriers in this study. Of 49 isolates typed via rep-PCR, 7 (14.3%) did not match types in the DiversiLabH library. Twenty (40.8%) isolates matched the CA strain, USA300. The other 22 isolates matched HA strains; 15 (30.6%) were USA100, 2 (4.1%) were USA500, 3 (6.1%) were USA800, 1 (2.0%) was USA200, and 1 (2.0%) was USA600. None of the non-typeable strains had a pattern that matched the livestock associated multi locus sequence type 398 strain in the DiversiLabH typing system [25]. CA and HA carriers were compared with controls using conditional logistic regression models (Table S3). Because there were only 20 CA isolates and 22 HA isolates in these analyses, the resulting OR estimates were imprecise. However, a higher proportion of CA carriers but a lower proportion of HA carriers were previously hospitalized with a past positive MRSA screen (OR: 7.67; 95% CI: 0.86–68.38; OR: 0.54; 95% CI: 0.10–2.90); had indoor pets (OR: 1.93; 95% CI: 0.49–7.58; OR: 0.60; 95% August 2013 \| Volume 8 \| Issue 8 \| e73595 PLOS ONE \| www.plosone.org 4 MRSA and Medical Household Exposures Table 2. Estimates of association of methicillin resistant Staphylococcus aureus (MRSA) nasal carriage identified by polymerase chain reaction with medical and household exposures from a multivariable logistic model conditioned on age and gender. No. Molecular typing Estimates of association of methicillin resistant Staphylococcus aureus (MRSA) nasal carriage identified by polymerase chain reaction with medical and household exposures from a multivariable logistic model conditioned on age and gender. No household members No household members Household members used antibiotics in the past 4 weeks and/or hospitalized in the past 12 mo. 25 21 breviation: months, mo.; odds ratio, OR; confidence interval, CI. , ; , ; , Non-white or Hispanic includes non-Hispanic black, Hispanic/Latino, Asian, American Indian, or other race/ethnicities. The gym visitation/sports participation variable reflects the 2 weeks prior to the hospital admission, even though 2 cases and 1 control were screened for MRSA 9 or more days before their current hospital admission. E d i h d l 3 l l i l i bl , ; , ; , aNon-white or Hispanic includes non-Hispanic black, Hispanic/Latino, Asian, American Indian, or other race/ethnicities. bThe gym visitation/sports participation variable reflects the 2 weeks prior to the hospital admission, even though 2 cases and 1 control were screened for MRSA 9 or more days before their current hospital admission. aNon-white or Hispanic includes non-Hispanic black, Hispanic/Latino, Asian, American Indian, or other race/ethnicities. bThe gym visitation/sports participation variable reflects the 2 weeks prior to the hospital admission, even though 2 cases more days before their current hospital admission. Entered into the model as a 3-level categorical variable. oi:10.1371/journal.pone.0073595.t002 doi:10.1371/journal.pone.0073595.t002 methodology has been shown to be more sensitive than selective media, which might account for some of the higher detection [22]. However, these data could also raise concerns about false positives among the cases identified by PCR, which might have detected methicillin sensitive S. aureus containing SCCmec fragments that lack the mecA gene, non-viable bacteria, or DNA residue that remained following decolonization [23,32,33,34]. The percentage of PCR positive swabs that did not grow colonies was similar to that reported in some but not all studies. For example, in a neonatal intensive care unit, MRSA was detected by PCR in 21 of 435 (4.8%) nasal swabs, and 11 (52.4%) of the PCR positive swabs grew MRSA colonies when cultured [35]. In another study, 123 of 599 nasal swabs were positive by PCR, of which 77 (62.6%) were positive by culture without enrichment [36]. Molecular typing In a third study, 606 nose, throat, and groin/perineum swabs were collected from 202 patients; 120 MRSA specimens were detected by PCR, of which 85 (70.8%) were detected by direct culture [33]. In our study, the delay from the time when the duplicate swabs from PCR positives were transported and plated onto selective media varied from 24 – 48 hours, which could have reduced subsequent culture yields. Additionally, a higher proportion of cases might have grown colonies on selective media had overnight enrichment been used, which has been shown to improve the sensitivity of cultures [37]. Of 49 culture positive isolates, 22 (44.9%) matched HA types, 20 (40.8%) matched CA types and 7 (14.3%) did not have a match in the DiversiLabH MRSA library. To explore potential differ- ences in the epidemiology of HA versus CA MRSA, we examined associations between carriage of each MRSA type and the exposure variables. Because of small numbers of participants in these analyses, the measures of association were imprecise. However, a higher proportion of CA carriers than controls were hospitalized and screened positive for MRSA previously. Other studies have reported that CA strains may be carried by those with a history of hospitalization [5]. Based on small numbers, the proportion of culture positive cases carrying CA strains was high compared to that reported by others [5,15]. The high proportion of cases carrying CA MRSA in our study could be attributable to the exclusion of patients over age 65, other unique characteristics of the study population, or the relatively low yield (45%) of cultures grown and typed from MRSA nasal carriers identified by PCR. methodology has been shown to be more sensitive than selective media, which might account for some of the higher detection [22]. However, these data could also raise concerns about false positives among the cases identified by PCR, which might have detected methicillin sensitive S. aureus containing SCCmec fragments that lack the mecA gene, non-viable bacteria, or DNA residue that remained following decolonization [23,32,33,34]. The percentage of PCR positive swabs that did not grow colonies was similar to that reported in some but not all studies. For example, in a neonatal intensive care unit, MRSA was detected by PCR in 21 of 435 (4.8%) nasal swabs, and 11 (52.4%) of the PCR positive swabs grew MRSA colonies when cultured [35]. Molecular typing ( %) Controls Cases OR 95% CI (n = 119) (n = 117) At least high school or general education development degree 106 89.1 93 79.5 1.00 - Less than high school or general education development degree 13 10.9 24 20.5 2.04 0.75–5.50 No cats or dogs inside the home 79 66.4 72 61.5 1.00 - Cats or dogs inside the home 40 33.6 45 38.5 1.79 0.88–3.63 Non-Hispanic white race or ethnicitya 58 48.7 54 46.2 1.00 - Hispanic and/or non-white race or ethnicity 61 51.3 63 53.9 1.48 0.70–3.11 Did not smoke tobacco cigarettes in the past 12 mo. 76 63.9 65 55.6 1.00 - Smoked tobacco cigarettes in the past 12 mo. 43 36.1 52 44.4 1.60 0.80–3.19 Did not visit a gym or participate in sports in the past 2 weeksb 114 95.8 108 92.3 1.00 - Visited a gym or participated in sports in the past 2 weeks 5 4.2 9 7.7 2.64 0.64–10.88 Prior hospitalization and MRSA nasal carriage in the past 12 mo.c Not hospitalized in the past 12 mo. 49 41.2 46 39.3 1.00 - Hospitalized and never screened positive for MRSA in the past 12 mo. 64 53.8 41 35.0 0.40 0.19–0.87 Hospitalized and screened positive for MRSA at least once in the past 12 mo. 6 5.0 30 25.6 3.21 1.12–9.23 Household members c No household members 23 19.3 15 12.8 1.00 - Household members did not use antibiotics in the past 4 weeks and not hospitalized in the past 12 mo. 71 59.7 58 49.6 1.42 0.59–3.45 Household members used antibiotics in the past 4 weeks and/or hospitalized in the past 12 mo. 25 21.0 44 37.6 3.27 1.24–8.57 Abbreviation: months, mo.; odds ratio, OR; confidence interval, CI. aNon-white or Hispanic includes non-Hispanic black, Hispanic/Latino, Asian, American Indian, or other race/ethnicities. bThe gym visitation/sports participation variable reflects the 2 weeks prior to the hospital admission, even though 2 cases and 1 control were screened for MRSA 9 or more days before their current hospital admission. cEntered into the model as a 3-level categorical variable. Table 2. Estimates of association of methicillin resistant Staphylococcus aureus (MRSA) nasal carriage identified by polymerase chain reaction with medical and household exposures from a multivariable logistic model conditioned on age and gender. Table 2. References 1. Deurenberg RH, Stobberingh EE (2008) The evolution of Staphylococcus aureus. Infect Genet Evol 8: 747–763. 12. Casey JA, Cosgrove SE, Stewart WF, Pollak J, Schwartz BS (2013) A population-based study of the epidemiology and clinical features of methicil- lin-resistant Staphylococcus aureus infection in Pennsylvania, 2001–2010. Epidemiol Infect 141: 1166–1179. 1. Deurenberg RH, Stobberingh EE (2008) The evolution of Staphylococcus aureus. Infect Genet Evol 8: 747–763. 2. Herold BC, Immergluck LC, Maranan MC, Lauderdale DS, Gaskin RE, et al. (1998) Community-acquired methicillin-resistant Staphylococcus aureus in children with no identified predisposing risk. JAMA 279: 593–598. 2. Herold BC, Immergluck LC, Maranan MC, Lauderdale DS, Gaskin RE, et al. (1998) Community-acquired methicillin-resistant Staphylococcus aureus in children with no identified predisposing risk. JAMA 279: 593–598. 13. Bhattacharya D, Carleton H, Tsai CJ, Baron EJ, Perdreau-Remington F (2007) Differences in clinical and molecular characteristics of skin and soft tissue methicillin-resistant Staphylococcus aureus isolates between two hospitals in Northern California. J Clin Microbiol 45: 1798–1803. 3. McDougal LK, Steward CD, Killgore GE, Chaitram JM, McAllister SK, et al. (2003) Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococ- cus aureus isolates from the United States: establishing a national database. J Clin Microbiol 41: 5113–5120. 14. Stenhem M, Ortqvist A, Ringberg H, Larsson L, Olsson-Liljequist B, et al. (2006) Epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in Sweden 2000–2003, increasing incidence and regional differences. BMC Infect Dis 6: 30. 4. Seybold U, Kourbatova EV, Johnson JG, Halvosa SJ, Wang YF, et al. (2006) Emergence of community-associated methicillin-resistant Staphylococcus aureus USA300 genotype as a major cause of health care-associated blood stream infections. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 42: 647–656. 15. Hidron AI, Kourbatova EV, Halvosa JS, Terrell BJ, McDougal LK, et al. (2005) Risk factors for colonization with methicillin-resistant Staphylococcus aureus (MRSA) in patients admitted to an urban hospital: emergence of community- associated MRSA nasal carriage. Clin Infect Dis 41: 159–166. 5. Peterson AE, Davis MF, Julian KG, Awantang G, Greene WH, et al. (2012) Molecular and Phenotypic Characteristics of Healthcare- and Community- Associated Methicillin-Resistant Staphylococcus aureus at a Rural Hospital. PLoS One 7: e38354. associated MRSA nasal carriage. Clin Infect Dis 41: 159–166. 16. Popovich KJ, Smith KY, Khawcharoenporn T, Thurlow CJ, Lough J, et al. (2012) Community-Associated Methicillin-Resistant Staphylococcus aureus Colonization in High-Risk Groups of HIV-Infected Patients. Clin Infect Dis 54: 1296–1303. 6. Author Contributions Conceived and designed the experiments: LS SW PDMM DR JS KMR DLN. Performed the experiments: LS KLA. Analyzed the data: LS KLA. Contributed reagents/materials/analysis tools: KMR LS. Wrote the paper: LS SW PDMM DR JS KLA DLN KMR. Analysis interpretation: DR JS SW LS KLA KMR. Conceived and designed the experiments: LS SW PDMM DR JS KMR DLN. Performed the experiments: LS KLA. Analyzed the data: LS KLA. Contributed reagents/materials/analysis tools: KMR LS. Wrote the paper: LS SW PDMM DR JS KLA DLN KMR. Analysis interpretation: DR JS SW LS KLA KMR. Conceived and designed the experiments: LS SW PDMM DR JS KMR DLN. Performed the experiments: LS KLA. Analyzed the data: LS KLA. Contributed reagents/materials/analysis tools: KMR LS. Wrote the paper: Acknowledgments We thank Ruth Ethridge (Vidant Medical Center Clinical Microbiology Department), who helped collect duplicate nasal swabs for culturing and typing. We thank Meghan Davis, Amy Peterson, Ellen Silbergeld, and Christopher Heaney for their recommendations during the preliminary planning and designing of this study. In addition, we are grateful to Devon Hall, Dothula Baron, Jennifer Hall, Clifton Smith, and Norma Mejia for their advice when we designed the structured interview questions. Community and household exposures may influence MRSA carriage and VMC’s hospital-based screening program may be detecting carriage among those exposed to MRSA. Further study of the relationship between MRSA carriage and community exposures, and continued surveillance for strains entering hospitals will help elucidate the dynamic epidemiology of MRSA. Molecular typing Therefore, results from this study might not be generalizable to members of other North Carolina communities, or to non-hospitalized populations. Because VMC only screens patients’ anterior nares, people with MRSA on other regions of the body could have been misidentified as controls. Additionally, although cases could have been carrying multiple MRSA strains [38], DNA of only 1 bacterial colony per culture-positive swabs was extracted and typed. Finally, partici- pants’ knowledge of their screening results could have influenced their interview responses. Table S2 Estimates of association of methicillin resis- tant Staphylococcus aureus (MRSA) nasal carriage identified by culture with medical and household exposures from a multivariable logistic model condi- tioned on age and gender. Table S3 Estimates of association of health care and community associated methicillin resistant Staphylococ- cus aureus (MRSA) nasal carriage with medical and household exposures from conditional logistic regres- sion models. This study has several strengths. One author (L.S.) conducted all interviews and abstracted data from medical records, which helped maintain internal consistency. By conducting in- hospital interviews, we collected information that would be unavailable by record review alone. By using information in patients’ medical records, the quality of the interview data was improved. Overall, there were small amounts of missing data and participation rates were high. Using the rapid PCR testing methodology to screen patients for MRSA allowed the interviewer to identify and approach eligible patients soon after they were admitted [22]. (DOCX) Molecular typing In another study, 123 of 599 nasal swabs were positive by PCR, of which 77 (62.6%) were positive by culture without enrichment [36]. In a third study, 606 nose, throat, and groin/perineum swabs were collected from 202 patients; 120 MRSA specimens were detected by PCR, of which 85 (70.8%) were detected by direct culture [33]. In our study, the delay from the time when the duplicate swabs from PCR positives were transported and plated onto selective media varied from 24 – 48 hours, which could have reduced subsequent culture yields. Additionally, a higher proportion of cases might have grown colonies on selective media had overnight enrichment been used, which has been shown to improve the sensitivity of cultures [37]. This study was non-randomized. In recent decades, emphasis on p-values and statistical tests of significance has been strongly discouraged in non-randomized study for several reasons [31]. Therefore, we have interpreted ORs based on their magnitude and biological plausibility, using 95% CIs as measures of precision rather than tests of significance. The 236 participants of this study were residents of 22 North Carolina counties. Based on United States Census data, percent- ages of Black residents in these counties ranged from 15% to 62% [18]. By comparison, 21% of all North Carolina residents are The 236 participants of this study were residents of 22 North Carolina counties. Based on United States Census data, percent- ages of Black residents in these counties ranged from 15% to 62% [18]. By comparison, 21% of all North Carolina residents are These results must be interpreted cautiously. The BD GenOhmH real-time PCR identified the 117 cases. After subculturing on selective media, MRSA was recovered from 49 of 108 available duplicate swabs of the anterior nares. The PCR August 2013 \| Volume 8 \| Issue 8 \| e73595 August 2013 \| Volume 8 \| Issue 8 \| e73595 PLOS ONE \| www.plosone.org 5 MRSA and Medical Household Exposures discharge summaries of methicillin resistant Staphylo- coccus aureus (MRSA) nasal carriers and controls. (DOCX) Black [18]. Therefore, results from this study might not be generalizable to members of other North Carolina communities, or to non-hospitalized populations. Black [18]. Therefore, results from this study might not be generalizable to members of other North Carolina communities, or to non-hospitalized populations. discharge summaries of methicillin resistant Staphylo- coccus aureus (MRSA) nasal carriers and controls. (DOCX) Black [18]. MRSA and Medical Household Exposures 23. French GL (2009) Methods for screening for methicillin-resistant Staphylococcus aureus carriage. Clin Microbiol Infect 15 Suppl 7: 10–16. 32. Huletsky A, Giroux R, Rossbach V, Gagnon M, Vaillancourt M, et al. (2004) New real-time PCR assay for rapid detection of methicillin-resistant Staphylo- coccus aureus directly from specimens containing a mixture of staphylococci. J Clin Microbiol 42: 1875–1884. 24. Wacholder S, Silverman DT, McLaughlin JK, Mandel JS (1992) Selection of controls in case-control studies. III. Design options. Am J Epidemiol 135:1042– 50. 33. Rossney AS, Herra CM, Fitzgibbon MM, Morgan PM, Lawrence MJ, et al. (2007) Evaluation of the IDI-MRSA assay on the SmartCycler real-time PCR platform for rapid detection of MRSA from screening specimens. Eur J Clin Microbiol Infect Dis 26: 459–466. 25. Grisold AJ, Zarfel G, Hoenigl M, Krziwanek K, Feierl G, et al. (2010) Occurrence and genotyping using automated repetitive-sequence-based PCR of methicillin-resistant Staphylococcus aureus ST398 in Southeast Austria. Diagnostic Microbiology and Infectious Disease 66: 217–221. 34. Lucke K, Hombach M, Hug M, Pfyffer GE (2010) Rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) in diverse clinical specimens by the BD GeneOhm MRSA assay and comparison with culture. J Clin Microbiol 48: 981–984. 26. Muto CA, Jernigan JA, Ostrowsky BE, Richet HM, Jarvis WR, et al. (2003) SHEA guideline for preventing nosocomial transmission of multidrug-resistant strains of Staphylococcus aureus and enterococcus. Infect Control Hosp Epidemiol 24: 362–386. 35. Sarda V, Molloy A, Kadkol S, Janda WM, Hershow R, et al. (2009) Active surveillance for methicillin-resistant Staphylococcus aureus in the neonatal intensive care unit. Infect Control Hosp Epidemiol 30: 854–860. 27. Gorwitz RJ, Kruszon-Moran D, McAllister SK, McQuillan G, McDougal LK, et al. (2008) Changes in the prevalence of nasal colonization with Staphylococcus aureus in the United States, 2001-2004. J Infect Dis 197: 1226–1234. 36. Farley JE, Stamper PD, Ross T, Cai M, Speser S, et al. (2008) Comparison of the BD GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) PCR assay to culture by use of BBL CHROMagar MRSA for detection of MRSA in nasal surveillance cultures from an at-risk community population. J Clin Microbiol 46: 743–746. 28. Gupta K, Martinello RA, Young M, Strymish J, Cho K, et al. (2013) MRSA nasal carriage patterns and the subsequent risk of conversion between patterns, infection, and death. PLoS One 8: e53674. , 29. References Klevens RM, Morrison MA, Nadle J, Petit S, Gershman K, et al. (2007) Invasive methicillin-resistant Staphylococcus aureus infections in the United States. Jama 298: 1763–1771. 17. Wing S, Cole D, Grant G (2000) Environmental injustice in North Carolina’s hog industry. Environ Health Perspect 108: 225–231. 7. Davis MF, Iverson SA, Baron P, Vasse A, Silbergeld EK, et al. (2012) Household transmission of meticillin-resistant Staphylococcus aureus and other staphylo- cocci. Lancet Infect Dis 12: 703–716. 18. The North Carolina Rural Center (2012) Rural Data Bank Custom Data Search. Available: http://www.ncruralcenter.org/Accessed March 2013. 19. Pofahl WE, Goettler CE, Ramsey KM, Cochran MK, Nobles DL, et al. (2009) Active Surveillance Screening of MRSA and Eradication of the Carrier State Decreases Surgical-Site Infections Caused by MRSA. J Am Coll Surg 208: 981– 988. 8. Uhlemann AC, Knox J, Miller M, Hafer C, Vasquez G, et al. (2011) The environment as an unrecognized reservoir for community-associated methicillin resistant Staphylococcus aureus USA300: a case-control study. PLoS One 6: e22407. 20. Tenover FC, Gay EA, Frye S, Eells SJ, Healy M, et al. (2009) Comparison of typing results obtained for methicillin-resistant Staphylococcus aureus isolates with the DiversiLab system and pulsed-field gel electrophoresis. J Clin Microbiol 47: 2452–2457. 9. Ferreira JP, Anderson KL, Correa MT, Lyman R, Ruffin F, et al. (2011) Transmission of MRSA between companion animals and infected human patients presenting to outpatient medical care facilities. PLoS One 6: e26978. 10. Weese JS, Dick H, Willey BM, McGeer A, Kreiswirth BN, et al. (2006) Suspected transmission of methicillin-resistant Staphylococcus aureus between domestic pets and humans in veterinary clinics and in the household. Vet Microbiol 115: 148–155. 21. Statistical Analysis Software (2012) Maps Online Zipcode Dataset. January 2012 ed. Available: http://support.sas.com/rnd/datavisualization/mapsonline/html/ misc2012.html. Accessed 2012 January. 22. Boyce JM, Havill NL (2008) Comparison of BD GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) PCR versus the CHROMagar MRSA assay for screening patients for the presence of MRSA strains. J Clin Microbiol 46: 350– 351. 11. Cohen PR (2008) The skin in the gym: a comprehensive review of the cutaneous manifestations of community-acquired methicillin-resistant Staphylococcus aureus infection in athletes. Clin Dermatol 26: 16–26. August 2013 \| Volume 8 \| Issue 8 \| e73595 August 2013 \| Volume 8 \| Issue 8 \| e73595 PLOS ONE \| www.plosone.org 6 MRSA and Medical Household Exposures Miller LG, Diep BA (2008) Clinical practice: colonization, fomites, and virulence: rethinking the pathogenesis of community-associated methicillin- resistant Staphylococcus aureus infection. Clin Infect Dis 46: 752–760. 37. Van Heirstraeten L, Cortinas Abrahantes J, Lammens C, Lee A, Harbarth S, et al. (2009) Impact of a short period of pre-enrichment on detection and bacterial loads of methicillin-resistant Staphylococcus aureus from screening specimens. J Clin Microbiol 47: 3326–3328. p y 30. 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https://openalex.org/W3010778519	https://ejournal.undip.ac.id/index.php/jgti/article/download/17843/16568	Indonesian	null	RANCANGAN DISTRIBUSI LPG 3 KG BERDASARKAN KEBIJAKAN DISTRIBUSI SISTEM TERTUTUP DI WILAYAH PEMASARAN KOTA BANDUNG	J@ti Undip	2,020	cc-by-sa	8,424	RANCANGAN DISTRIBUSI LPG 3 KG BERDASARKAN KEBIJAKAN DISTRIBUSI SISTEM TERTUTUP DI WILAYAH PEMASARAN KOTA BANDUNG Sofiani Nalwin Nurbani* Prodi Teknik Industri, Fakultas Teknik, Universitas Sangga Buana Bandung Jl. PHH Mustofa N0. 68, Bandung 40124 (Received: March 2, 2018/ Accepted: November 21, 2019) Abstrak Pada tahun 2007 Pemerintah mulai melakukan konversi dari minyak tanah ke LPG 3 kg. Untuk mengatasi kelangkaan LPG 3 kg, Pertamina dan Pemerintah mulai melakukan perbaikan yang salah satunya adalah merubah sistem distribusi LPG 3 kg yang awalnya distribusi Sistem Terbuka menjadi distribusi Sistem Tertutup. Optimasi distribusi LPG 3kg dengan sistem terbuka dan sistem tertutup dilakukan dengan menggunakan metode Transportasi dengan linear programming. Perhitungan optimasi maka perhitungan dilakukan dengan bantuan software excel Analytic Solver Platform, sehingga didapatkan bahwa Ongkos Transportasi LPG 3kg berdasarkan Sistem Terbuka sebesar Rp.8.319.765.767,07 sedangkan untuk Sistem Tertutup sebesar Rp.8.724.410.280,30. Hasil akhir pengelompokan coverage area SPPBE berdasarkan sistem tertutup adalah SPPBE 1 memiliki wilayah pemasaran sejumlah 12 Agen dengan kapasitas SPPBE yang terpakai sebesar 867.473 tabung per bulan. SPPBE 2 memiliki wilayah pemasaran sejumlah 12 Agen dengan kapasitas SPPBE 2 yang terpakai yaitu sebesar 1.053.607 tabung per bulan. SPPBE 4 memiliki wilayah pemasaran sebanyak 7 Agen dengan kapasitas yang terpakai yaitu sebesar sebesar 802.747 tabung per bulan. Penelitian secara kualitatif dilakukan bertujuan untuk mengetahui kriteria-kriteria yang dibutuhkan dalam menentukan lokasi pendirian SPPBE baru. Perhitungan berdasarkan metode AHP dilakukan dengan software expertchoise. Hasil akhir pengukuran metode AHP diperoleh bahwa alternatif lokasi yang memiliki prioritas tertinggi adalah wilayah Kecamatan Gedebage. Kata Kunci: distribusi sistem tertutup; linear programming; software excel Analytic Solver Platform )Penulis Korespondensi. E-mail: sofiani.nalwin@usbypkp.ac.id/ sofianinalwinnurbani@gmail.com programming; excel Analytic Solver Platform programming; excel Analytic Solver Platform Abstract [3kg LPG Distribution Design Based On Closed System Distribution Policy In The Marketing Area Of Bandung City] In 2007 the Government started to convert from kerosene to LPG 3 kg. To overcome the scarcity of LPG 3kg, Pertamina and Government started to make improvements which one of them is to change the distribution system of LPG 3kg which initially the Open Systems distribution into the distribution of Closed Systems. Optimization of 3kg LPG distribution with open system and closed system is done by using Transportation method with linear programming. To simplify the calculation of optimization, the calculation is done with the help of software excel Analytic Solver Platform, so it is found that 3kg LPG Transportation Costs based on Open System Rp.8.319.765.767,07 while for Closed System Rp.8.724.410.280,30. The final result of SPPBE coverage area coverage based on closed system is SPPBE 1 has marketing area of 12 Agent with SPPBE capacity which is used is 867.473 tubes per month. SPPBE 2 has a marketing area of 12 Agents with SPPBE 2 capacity of 1,053,607 tubes per month. And SPPBE 4 has a marketing area of 7 Agents with SPPBE 4 capacity used, which amounted to 802,747 tubes per month. The qualitative research is aimed to find out the criteria needed to determine the location of new SPPBE establishment. Calculations based on the AHP method are performed with the help of expert choise software. Based on the final result of measurement of AHP method, it is found that the location alternative that has the highest priority is Gedebage sub-district. Keywords: loseloop distribution; linear )Penulis Korespondensi. E-mail: sofiani.nalwin@usbypkp.ac.id/ sofianinalwinnurbani@gmail.com 149 J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 1. Bahan Dan Metode p 3. Menentukan perkiraan lokasi alternatif SPPBE baru berdasarkan perkiraan permintaan sampai tahun 2030. Rancangan penelitian yang penulis lakukan dapat dilihat jelas pada gambar diagram alir penelitian secara umum di bawah ini. Selanjutnya peneliti akan menjabarkan setiap langkah yang dilakukan pada penelitian ini seperti yangakan dibahas pada subbab berikutnya. Pendahuluan Banyaknya warga berkategori mampu tetapi memakai gas ukuran 3 kg, Asumsi yang dilakukan dipenelitian ini di antaranya adalah : Asumsi yang dilakukan dipenelitian ini di antaranya adalah : g g 4. Restoran maupun hotel yang menggunakan gas ukuran 3 kg. 4. Restoran maupun hotel yang menggunakan gas ukuran 3 kg. 4. Restoran maupun hotel yang menggunakan gas ukuran 3 kg. 1) Data permintaan/ demand berdasarkan banyaknya Kepala Keluarga di Kota Bandung, dengan asumsi kebutuhan LPG 3 kg di tiap kelurahan sebesar 70%. Berdasarkan data penelitian ESDM, bahwa kebutuhan akan gas bumi akan mengalami peningkatan dari tahun ke tahun. Di mana kebutuhan akan gas bumi untuk tahun 2015-2020 akan meningkat sebesar 6% per tahun. Sedangkan pada tahun 2020- 2025 kebutuhan akan gas bumi meningkat sebesar 7% dan pada tahun 2025-2030 meningkat sebesar 5% pertahun. (Berita ESDM, 2014). 2) Kapasitas Truk Agen dibagi menjadi dua yaitu truk besar adalah 560 tabung dan truk kecil sebesar 360 tabung, maka kapastas Agen diasumsikan minimal sebesar 920 per bulan, 2) Kapasitas Truk Agen dibagi menjadi dua yaitu truk besar adalah 560 tabung dan truk kecil sebesar 360 tabung, maka kapastas Agen diasumsikan minimal sebesar 920 per bulan, 3) 1 liter premium/ solar untuk 12 km sehingga ongkos transportasi dari Agen ke Demand point untuk sistem tertutup sebesar Rp. 616,67, p Hal tersebut menandakan terjadinya peningkatan permintaan akan gas LPG 3 kg setiap tahunnya, oleh karena itu perlu dilakukan analisis penerapan sistem terbuka dan tertutup dalam pemsaran LPG 3 kg yang akan diterapkan oleh Pertamina. 4) Proyeksi pertumbuhan penduduk diasumsikan sama dengan kebutuhan akan gas bumi berdasarkan data dari ESDM yaitu untuk tahun 2015-2020 akan meningkat sebesar 6% per tahun, tahun 2020-2025 meningkat sebesar 7% dan pada tahun 2025-2030 meningkat sebesar 5% pertahun, 𝐷12 = 69√(𝑙𝑜𝑛𝑔1 −𝑙𝑜𝑛𝑔2)2 + (𝑙𝑎𝑡1 −𝑙𝑎𝑡2)2 6) Data penjualan yang digunakan adalah data penjualan di BPS Kota Bandung tahun 2013, 1.1 Tujuan Penelitian 5) Kapasitas SPPBE tak terbatas (Pertamina berhak menaikkan kuota (kapasitas) SPPBE), 5) Kapasitas SPPBE tak terbatas (Pertamina berhak menaikkan kuota (kapasitas) SPPBE), Sehubungan dengan tujuan dari distribusi LPG 3 kg adalah terpenuhinya permintaan akan LPG 3 kg di setiap tempat yang ada di Bandung, maka tujuan dari penelitian ini adalah sebagai berikut: 6) Kapasitas SPPBE ditentukan sebesar 12.000 MT pertahun, (Muchlis, Stefanus Eko Wiratno, 2009)) 1. Mengidentifikasi wilayah pemasaran (coverage area) untuk setiap SPPBE di Kota Bandung, 7) Wilayah alternatif berdasarkan wilayah Kecamatan. 2. Menghitung biaya distribusi LPG 3 kg berdasarkan sistem tertutup, 1.2 Pembatasan Dan Asumsi Penelitian Untuk mencari solusi dari suatu permasalahan maka diperlukannya batasan serta asumsi, dengan tujuan agar dapat memberikan arah serta proses penyelesaian masalah. Pembatasan dalam penelitian ini di antaranya adalah : Pendahuluan Bandung Raya merupakan salah satu wilayah metropolitan yang meliputi: Kota Bandung, Kabupaten Bandung, Kabupaten Bandung Barat dan Kota Cimahi. Kota Bandung merupakan Ibukota Propinsi Jawa Barat. Kota Bandung terdiri atas 30 Kecamatan dan 151 Kelurahan dengan jumlah penduduk sebanyak 2.483.977 jiwa. Elpiji merupakan salah satu produk andalan PT. Pertamina untuk bahan bakar rumah tangga maupun usaha kecil, usaha menengah dan usaha besar yang berbentuk LPG (Liquefied Petroleum Gas). Pada tahun 2009 pemerintah mengeluarkan ketentuan mengenai produksi dan distribusi LPG 3 Kg yang telah diatur oleh Pemerintah dalam hal ini Kementrian ESDM melalui Permen Nomor 26 Tahun 2009 Tentang Penyediaan dan Pendistribusian Liquefied Petroleum Gas. Peraturan tersebut mengartikan bahwa sistem distribusi LPG 3 kg mengalami perubahan sistem dari sistem distribusi terbuka (open system) menjadi sistem distribusi tertutup (closed system). SPPBE yang tersebar di Wilayah Metropolitan Bandung terdapat 31 SPPBE yang tersebar di setiap Wilayah Metropolitan Bandung. Dari gambar dapat dilihat bahwa SPBE yang ada di wilayah Bandung Kota ada 6 (enam) SPPBE yaitu Patra Trading Depot Ujung Berung, PT. Limagas Jaya Mandiri, PT. Purnatarum Murni Rahayu 1, Patra Ujung Berung, PT. Mandalagiri Kaya Raya, dan PT. Purnatarum Murni Rahayu 2. Sistem distribusi terbuka (open system) diartikan bahwa pangkalan/sub agen boleh mengambil LPG dari sejumlah agen mana saja, bahkan pengecer bisa mengambil LPG langsung ke sejumlah agen dan agen boleh mengambil LPG dari SPPBE mana saja. Sedangkan distribusi tertutup (closed system) diartikan bahwa suatu sistem distribusi yang diterapkan untuk pengisian LPG di mana Agen LPG di satu wilayah hanya bisa mengisi LPG di stasiun (SPPBE) yang ada di wilayah itu. Mereka juga hanya diperbolehkan mendistribusikan LPG di wilayah yang sudah ditetapkan. Dari hasil pengamatan maka diperlukan sebuah rancangan distribusi LPG 3 kg jika sistem tertutup diterapkan di wilayah Kota Bandung. Serta kebutuhan akan SPPBE yang dikarenakan oleh permintaan LPG 3kg yang selalu mengalami peningkatan tiap tahunnya. Beberapa hal yang mengakibatkan peningkatan permintaan akan LPG 3kg di antaranya adalah: (Siagian, 2014), 1. Ekspansi penggunaan LPG 12 kg ke LPG 3kg akibat dari kenaikan harga LPG 12kg, 2. Meningkatnya usaha kecil dan menengah pengguna LPG 3kg, J@ti Undip: Jurnal Teknik Industri Vol 14 No 3 September 2019 1 Gambar 1 Peta Lokasi Penyebaran SPPBE untuk Wilayah Bandung Kota Gambar 1 Peta Lokasi Penyebaran SPPBE untuk Wilayah Bandung Kota 150 J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 3. Banyaknya warga berkategori mampu tetapi memakai gas ukuran 3 kg, 3. Perhitungan Jarak Perhitungan ini dilakukan untuk menghitung jarak antara SPPBE ke Agen dan Agen ke Demand Point (kecamatan). Jarak antara SPPBE dengan Agen serta Agen dengan demand point (kecamatan) akan dihitung dengan menggunakan metode Euclidean. Ada beberapa data jarak yang digunakan adalah koordinat lokasi berupa longitude dan latitude, , maka akan dihitung jarak antar lokasi dengan persamaan yang ditentukan oleh Simchi, 1997. Persamaan tersebut digunakan untk menghitung jarak antar dua titik di bumi. Berikut ini merupakan rumus perhitungannya: (Anggaini, 2010) 1) Penelitian hanya dilakukan dari SPPBE, Agen dan Konsumen (demand point), 2) Konsumen (demand point) yang digunakan berdasarkan wilayah Kelurahan, 3) Data jumlah penduduk diambil dari BPS (Badan Pusat Statistik) dari data Bandung Dalam Angka tahun 2014, 4) Demand point SPPBE berdasarkan jumlah Kepala Keluarga (KK) per Kelurahan. 5) Perancangan tidak memperhatikan alat transportasi (truck/pickup) yang digunakan dalam mengangkut LPG dari SPPBE ke Agen dan dari Agen ke Kelurahan (demand point), 𝐷12 = 69√(𝑙𝑜𝑛𝑔1 −𝑙𝑜𝑛𝑔2)2 + (𝑙𝑎𝑡1 −𝑙𝑎𝑡2)2 Perhitungan Kebutuhan LPG per Kelurahan 7) Metode peramalan yang digunakan pada penelitian ini adalah Metode Konstan, Regresi Linear, Single Exponencial Smoothing dengan α sebesar 0,2 , 0,5 , dan 0,9, Double Exponencial Smoothing dengan α sebesar 0,2 , 0,5 , dan 0,9, dan metode Moving Average Periode 3 bulan dan 5 bulan. Standar error yang digunakan adalah SEE. Untuk perancangan distribusi untuk sistem tertutup ada beberpa hal yang harus dilakukan yaitu menghitung permintaan. Perhitungan demand point merupakan banyaknya permintaan akan LPG 3kg berdasarkan banyaknya penduduk tiap Kelurahan. Data-data yang mempengaruhi permintaan akan LPG 3kg di antaranya adalah sebagai berikut ini: Jumlah J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 151 Kepala Keluarga (KK) per kelurahan dan Jumlah warung makan dan rumah makan per kelurahan. Kepala Keluarga (KK) per kelurahan dan Jumlah warung makan dan rumah makan per kelurahan. kg yang terjadi saat ini (eksisting-sistem terbuka) adalah pasokan bulk LPG dipasok oleh Depot area Pertamina, lalu Agen datang membeli LPG dalam kemasan tabung 3 kg dengan cara melakukan pengisian ulang tabung LPG 3 kg ke SPPBE. Agen melakukan pemasaran LPG ke wilayah mana saja tetetapi masih dalam wilayah pemasaran (Kota Bandung bukan Kebupaten Bandung). Gambar 3 akan menggambarkan skema pendistribusian LPG 3 kg eksisting . Maka rumus untuk melakukan perhitungan kebutuhan akan LPG 3 kg per bulan adalah sebagai berikut : 𝑑𝑘= 𝑘𝑜𝑛𝑠𝑢𝑚𝑒𝑛= 31 ℎ𝑎𝑟𝑖 𝑙𝑎𝑚𝑎 𝑝𝑒𝑚𝑎𝑘𝑎𝑖𝑎𝑛 𝑑𝑘= 𝑘𝑜𝑛𝑠𝑢𝑚𝑒𝑛= 31 ℎ𝑎𝑟𝑖 𝑙𝑎𝑚𝑎 𝑝𝑒𝑚𝑎𝑘𝑎𝑖𝑎𝑛 𝑑𝑘= 𝑘𝑜𝑛𝑠𝑢𝑚𝑒𝑛= 31 ℎ𝑎𝑟𝑖 𝑙𝑎𝑚𝑎 𝑝𝑒𝑚𝑎𝑘𝑎𝑖𝑎𝑛 𝑑𝑘= 𝑘𝑜𝑛𝑠𝑢𝑚𝑒𝑛= 31 ℎ𝑎𝑟𝑖 𝑙𝑎𝑚𝑎 𝑝𝑒𝑚𝑎𝑘𝑎𝑖𝑎𝑛 Maka banyaknya kebutuhan (demand) akan LPG 3 kg per bulan per kelurahan adalah : g Sedangkan skema pendistribusian LPG 3 kg berdasarkan distribusi sistem tertutup (closed system) di mana wilayah pemasaran setiap SPPBE berbeda dan tiap Agen pun memiliki wilayah pemasaran yang berbeda. p p 𝑑𝑡𝑜𝑡= (𝑑𝑘∗𝑗𝑢𝑚𝑙𝑎ℎ 𝑅𝑇) + (𝑑𝑘∗𝑗𝑢𝑚𝑙𝑎ℎ 𝑤𝑎𝑟𝑢𝑛𝑔 𝑚𝑎𝑘𝑎𝑛) + (𝑑𝑘∗𝑗𝑢𝑚𝑙𝑎ℎ 𝑤𝑎𝑟𝑢𝑛𝑔 𝑚𝑎𝑘𝑎𝑛) J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 Optimasi Distribusi LPG Parameter Penelitian Penelitian ini dilakukan di wilayah Bandung kota dengan skema penyediaan pendistribusian LPG 3 Supply Point SPPBE A Agen A (Utama) Agen B Agen C Sub Agen Sub Agen Pangkalan Sub Agen Pangkalan Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN SPPBE B Supply Point WILAYAH PEMASARAN (COVERAGE AREA) Gambar 2 Skema Distribusi Sistem Terbuka WILAYAH PEMASARAN (COVERAGE AREA) Retailer / Pengecer Agen B Agen A (Utama) SPPBE A Pangkalan Supply Point Pangkalan Gambar 2 Skema Distribusi Sistem Terbuka J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 152 J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 Supply Point SPPBE A Agen A (Utama) Agen B Agen C Sub Agen Sub Agen Pangkalan Sub Agen Pangkalan Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN SPPBE B Supply Point WILAYAH PEMASARAN A (COVERAGE AREA) WILAYAH PEMASARAN B (COVERAGE AREA) Gambar 3 Skema Distribusi Sistem Tertutup KETERANGAN : Demand Point SPPBE Agen Sub Agen/Pangkalan Retailer/Pengecer Gambar 4 Skema Distribusi LPG 3 kg Berdasarkan Closed System Model distribusi tertutup yang menjadi kajian ada penelitian ini dapat digambarkan lebih jelas pada ambar 4 skema distribusi closed system berikut ini. Tujuan Pemerintah melakukan di sistem tertutup adalah untuk mentertibkan pe dan pendistribusian LPG 3 kg. Optimasi Distribusi LPG Parameter Penelitian Sedangkan tuju setiap distribusi adalah meminimasi ongkos di Supply Point SPPBE A Agen A (Utama) Agen B Agen C Sub Agen Sub Agen Pangkalan Sub Agen Pangkalan Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN SPPBE B Supply Point WILAYAH PEMASARAN A (COVERAGE AREA) WILAYAH PEMASARAN B (COVERAGE AREA) Gambar 3 Skema Distribusi Sistem Tertutup Supply Point SPPBE A Agen A (Utama) Agen B Agen C Sub Agen Sub Agen Pangkalan Sub Agen Pangkalan Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN SPPBE B Supply Point WILAYAH PEMASARAN A (COVERAGE AREA) WILAYAH PEMASARAN B (COVERAGE AREA) Gambar 3 Skema Distribusi Sistem Tertutup WILAYAH PEMASARAN A (COVERAGE AREA) WILAYAH PEMASARAN A (COVERAGE AREA) Retailer / Pengecer Sub Agen Supply Point Agen B Retailer / Pengecer SPPBE A Retailer / Pengecer Agen A (Utama) Retailer / Pengecer Pangkalan Retailer / Pengecer Sub Agen Retailer / Pengecer Agen C Pangkalan Retailer / Pengecer Supply Point WILAYAH PEMASARAN B (COVERAGE AREA) ( Gambar 3 Skema Distribusi Sistem Tertutup ( Gambar 3 Skema Distribusi Sistem Tertutup Gambar 3 Skema Distribusi Sistem Tertutup KETERANGAN : Demand Point SPPBE Agen Sub Agen/Pangkalan Retailer/Pengecer Gambar 4 Skema Distribusi LPG 3 kg Berdasarkan Closed System KETERANGAN : Demand Point SPPBE Agen Sub Agen/Pangkalan Retailer/Pengecer Gambar 4 Skema Distribusi LPG 3 kg Berdasarkan Closed System Gambar 4 Skema Distribusi LPG 3 kg Berdasarkan Closed System Model distribusi tertutup yang menjadi kajian pada penelitian ini dapat digambarkan lebih jelas pada gambar 4 skema distribusi closed system berikut ini. Model distribusi tertutup yang menjadi kajian pada penelitian ini dapat digambarkan lebih jelas pada gambar 4 skema distribusi closed system berikut ini. Tujuan Pemerintah melakukan distribusi sistem tertutup adalah untuk mentertibkan pengisian dan pendistribusian LPG 3 kg. Sedangkan tujuan dari setiap distribusi adalah meminimasi ongkos distribusi. J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 153 Jika dilihat dari permasalahan yang terjadi pada distribusi LPG 3 kg berdasarkan sistem tertutup, maka penentuan wilayah pemasaran tiap SPPBE yang menjadi permasalahannya. Metode transportasi merupakan metode yang bertujuan untuk menetapkan suplai yang cukup untuk beberapa lokasi tujuan dari beberapa dengan minimum biaya untuk pabrik dan kapasitas DC. menyelesaikan permasalahan dari distribusi sistem tertutup LPG 3 kg. Karena dengan metode transportasi, peneliti dapat memperoleh hasil akhir berupa wilayah pemasaran dan jumlah tabung yang didistribusikan oleh Agen berdasarkan minimasi ongkos dan serta jarak. Pada gambar 5 dapat dilihat variabel yang mempengaruhi dalam permasalahan distribusi sistem tertutup. Dilihat dari skema distribusi sistem tertutup LPG 3 kg maka metode transportasi dipandang dapat SPPBE A Agen A Agen B Agen C Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN KONSUMEN SPPBE A WILAYAH PEMASARAN A (COVERAGE AREA) WILAYAH PEMASARAN B (COVERAGE AREA) ij ij ij d x c i j k jk jk jk d x c ij ij ij d x c jk jk jk d x c ij ij ij d x c jk jk jk d x c Gambar 5. Variabel-variabel yang Mempengaruhi Distribusi LPG 3 Kg Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer Agen A Retailer / Pengecer Retailer / Pengecer Retailer / Pengecer k Gambar 5. Variabel-variabel yang Mempengaruhi Distribusi LPG 3 Kg ∑∑𝑐𝑖𝑗𝑥𝑖𝑗𝑑𝑖𝑗 𝑚 𝑗=1 𝑛 𝑖=1 + ∑∑𝑐𝑗𝑘𝑥𝑗𝑘𝑑𝑗𝑘 𝑝 𝑘=1 𝑚 𝑗=1 Pada permasalahan distribusi system tertutup LPG 3 kg terdapat beberapa variabel yang mempengaruhinya di antaranya adalah : 1. Cij : biaya transportasi dari SPPBE ke Agen 1. Cij : biaya transportasi dari SPPBE ke Agen a) Faktor Pembatas (Constraint) a) Faktor Pembatas (Constraint) a. Untuk sistem terbuka : a) Faktor Pembatas (Constrain a. Untuk sistem terbuka : a) Faktor Pembatas (Constraint) a. Untuk sistem terbuka : a. Untuk sistem terbuka : a. Untuk sistem terbuka : 2. Xij : Jumlah unit yang dikirimkan = banyaknya yang dikirim dari SPPBE tidak melebihi kapasitas SPPBE 3. Keterangan Notasi : Keterangan Notasi : Keterangan Notasi : Keterangan Notasi : i = Index untuk SPPBE (1,2,...n) yij = 1 bila SPPBE dipilih dan 0 jika tidak j = Indeks untuk Agen (1,2,..m) yj = 1 bila Agen dipilih dan 0 jika tidak k = Indeks untuk demand point (1,2,...p) xij = volume yang dikirim dari SPPBE ke Agen Dk = permintaan bulanan dari demand point xjk = volume yang dikirim Agen ke Demand point djk = Jarak antara Agen ke Demand point xik = volume yang dikirim SPBBE ke demand point dik = jarak antara SPBBE ke demand point Kj =Kapasitas bulanan Agen dij = jarak antara SPPBE ke Agen cij=ongkos transportasi SPPBE-Agen Ki = Kapasitas bulanan SPPBE cjk = biaya transportasi Agen- Demand point cik= biaya transportasi SPPBE – Demand point B = banyaknya Agen = 31 R = Banyaknya Kelurahan = 151 yij = 1 bila SPPBE dipilih dan 0 jika tidak i = Index untuk SPPBE (1,2,...n) = total binary penugasan SPPBE- Agen sama dengan 1, artinya satu Agen hanya dilayani oleh satu SPPBE j = Indeks untuk Agen (1,2,..m) yj = 1 bila Agen dipilih dan 0 jika tidak = banyaknya yang dikirim dari SPPBE sama dengan Kapasitas Agen dikalikan dengan binary alokasi untuk SPPBE - Agen k = Indeks untuk demand point (1,2,...p) xij = volume yang dikirim dari SPPBE ke Agen xjk = volume yang dikirim Agen ke Demand point Dk = permintaan bulanan dari demand point = banyaknya yang dikirim dari SPPBE tidak melebihi kapasitas SPPBE djk = Jarak antara Agen ke Demand point xik = volume yang dikirim SPBBE ke demand point = banyaknya binary penugasan tidak melebihi jumlah Kelurahan, artinya satu Agen boleh melayani lebih dari satu Kelurahan tetapi tidak boleh melebihi dari jumlah total Kelurahan = total binary penugasan Agen- Kelurahan sama dengan 1, artinya satu Kelurahan hanya dilayani oleh satu Agen = banyaknya yang dikirim dari Agen sama dengan demand dikalikan dengan binary alokasi untuk Agen – Kelurahan = banyaknya yang dikirim dari Agen sama dengan demand dikalikan dengan binary alokasi untuk Agen – Kelurahan = jumlah yang dikirim dari SPBBE sama dengan jumlah yang dikirim Agen ke demand point Untuk mengidentifikasi lokasi alternatif pendirian SPPBE baru, pendekatan kualitatif harus diterapkan pada tahap ini karena setelah dilakukan perancangan distribusi LPG 3kg maka perlu diketahui lokasi (coverage area) yang memiliki hubungan terbesar terhadap kriteria pemilihan lokasi. J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 dij : Jarak dari SPPBE ke Agen di mana ; i= SPPBE, j = Agen, dan, k = Retailer (demand point berdasarkan kelurahan) Maka rumusan Metode transportasi dengan linear programming dapat ditulis sebagai berikut : Maka rumusan Metode transportasi dengan linear programming dapat ditulis sebagai berikut : = Jumlah yang dikirim dari SPPBE kesetiap agen lebih kecil atau sama dengan kapasitas pada agen Fungsi Objektif : Sistem Terbuka = Jumlah yang dikirim dari Agen ke setiap demand point bisa lebih besar atau sama dengan demand di demand point. Minimasi Ongkos : = Jumlah yang dikirim ke Agen dari SPPBE sama dengan jumlah yang dikirim ke setiap demand point = Jumlah yang dikirim ke Agen dari SPPBE sama dengan jumlah yang dikirim ke setiap demand point 𝑗 Sistem Tertutup p Minimasi Ongkos SPPBE ke Demand point: J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 154 𝑥𝑖𝑗, 𝑥𝑗𝑘 ≥0 𝐾𝑗≥920 = kapasitas minimal agen sebesar 920 tabung 𝑥𝑖𝑗, 𝑥𝑗𝑘 ≥0 𝑦𝑖𝑗, 𝑦𝑗, 𝑦𝑗𝑘, 𝑦𝑘= 𝐵𝑖𝑛𝑎𝑟𝑦 (0,1) 𝑥𝑖𝑗, 𝑥𝑗𝑘= 𝑖𝑛𝑡𝑒𝑔𝑒𝑟 Keterangan Notasi : Berikut ini kriteria dan faktor keputusan yang menjadi bahan kajian didalam kuesioner. = total yang dikirim dari Agen ke Kelurahan menjadi kapasitas agen = total yang dikirim dari Agen ke Kelurahan menjadi kapasitas agen g ∑𝑥𝑗𝑘= 𝐾𝑗 𝑝 = banyaknya yang dikirim dari Agen menjadi kapasitas agen g = banyaknya yang dikirim dari Agen menjadi kapasitas agen Tabel 1 Kriteria dan Faktor Keputusan Penentuan Lokasi SPPBE Tabel 1 Kriteria dan Faktor Keputusan Penentuan Lokasi SPPBE Tabel 1 Kriteria dan Faktor Keputusan Penentuan Lokasi SPPBE No. Kriteria Faktor Keputusan 1 Keadaan populasi Jumlah populasi, tingkat pendapatan 2 Kondisi transportasi Kestrategisan, jumlah transportasi umum, jumlah pejalan kaki, arus lalu lintas, tingkat kemacetan lalu lintas, ketersediaan transportasi umum, jalan raya yang besar 3 Kondisi Pasar Jumlah kepala keluarga, jumlah Warung makan, jumlah PKL, jumlah Pasar Tradisional, kedekatan dengan pusat perbelanjaan 4 Kondisi lokasi Luas lokasi, mudah dilihat. kedekatan dengan depot Pertamina, kedekatan dengan pemukiman warga, kedekatan dengan listrik tekanan tinggi 5 Biaya yang terkait Biaya tanah, pajak, biaya produksi, status kepemilikan lahan, profit Tabel 1 Kriteria dan Faktor Keputusan Penentuan Lokasi SPPBE No. Kriteria Faktor Keputusan 1 Keadaan populasi Jumlah populasi, tingkat pendapatan 2 Kondisi transportasi Kestrategisan, jumlah transportasi umum, jumlah pejalan kaki, arus lalu lintas, tingkat kemacetan lalu lintas, ketersediaan transportasi umum, jalan raya yang besar 3 Kondisi Pasar Jumlah kepala keluarga, jumlah Warung makan, jumlah PKL, jumlah Pasar Tradisional, kedekatan dengan pusat perbelanjaan 4 Kondisi lokasi Luas lokasi, mudah dilihat. kedekatan dengan depot Pertamina, kedekatan dengan pemukiman warga, kedekatan dengan listrik tekanan tinggi 5 Biaya yang terkait Biaya tanah, pajak, biaya produksi, status kepemilikan lahan, profit 𝑥𝑖𝑗, 𝑥𝑗𝑘= 𝑖𝑛𝑡𝑒𝑔𝑒𝑟 b. Untuk sistem tertutup: = banyaknya binary penugasan tidak melebihi jumlah Agen, artinya satu SPPBE boleh melayani lebih dari satu Agen tetapi tidak boleh melebihi dari jumlah total Agen 𝑦𝑖𝑗, 𝑦𝑗, 𝑦𝑗𝑘, 𝑦𝑘= 𝐵𝑖𝑛𝑎𝑟𝑦 (0,1) 𝑥𝑖𝑗, 𝑥𝑗𝑘= 𝑖𝑛𝑡𝑒𝑔𝑒𝑟 Keterangan Notasi : Faktor Keputusan b) Sistem Tertutup enam buah SPPBE tetapi hanya tiga SPPBE yang ditugaskan untuk pengisian tabung LPG 3 kg di antaranya adalah : Faktor Keputusan 155 3. Hasil Dan Pembahasan Pada tahap pengolahan hasil penelitian, peneliti melakukan dua tahapan penyelesian permasalahan yaitu : 1) Perhitungan Ongkos Distribusi Perhitungan ongkos distribusi dilakukan untuk mempermudah perhitungan dengan menggunakan excel solver. Di Kota Bandung terdapat enam buah SPPBE tetapi hanya tiga SPPBE yang ditugaskan untuk pengisian tabung LPG 3 kg di antaranya adalah : 1. SPPBE Wira Bangun Sarana (SPPBE 1) 2. SPPBE Patra Trading Ujung Berung (SPPBE 2), 3. SPPBE Purnatarum Murni Rahayu Putra (SPPBE4) Tabel 2 Contoh Hasil Perhitungan Jarak SPPBE - KELURAHAN KELURAHAN SPPBE Koordinat 1 2 3 SPPBE Wira Bangun Sarana SPPBE 1 6,97 10,176 9,475 9,424 107,64 SPPBE Patra Trading Ujung Berung SPPBE 2 6,94 9,885 9,312 9,655 107,64 107,69 SPPBE Purnatarum Murni Rahayu Putra SPPBE 4 6,94 9,969 9,396 9,739 107,64 107,68 Sumber: Hasil Pengolahan Data Tabel 3 Contoh Hasil Perhitungan Ongkos Distribusi dari SPPBE ke Kelurahan Kel. SPPBE Trans. Fee 1 2 3 4 5 SPPBE 1 Rp 390,10 2.326,15 2.153,36 2.404,81 2.360,25 1.547,66 SPPBE 2 Rp 390,10 1.949,71 1.790,55 2.071,10 2.051,34 1.169,85 SPPBE 4 Rp 390,10 1.326,33 1.134,59 1.395,74 1.382,71 572,04 Sumber: Hasil Pengolahan Data Optimasi Ongkos Distribusi LPG 3 kg Optimasi ongkos distribusi yang akan dibahas pada penelitian ini adalah ongkos distribusi LPG dengan sistem Terbuka dan Sistem Tertutup. a) Sistem Terbuka Tabel 4. Hasil Perhitungan Optimasi SPPBE - Agen (dalam satuan tabung), Agen SPPBE Agen 1 Agen 2 Agen 3 ..... Agen 30 Agen 31 TOTAL KAP. SPPBE SPPBE 1 0 27881 0 ..... 0 155574 866927 930.000 SPPBE 2 20929 0 76658 ..... 98749 0 1054000 1.054.000 SPPBE 4 0 0 0 ..... 0 0 802900 802.900 TOTAL 20.929 27.881 76.658 ..... 98.749 155.574 KAP. Agen 20.929 27.881 76.658 ..... 98.749 155.574 - Sumber: Hasil Pengolahan Data Tabel 5. Hasil Perhitungan Optimasi Agen – Kelurahan (dalam satuan tabung) KELURAHAN AGEN 1 2 3 ..... 151 TOTAL KAP. Agen Agen 1 0 0 0 ..... 0 20929 920 Agen 2 0 0 0 ..... 0 27881 920 Agen 3 0 0 0 ..... 0 76658 920 : : : : : : : : ..... : : : : : : Agen 31 0 0 10741 ..... 0 155574 920 TOTAL 21459 31483 10741 ..... 12486 DEMAND 21459 31483 10741 ..... 12486 Berdasarkan hasil perhitungan dengan menggunakan Excel Analytic Solver Platform diperoleh Ongkos Transportasi LPG 3 kg berdasarkan Sistem Terbuka adalah sebesar Rp 8.319.765.767,07. 3. Hasil Dan Pembahasan Pada tahap pengolahan hasil penelitian, peneliti melakukan dua tahapan penyelesian permasalahan yaitu : Tabel 2 Contoh Hasil Perhitungan Jarak SPPBE - KELURAHAN KELURAHAN SPPBE Koordinat 1 2 3 SPPBE Wira Bangun Sarana SPPBE 1 6,97 10,176 9,475 9,424 107,64 SPPBE Patra Trading Ujung Berung SPPBE 2 6,94 9,885 9,312 9,655 107,64 107,69 SPPBE Purnatarum Murni Rahayu Putra SPPBE 4 6,94 9,969 9,396 9,739 107,64 107,68 Sumber: Hasil Pengolahan Data Tabel 3 Contoh Hasil Perhitungan Ongkos Distribusi dari SPPBE ke Kelurahan Kel. SPPBE Trans. Fee 1 2 3 4 5 SPPBE 1 Rp 390,10 2.326,15 2.153,36 2.404,81 2.360,25 1.547,66 SPPBE 2 Rp 390,10 1.949,71 1.790,55 2.071,10 2.051,34 1.169,85 SPPBE 4 Rp 390,10 1.326,33 1.134,59 1.395,74 1.382,71 572,04 Sumber: Hasil Pengolahan Data J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 Optimasi Ongkos Distribusi LPG 3 kg Optimasi Ongkos Distribusi LPG 3 kg Optimasi ongkos distribusi yang akan dibahas pada penelitian ini adalah ongkos distribusi LPG dengan sistem Terbuka dan Sistem Tertutup. Tabel 4. Hasil Perhitungan Optimasi SPPBE - Agen (dalam satuan tabung), Tabel 4. Hasil Perhitungan Optimasi SPPBE - Agen (dalam satuan tabung), Agen SPPBE Agen 1 Agen 2 Agen 3 ..... Agen 30 Agen 31 TOTAL KAP. SPPBE SPPBE 1 0 27881 0 ..... 0 155574 866927 930.000 SPPBE 2 20929 0 76658 ..... 98749 0 1054000 1.054.000 SPPBE 4 0 0 0 ..... 0 0 802900 802.900 TOTAL 20.929 27.881 76.658 ..... 98.749 155.574 KAP. Agen 20.929 27.881 76.658 ..... 98.749 155.574 - Sumber: Hasil Pengolahan Data Tabel 5. Hasil Perhitungan Optimasi Agen – Kelurahan (dalam satuan tabung) KELURAHAN AGEN 1 2 3 ..... 151 TOTAL KAP. Agen Agen 1 0 0 0 ..... 0 20929 920 Agen 2 0 0 0 ..... 0 27881 920 Agen 3 0 0 0 ..... 0 76658 920 : : : : : : : : ..... : : : : : : Agen 31 0 0 10741 ..... 0 155574 920 TOTAL 21459 31483 10741 ..... 12486 DEMAND 21459 31483 10741 ..... 12486 Berdasarkan hasil perhitungan dengan menggunakan Excel Analytic Solver Platform diperoleh Ongkos Transportasi LPG 3 kg berdasarkan Sistem Terbuka adalah sebesar Rp 8.319.765.767,07. b) Sistem Tertutup Tabel 5. Hasil Perhitungan Optimasi Agen – Kelurahan (dalam satuan tabung) Transportasi LPG 3 kg berdasarkan Sistem Terbuka adalah sebesar Rp 8.319.765.767,07. b) Sistem Tertutup J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 156 Tabel 6. Penugasan SPPBE ke Agen AGEN SPPBE Agen 1 Agen 2 Agen 3 .... Agen 30 Agen 31 TOTAL Total SPPBE SPPBE 1 0 0 0 .... 0 1 12 31 SPPBE 2 1 1 0 .... 1 0 12 31 SPPBE 4 0 0 0 .... 0 0 7 31 Total Agen 1 1 1 .... 1 1 TOTAL 1 1 1 .... 1 1 Sumber: Hasil Pengolahan Data Tabel 7. Kapasitas Tiap Agen (satuan tabung) AGEN SPPBE Agen 1 Agen 2 Agen 3 ... Agen 30 Agen 31 SPPBE 1 21.781 15.305 62.189 ... 132.593 107.741 SPPBE 2 21.781 15.305 62.189 ... 132.593 107.741 SPPBE 3 21.781 15.305 62.189 ... Optimasi Ongkos Distribusi LPG 3 kg 1 0 12 31 SPPBE 4 0 0 0 .... 0 0 7 31 Total Agen 1 1 1 .... 1 1 TOTAL 1 1 1 .... 1 1 Sumber: Hasil Pengolahan Data Tabel 6. Penugasan SPPBE ke Agen Tabel 7. Kapasitas Tiap Agen (satuan tabung) AGEN SPPBE Agen 1 Agen 2 Agen 3 ... Agen 30 Agen 31 SPPBE 1 21.781 15.305 62.189 ... 132.593 107.741 SPPBE 2 21.781 15.305 62.189 ... 132.593 107.741 SPPBE 3 21.781 15.305 62.189 ... 132.593 107.741 SPPBE 4 21.781 15.305 62.189 ... 132.593 107.741 SPPBE 5 21.781 15.305 62.189 ... 132.593 107.741 SPPBE 6 21.781 15.305 62.189 ... 132.593 107.741 Sumber: Hasil Pengolahan Data Tabel 8. Hasil Perhitungan Optimasi SPPBE - Agen (satuan tabung) AGEN SPPBE Agen 1 Agen 2 Agen 3 ..... Agen 31 TOTAL KAP. SPPBE SPPBE 1 0 0 0 ..... 107741 867473 930.000 SPPBE 2 21781 15305 0 ..... 0 1.053.607 1.054.000 SPPBE 4 0 0 0 ..... 0 802.747 802.900 TOTAL 21781 15305 62189 ..... 107741 KAP. Agen 21781 15305 62189 ..... 107741 Sumber: Hasil Pengolahan Data Tabel 9. Penugasan Agen-Kelurahan KEL. AGEN 1 2 3 .... 151 TOTAL KAP. Agen Agen 1 0 0 0 .... 0 2 151 Agen 2 0 0 0 .... 0 2 151 Agen 3 0 0 0 .... 0 4 151 Agen 4 1 1 1 .... 0 13 151 ..... ..... ..... ..... .... ..... ..... ..... Agen 30 0 0 0 .... 0 6 151 Agen 31 0 0 0 .... 0 6 151 TOTAL Kel. 1 1 1 .... 1 TOTAL 1 1 1 .... 1 Sumber: Hasil Pengolahan Data Sumber: Hasil Pengolahan Data Tabel 10. Hasil Perhitungan Optimasi SPPBE - Agen (satuan tabung) KEL. AGEN 1 2 3 .... 151 TOTAL KAP. Agen Agen 1 0 0 0 .... 0 21781 920 Agen 2 0 0 0 .... 0 15305 920 Agen 3 0 0 0 .... 0 62189 920 Agen 4 21459 31483 10741 .... 0 298665 920 ..... ..... ..... ..... .... ..... ..... ..... Agen 30 0 0 0 .... 0 132593 920 Agen 31 0 0 0 .... 0 107741 920 TOTAL 21459 31483 10741 .... 12486 DEMAND 21459 31483 10741 .... 12486 Sumber: Hasil Pengolahan Data distribusi terbuka yaitu sebesar Rp. 404.644.513,23. Optimasi Ongkos Distribusi LPG 3 kg 132.593 107.741 SPPBE 4 21.781 15.305 62.189 ... 132.593 107.741 SPPBE 5 21.781 15.305 62.189 ... 132.593 107.741 SPPBE 6 21.781 15.305 62.189 ... 132.593 107.741 Sumber: Hasil Pengolahan Data Tabel 8. Hasil Perhitungan Optimasi SPPBE - Agen (satuan tabung) AGEN SPPBE Agen 1 Agen 2 Agen 3 ..... Agen 31 TOTAL KAP. SPPBE SPPBE 1 0 0 0 ..... 107741 867473 930.000 SPPBE 2 21781 15305 0 ..... 0 1.053.607 1.054.000 SPPBE 4 0 0 0 ..... 0 802.747 802.900 TOTAL 21781 15305 62189 ..... 107741 KAP. Agen 21781 15305 62189 ..... 107741 Sumber: Hasil Pengolahan Data Tabel 9. Penugasan Agen-Kelurahan KEL. AGEN 1 2 3 .... 151 TOTAL KAP. Agen Agen 1 0 0 0 .... 0 2 151 Agen 2 0 0 0 .... 0 2 151 Agen 3 0 0 0 .... 0 4 151 Agen 4 1 1 1 .... 0 13 151 ..... ..... ..... ..... .... ..... ..... ..... Agen 30 0 0 0 .... 0 6 151 Agen 31 0 0 0 .... 0 6 151 TOTAL Kel. 1 1 1 .... 1 TOTAL 1 1 1 .... 1 Sumber: Hasil Pengolahan Data Tabel 10. Hasil Perhitungan Optimasi SPPBE - Agen (satuan tabung) KEL. AGEN 1 2 3 .... 151 TOTAL KAP. Agen Agen 1 0 0 0 .... 0 21781 920 Agen 2 0 0 0 .... 0 15305 920 Agen 3 0 0 0 .... 0 62189 920 Agen 4 21459 31483 10741 .... 0 298665 920 ..... ..... ..... ..... .... ..... ..... ..... Agen 30 0 0 0 .... 0 132593 920 Agen 31 0 0 0 .... 0 107741 920 TOTAL 21459 31483 10741 .... 12486 DEMAND 21459 31483 10741 .... 12486 Sumber: Hasil Pengolahan Data Berdasarkan hasil perhitungan dengan menggunakan Excel Analytic Solver Platform diperoleh Ongkos Transportas LPG 3kg berdasarkan Sistem Tertutup adalah sebesar Rp 8.724.410.280,30. Besarnya ongkos transportasi dengan sistem tertutup lebih besar dibandingkan dengan ongkos distribusi terbuka yaitu sebesar Rp. 404.644.513,23. Hal ini dikarenakan pada sistem tertutup satu Agen hanya boleh menerima LPG dari satu SPPBE, sedangkan pada sistem terbuka Agen boleh mengambil LPG dari lebih dari satu SPPBE. Begitu Tabel 6. Penugasan SPPBE ke Agen AGEN SPPBE Agen 1 Agen 2 Agen 3 .... Agen 30 Agen 31 TOTAL Total SPPBE SPPBE 1 0 0 0 .... 0 1 12 31 SPPBE 2 1 1 0 .... J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 2) Coverage Area 2) Coverage Area Coverage area untuk distribusi sistem tertutup dibagi menjadi dua yaitu coverage area SPPBE ke Agen dan Agen ke Kelurahan (konsumen). Untuk coverage area SPPBE ke Agen, adalah sebagai berikut : valuasi Kebutuhan SPPBE Hingga Tahun 2030 • SPPBE 1 memiliki wilayah pemasaran untuk Agen 4, Agen 6, Agen 7,Agen 10, Agen 11, Agen 16, Agen 20, Agen 21, Agen 23, Agen 24, Agen 25, dan Agen 31 dengan kapasitas SPPBE yang terpakai sebesar 867.473 tabung per bulan dari kapasitas total sebesar 930.000 tabung per bulan. Evaluasi kebutuhan dilakukan untuk mengetahui perkiraan permintaan dari tahun 2015- 2030. Pada bagian evaluasi ini pengerjaan dibagi menjadi tiga bagian, yaitu : • SPPBE 2 memiliki wilayah pemasaran untuk Agen 1, Agen 2, Agen 5,Agen 8, Agen 11, Agen 12, Agen 15, Agen 17, Agen 18, Agen 19, Agen 26 dan Agen 30 dengan kapasitas SPPBE 2 yang terpakai 1. Perkiraan permintaan LPG 3 kg berdasarkan prediksi ESDM, 2. Perkiraan permintaan LPG 3 kg berdasarkan metode Forecasting, 3. Perkiraan Lokasi untuk pendiriaan SPPBE baru. 3. Perkiraan Lokasi untuk pendiriaan SPPBE baru. Gambar 5 Peta Coverage Area SPPBE untuk Distribusi Sistem Tertutup Gambar 5 Peta Coverage Area SPPBE untuk Distribusi Sistem Tertutup Perkiraan Permintaan LPG 3 kg Berdasarkan Prediksi ESDM Perkiraan Permintaan LPG 3 kg Berdasarkan Prediksi ESDM Perkiraan Permintaan LPG 3kg Berdasarkan Metode Forecasting Optimasi Ongkos Distribusi LPG 3 kg Hal ini dikarenakan pada sistem tertutup satu Agen hanya boleh menerima LPG dari satu SPPBE, sedangkan pada sistem terbuka Agen boleh mengambil LPG dari lebih dari satu SPPBE. Begitu distribusi terbuka yaitu sebesar Rp. 404.644.513,23. Hal ini dikarenakan pada sistem tertutup satu Agen hanya boleh menerima LPG dari satu SPPBE, sedangkan pada sistem terbuka Agen boleh mengambil LPG dari lebih dari satu SPPBE. Begitu Berdasarkan hasil perhitungan dengan menggunakan Excel Analytic Solver Platform diperoleh Ongkos Transportas LPG 3kg berdasarkan Sistem Tertutup adalah sebesar Rp 8.724.410.280,30. Besarnya ongkos transportasi dengan sistem tertutup lebih besar dibandingkan dengan ongkos J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 157 pula dengan penjualan yang dilakukan oleh satu kelurahan hanya boleh dilayani oleh satu Agen. yaitu sebesar 1.053.607 dari total kapasitas 1.054.000 tabung per bulan. yaitu sebesar 1.053.607 dari total kapasitas 1.054.000 tabung per bulan. • SPPBE 4 memiliki wilayah pemasaran untuk Agen 3, Agen 9, Agen 14, Agen 22, Agen 27, Agen 28, dan Agen 29 dengan kapasitas SPPBE 4 yang terpakai yaitu sebesar sebesar 802.747 dari total kapasitas 802.900 tabung per bulan. Perkiraan Permintaan LPG 3kg Berdasarkan Metode Forecasting Perkiraan Lokasi Pendirian SPPBE Pada subbab berikut ini penelitian dilakukan secara kualitatif yang bertujuang untuk mengtahui hubungan keterkaitan antara tiap kriteria yang menjadi kriteria yang diperlukan dalam menentukan lokasi pendirian fasilitas/ pabrik/ gudang baru. Dari semua hasil perhitungan AHP dengan bantuan expert choise maka penentuan kriteria yang mampengaruhi keputusan dalam memilih lokasi SPPBE baru dapat diambil kesimpulan bahwa: 1) Lokasi SPPBE harus memiliki luas lokasi yang memenuhi persyaratan yang Pertamina berikan, 2) Lokasi SPPBE harus berdekatan dengan Depot LPG Pertamina. 3) Lokasi SPPBE harus memiliki jalan transportasi yang besar, 4) Status kepemilikan lahan SPPBE harus diperhatikan, 5) Biaya produksi berpengaruh pada keuntungan yang diperoleh, sehingga harus memiliki harga tanah yang bisa lebih menguntungkan. 6) Lokasi SPPBE harus berdekatan dengan jumlah Kepala Keluarga terbanyak dan jumlah warung makan terbanyak. Dari hasil kriteria di atas maka peneliti mencoba untuk memberikan alternatif lokasi SPPBE berdasarkan kedekatan dengan Depot, Jumlah KK dan warung makan terbesar. Peneliti menentukan beberapa alternatif lokasi yang cocok untuk mendirikan SPPBE baru dengan asumsi kedekatan berada pada radius 2 km adalah: Tabel 12 Alternatif Lokasi SPPBE No Alternatif Lokasi Alasan 1 Kecamatan Buah Batu Berada di antara radius kedekatan dengan Depot dan warung makan dan KK 2 Kecamatan Bandung kidul Berada di antara radius kedekatan dengan Depot dan Jumlah KK dan warung makan terbanyak. 3 Kecamatan Gedebage Berada di radius kedekatan dengan Depot 4 Kecamatan Cinambo Berada di radius kedekatan dengan Depot 5 Kecamatan Arcamanik Berada di radius kedekatan dengan Depot Langkah selanjutnya peneliti melakukan survey dengan menggunakan kuesioner ke berbagai pihak yang terlibat dalam bisnis LPG 3 kg. Setelah itu data hasil kuesioner diolah dengan menggunakan Tabel 11 Kesimpulan Hasil Akhir Perhitungan Kecukupan dan Kekurangan SPPBE Berdasarkan Hasil Perkiraan ESDM (dalam satuan tabung) Th. Total / Bulan Total / Tahun Kap. Tabel 12 Alternatif Lokasi SPPBE y 3) Hingga tahun 2030 ternyata dibutuhkan 4 SPPBE baru yaitu pada tahun 2015, 2019, 2024 dan 2029. y 3) Hingga tahun 2030 ternyata dibutuhkan 4 SPPBE baru yaitu pada tahun 2015, 2019, 2024 dan 2029. Tabel 12 Alternatif Lokasi SPPBE No Alternatif Lokasi Alasan 1 Kecamatan Buah Batu Berada di antara radius kedekatan dengan Depot dan warung makan dan KK 2 Kecamatan Bandung kidul Berada di antara radius kedekatan dengan Depot dan Jumlah KK dan warung makan terbanyak. 3 Kecamatan Gedebage Berada di radius kedekatan dengan Depot 4 Kecamatan Cinambo Berada di radius kedekatan dengan Depot 5 Kecamatan Arcamanik Berada di radius kedekatan dengan Depot Perkiraan Permintaan LPG 3kg Berdasarkan Metode Forecasting SPPBE Selisih (3)-(4) Alternatif 1 Alternatif 2 (-) SPPB E (+) Kapasita s Selisih (3)-(7) Penambahan Kuota Tiap SPPBE/Th Penambahan Kuota Tap SPPBE/bln Penambahan Kuota SPPBE/hari 2014 2.723.82 7 32.685.92 4 36.000.0 00 3.314.076 2015 2.887.32 3 34.647.87 6 36.000.0 00 1.352.124 2016 3.060.63 4 36.727.60 8 36.000.0 00 -727.608 1 48.000.0 00 11.272.3 92 242.536 20.211 674 : : : : : : : : : : : 2027 5.976.04 7 71.712.56 4 36.000.0 00 - 35.712.56 4 72.000.0 00 287.436 11.904.188 992.016 33.067 2028 6.274.92 5 75.299.10 0 36.000.0 00 - 39.299.10 0 1 84.000.0 00 8.700.90 0 13.099.700 1.091.642 36.388 2029 6.588.73 9 79.064.86 8 36.000.0 00 - 43.064.86 8 84.000.0 00 4.935.13 2 14.354.956 1.196.246 39.875 2030 6.918.25 1 83.019.01 2 36.000.0 00 - 47.019.01 2 84.000.0 00 980.988 15.673.004 1.306.084 43.536 Hasil akhir dari perkiraan demand berdasarkan metode forecating dari tahun 2015 hingga 2030 dapat disimpulkan sebagai berikut: Hasil akhir dari perkiraan demand berdasarkan metode forecating dari tahun 2015 hingga 2030 dapat disimpulkan sebagai berikut: 5) Biaya produksi berpengaruh pada keuntungan yang diperoleh, sehingga harus memiliki harga tanah yang bisa lebih menguntungkan. 5) Biaya produksi berpengaruh pada keuntungan yang diperoleh, sehingga harus memiliki harga tanah yang bisa lebih menguntungkan. 6) Lokasi SPPBE harus berdekatan dengan jumlah Kepala Keluarga terbanyak dan jumlah warung makan terbanyak. 1) Jika Pemerintah dan Pertamina masih memberlakukan kebijakan kalau 1 SPPBE kapasitas maksimum 12.000 MT per tahun maka pada tahun 2015 akan terjadi kekurangan sebanyak 1.967.617 tabung per tahun. Maka idealnya adalah dibutuhkan pendirian 1 SPPBE baru, atau Dari hasil kriteria di atas maka peneliti mencoba untuk memberikan alternatif lokasi SPPBE berdasarkan kedekatan dengan Depot, Jumlah KK dan warung makan terbesar. Peneliti menentukan beberapa alternatif lokasi yang cocok untuk mendirikan SPPBE baru dengan asumsi kedekatan berada pada radius 2 km adalah: 2) Dibutuhkan penambahan kuota tiap SPPBE sebanyak 655.872 per tahun setara dengan 54.656 per bulan atau 1.822 per hari untuk tiap SPPBE. Dan seterusnya. Tabel 12 Alternatif Lokasi SPPBE J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 Perkiraan Permintaan LPG 3kg Berdasarkan Metode Forecasting Perhitungan dilakukan berdasarkan prediksi peningkatan kebutuhan migas oleh ESDM. Di mana peningkatan permintaan yang telah diprediksi oleh ESDM, adalah sebagai berikut : Perhitungan dilakukan berdasarkan prediksi peningkatan kebutuhan migas oleh ESDM. Di mana peningkatan permintaan yang telah diprediksi oleh ESDM, adalah sebagai berikut : Untuk perkiraan permintaan berdasarkan metode forecasting dilakukan dengan menggunakan data permintaan penjualan LPG 3kg pada tahun 2013 yang diperoleh dari BPS Kota Bandung. Berdasarkan hasil perhitungan peramalan maka Standar Error (SEE) yang terkecil adalah berdasarkan metode Regresi Linear dengan nilai SEE sebesar 115129. 1) Pada tahun 2015-2020 akan meningkat sebesar 6 % per tahun, 2) Pada tahun 2020-2025 kebutuhan akan gas bumi meningkat sebesar 7%, dan 3) Pada tahun 2025-2030 meningkat sebesar 5% pertahun. J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 158 Tabel 11 Kesimpulan Hasil Akhir Perhitungan Kecukupan dan Kekurangan SPPBE Berdasarkan Hasil Perkiraan ESDM (dalam satuan tabung) Th. Total / Bulan Total / Tahun Kap. SPPBE Selisih (3)-(4) Alternatif 1 Alternatif 2 (-) SPPB E (+) Kapasita s Selisih (3)-(7) Penambahan Kuota Tiap SPPBE/Th Penambahan Kuota Tap SPPBE/bln Penambahan Kuota SPPBE/hari 2014 2.723.82 7 32.685.92 4 36.000.0 00 3.314.076 2015 2.887.32 3 34.647.87 6 36.000.0 00 1.352.124 2016 3.060.63 4 36.727.60 8 36.000.0 00 -727.608 1 48.000.0 00 11.272.3 92 242.536 20.211 674 : : : : : : : : : : : 2027 5.976.04 7 71.712.56 4 36.000.0 00 - 35.712.56 4 72.000.0 00 287.436 11.904.188 992.016 33.067 2028 6.274.92 5 75.299.10 0 36.000.0 00 - 39.299.10 0 1 84.000.0 00 8.700.90 0 13.099.700 1.091.642 36.388 2029 6.588.73 9 79.064.86 8 36.000.0 00 - 43.064.86 8 84.000.0 00 4.935.13 2 14.354.956 1.196.246 39.875 2030 6.918.25 1 83.019.01 2 36.000.0 00 - 47.019.01 2 84.000.0 00 980.988 15.673.004 1.306.084 43.536 Hasil akhir dari perkiraan demand berdasarkan metode forecating dari tahun 2015 hingga 2030 dapat disimpulkan sebagai berikut: 1) Jika Pemerintah dan Pertamina masih memberlakukan kebijakan kalau 1 SPPBE kapasitas maksimum 12.000 MT per tahun maka pada tahun 2015 akan terjadi kekurangan sebanyak 1.967.617 tabung per tahun. Maka idealnya adalah dibutuhkan pendirian 1 SPPBE baru, atau 2) Dibutuhkan penambahan kuota tiap SPPBE sebanyak 655.872 per tahun setara dengan 54.656 per bulan atau 1.822 per hari untuk tiap SPPBE. Dan seterusnya. 3) Hingga tahun 2030 ternyata dibutuhkan 4 SPPBE baru yaitu pada tahun 2015, 2019, 2024 dan 2029. Perkiraan Lokasi Pendirian SPPBE Pada subbab berikut ini penelitian dilakukan secara kualitatif yang bertujuang untuk mengtahui hubungan keterkaitan antara tiap kriteria yang menjadi kriteria yang diperlukan dalam menentukan lokasi pendirian fasilitas/ pabrik/ gudang baru. Dari semua hasil perhitungan AHP dengan bantuan expert choise maka penentuan kriteria yang mampengaruhi keputusan dalam memilih lokasi SPPBE baru dapat diambil kesimpulan bahwa: 1) Lokasi SPPBE harus memiliki luas lokasi yang memenuhi persyaratan yang Pertamina berikan, 2) Lokasi SPPBE harus berdekatan dengan Depot LPG Pertamina. 3) Lokasi SPPBE harus memiliki jalan transportasi yang besar, Langkah selanjutnya peneliti melakukan survey dengan menggunakan kuesioner ke berbagai pihak yang terlibat dalam bisnis LPG 3 kg. Setelah itu data hasil kuesioner diolah dengan menggunakan 4) Status kepemilikan lahan SPPBE harus diperhatikan, J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 159 Kec. Bandung Kidul sebesar 0,08 dan Kec. Buah Batu sebesar 0,06. Dari hasil akhir tersebut dapat disimpulkan bahwa wilayah Kecamatan Gedebage lebih banyak diminati oleh para sumber untuk mendirikan SPPBE baru. metode AHP dengan bantuan software expert choise. Hasil perhitungan expert choise diperoleh overall inconsistency sebesar 0,1 dengan hasil akhir untuk wilayah kec Gedebage sebesar 0,4, wilayah Kec. Cinambo sebesar 0,3, Kec. Arcamanik sebesar 0,1, Gambar 6 Lokasi Alternatif pendirian SPPBE Baru Gambar 6 Lokasi Alternatif pendirian SPPBE Baru J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 4. Kesimpulan Untuk coverage area SPPBE ke Agen sistem terbuka, adalah sebagai berikut: Metode transportasi merupakan salah satu metode yang digunakan untuk menentukan alokasi barang dan letak lokasi fasilitas. Metode transportasi yang terjadi pada sistem distribusi LPG 3 kg merupakan metode transpotasi tsTP atau metode transportasi dua tahap yaitu dari SPPBE ke Agen dan Agen ke Konsumen. Metode tsTP yang peneliti lakukan adalah berdasarkan linear programming dengan tujuan fungsi objektif adalah ongkos transportasi yang minimum. • SPPBE 1 memiliki wilayah pemasaran untuk Agen 2, Agen 4, Agen 7, Agen 10, Agen 11, Agen 13, Agen 16, Agen 20, Agen 23, Agen 24, dan Agen 31 dengan kapasitas SPPBE yang terpakai sebesar 866.927 tabung per bulan dari kapasitas total sebesar 930.000 tabung per bulan. • SPPBE 2 memiliki wilayah pemasaran untuk Agen 1, Agen 3, Agen 5, Agen 6, Agen 8, Agen 11, Agen 12, Agen 15, Agen 17, Agen 18, Agen 19, Agen 26, Agen 27, Agen 28, Agen 29 dan Agen 30 dengan kapasitas SPPBE 2 semuanya terpakai yaitu sebesar 1.054.000 tabung per bulan. Dengan menggunakan software excel Analytic Solver Platform penyelesaian permasalahan linear programing menjadi lebih ringan dan dapat menghasilkan solusi yang mendekati sempurna. Hasil akhir biaya distribusi sistem tertutup lebih besar dibandingkan biaya distribusi sistem terbuka di mana ongkos transportasi sistem terbuka sebesar Rp8.319.765.767,07 sedangkan ongkos trasnportasi sistem tertutup sebesar Rp8.724.410.280,30. dikarenakan lokasi SPPBE lebih terbusat dalam satu titik yang berdekatan dengan depot LPG sedangkan cakupan penjulan sistem distribusi tertutup menyebar. • 4 memiliki wilayah pemasaran untuk Agen 8, Agen 9, Agen 14 dan Agen 22 dengan kapasitas SPPBE 4 semuanya terpakai yaitu sebesar sebesar 802.900 tabung per bulan. Coverage area untuk distribusi sistem tertutup dibagi menjadi dua yaitu coverage area SPPBE ke Agen dan Agen ke Kelurahan (konsumen). Untuk coverage area SPPBE ke Agen, adalah sebagai berikut: J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 160 Prog. Studi Pascasarjana Teknik Industri, Universitas Indonesia, Jakarta. • SPPBE 1 memiliki wilayah pemasaran untuk Agen 4, Agen 6, Agen 7, Agen 10, Agen 11, Agen 16, Agen 20, Agen 21, Agen 23, Agen 24, Agen 25, dan Agen 31 dengan kapasitas SPPBE yang terpakai sebesar 867.473 tabung per bulan dari kapasitas total sebesar 930.000 tabung per bulan. Chopra, S. (2007). Supply Chain Management : Strategy, Planning, and Operation. 3rd edition. Singapore: Prentice Hall, 31, 495. Dikutip dari pernyataan Tua Siagian, Perwakilan Hisna Migas di inilahKoranCom. 4. Kesimpulan pada tanggal 26 Februari 2014. • SPPBE 2 memiliki wilayah pemasaran untuk Agen 1, Agen 2, Agen 5, Agen 8, Agen 11, Agen 12, Agen 15, Agen 17, Agen 18, Agen 19, Agen 26 dan Agen 30 dengan kapasitas SPPBE 2 yang terpakai yaitu sebesar 1.053.607 dari total kapasitas 1.054.000 tabung per bulan. Dystian Anggaini, I Nyoman Pujawan, dan Niniet Indah Arvitrida, 2010, Jurnal Tugas Akhir : “Optimasi Konfigurasi Jaringan Supply Chain Hulu Gas LPG di Indonesia”, Jurusan Teknik Industri, Institut Teknologi Sepuluh November (ITS), Surabaya. • SPPBE 4 memiliki wilayah pemasaran untuk Agen 3, Agen 9, Agen 14, Agen 22, Agen 27, Agen 28, dan Agen 29 dengan kapasitas SPPBE 4 yang terpakai yaitu sebesar sebesar 802.747 dari total kapasitas 802.900 tabung per bulan. Faizatul Widad, (2010),Jurnal Tugas Akhir: RancanganKonfigurasiJaringanLogistik DenganPendekatanJaringanLogistikDen ganPendekatanSistemTertutup (Studi Kasus :Distribusi LPG 3kg di Kab./Kota Malang dan Kota Batu).Jurusan Teknik Industri, Institut Teknologi Sepuluh November (ITS), Surabaya. Berdasarkan pada hasil perhitungan expert choise diperoleh overall inconsistency sebesar 0,1 dengan hasil akhir untuk wilayah kec Gedebage sebesar 0,4, wilayah Kec. Cinambo sevesar 0,3, Kec. Arcamanik sebesar 0,1, Kec. Bandung Kidul sebesar 0,08 dan Kec. Buah Batu sebesar 0,06. Dari hasil akhir tersebut dapat disimpulkan bahwa wilayah Kecamatan Gedebage lebih banyak diminati oleh para sumber untuk mendirikan SPPBE baru. Heragu, s. S. (2008). Facility Design. CRC Press. Heragu, s. S. (2008). Facility Design. CRC Press. http://www.republika.co.id/berita/breaking- news/ekonomi/10/12/15/152324-2014- distribusi-lpg-3-kg-sistem-tertutup-se- indonesia http://www.solver.com/solver-tutorial-using- risk-solver I Dewa Gd. EkaWiryaG., 2010. Tesis "Penentuan Lokasi Warehousing Baru Dengan Pendekatan Multi Criteria Goal Progamming Untuk Mencapai Efisiensi Rute (Studikasus: P T. C o ca-Cola AmatilIndonesia unit Balinusa), Institut Teknologi Sepuluh November (ITS), Surabaya. J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 5. Daftar Pustaka Alternatif BBM, achive for SPPBE, Satsiun Bahan Bakar Gas, kutipan pada tanggal 8 November 2012 pada pukul 01.43 WIB. Diakses dari http://Bahanbakarminyak.wordpres.com/ category/ sppbe/, pada tanggal 16 oktober 2014, pukul 00.26 WIB. Indrajit R.E, d. R. (2002). Konsep dan Aplikasi Bussines Process Reengineering. Jakarta: Gramedia. Adityawati, A. (2008). Jurnal Tesis : Analisis Jumlah Permintaan Minyak Tanah Berkaitan dengan Kebijakan Pemerintah Dalam Penghematan APBN Melalui Kebijakan Konversi Penggunaan Minyak Tanah Ke LPG. Jakarta: FE-UI. Jabidi. (2012). Jurnal Tesis : Optimasi Distribusi LPG Di Jakarta. Jakarta: Fak. Teknik, Prog. Magister Manajemen Gas, Universitas Indonesia. Berita ESDM, (24 Oktober 2014), (http//www.migas.esdm.go.id) Khwanruthai BUNRUAMKAEW (D3), ( March 1st, 2012), How to do AHP analysis in Excel, Division of Spatial Information ScienceGraduate School of Life and Environmental Sciences. University of Tsukuba. BPS Kota Bandung, “ Kota Bandung Dalam Angka Tahun 2014”, http://bandungkota.bps.go .id/ publikasi / kota – bandung -dalam-angka-tahun- 2014, Kompas (2009). Rayonisasi LPG 3 Kilogram, URL:http://cetak. kompas.com/read/xml/2009/08/18/04064 552/rayonisasi,LPG.tiga.kilogram> Budi Yanto Pargaulan Hutagaol, 2011, Jurnal Tesis : Optimasi Jaringan Sistribusi dari Depot/Terminal LPG ke SPBE/SPPBE Di Pulau Jawa Dengan Menggunakan Algoritma Tabu Search, Fak. Teknik, Mitsuo Gen, R. C. (2008). Network Models and Optimization : Multiobjective Genetic Algorithm Approach. Springer. J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019 161 Ni Putu Ayu Nariswari, (2009), Simulasi Penerapan Cosed System pada distribusi LPG 3 kg. Studi Kasus Distribusi LPG 3kg Kec. Klojen Malang.Jurusan Teknik Industri, Institut Teknologi Sepuluh November (ITS), Surabaya. Sofa. 2008. Teori Lokasi. Tersedia di http://massofa.wordpress.com [03 Agustus 2008] Muchlis, Stefanus Eko Wiratno, 2010, Jurnal Tugas Akhir: Perancangan Sistem Distribusi LPG 3Kg di Kota Surabaya Dengan Mempertimbangkan Pertumbuhan Demand, Jurusan Teknik Industri, Institut Teknologi Sepuluh November (ITS), Surabaya. Ni Putu Ayu Nariswari, (2009), Simulasi Penerapan Cosed System pada distribusi LPG 3 kg. Studi Kasus Distribusi LPG 3kg Kec. Klojen Malang.Jurusan Teknik Industri, Institut Teknologi Sepuluh November (ITS), Surabaya. ( ), y Sofa. 2008. Teori Lokasi. Tersedia di http://massofa.wordpress.com [03 Agustus 2008] 162 J@ti Undip: Jurnal Teknik Industri, Vol. 14, No. 3, September 2019
https://openalex.org/W2551418535	https://strategicjournals.com/index.php/journal/article/download/375/402	English	null	CHALLENGES FACING TIMELY COMPLETION OF ROAD PROJECTS MANAGED BY KENYA NATIONAL HIGHWAYS AUTHORITY	The strategic journal of business & change management	2,016	cc-by	8,340	CHALLENGES FACING TIMELY COMPLETION OF ROAD PROJECTS MANAGED BY KENYA NATIONAL HIGHWAYS AUTHORITY CHALLENGES FACING TIMELY COMPLETION OF ROAD PROJECTS MANAGED BY KENYA NATIONAL HIGHWAYS AUTHORITY CHALLENGES FACING TIMELY COMPLETION OF ROAD PROJECTS MANAGED BY KENYA NATIONAL HIGHWAYS AUTHORITY ROBERT ONYANGO OLOO, DR. PATRICK KARANJA NGUGI Vol. 3, Iss. 4 (62), pp 1210-1225, Oct 27, 2016, www.strategicjournals.com, ©strategic Journals Key Words: Budgetary Provisions, Procurement Procedures, Road Projects 1 Robert Onyango Oloo, 2Dr. Patrick Karanja Ngugi 1Msc. Student, Jomo Kenyatta University of Agriculture & Technology (JKUAT), Kenya 2Jomo Kenyatta University of Agriculture & Technology (JKUAT), Kenya 1211 - \| The Strategic Journal of Business & Change Management. ISSN 2312-9492(Online) 2414-8970(Print). www.str INTRODUCTION factors will ensure the construction industry realizes its efficient goals. Realizing these aspects, the present study was undertaken to suggest ways to improve efficient as well as to take care of certain critical factors that may lead to loss of efficient (KeNHA, 2011). According to Greer (2009), a project is successful if it satisfies all three legs of the triple constraint, namely, performance (specification), cost and time. Thomsett (2002) in an extensive examination of 20 failing projects over a period of 18 years expanded this criterion of success as: Although the causes for project success and failure have been the focus of numerous research studies, there has been no consensus on the issue. In a survey of west Bank in Palestinia, Mahamid(2011) indicated that the most severe factors affecting time delay in road construction projects from owners perspective are poor communication between construction parties. Examining the factors that cause delay in construction projects in Malaysia, Alghbari et al.(2007) tested 31 variables. The main finding of the study was financial factors. Faridi&El – Sayeh (2004) concluded the most significant causes of road construction delay are approval of drawings, inadequate early planning and delays in owners’ decision making process Satisfies stakeholder groups, meets functional requirements, meets quality expectations and requirements, within cost, within deadline, delivers sustained and actual benefits and provides the team with professional satisfaction and learning. The time required for the completion of road construction projects is influenced by a variety of factors both external to internal. Completion of a project within time stipulated at the tender a ward stage is a universally accepted measure of project success. Most projects would delay before they eventually take off(Samasian& soon,2007). This study presents the background of the statement of the problem, purpose of the study, research objectives and questions, study justification and significance, limitations and delimitations of the study of delay of completion of road projects in Kenya. Project success and failures have become a contemporary topic within the construction industry. Research shows that construction companies are vulnerable to bankruptcy (Wong & Ng. 2010). For example only about 43% of construction firms that began operations in 2008, survived after four years of operations in the US (Ganaway, ). Similarly delays and time overrun are prevalent on construction projects (Al-Momani, 2000). ABSTRACT The time required for the completion of road construction projects is influenced by a variety of factors both external to internal. Completion of a project within time stipulated at the tender a ward stage is a universally accepted measure of project success. Most projects would delay before they eventually take off. Helping the construction companies to identify the critical attributes responsible for achieving the desired efficient level (success factors) and also to find the attributes adversely affecting the project efficient (failure factors) has been the motivating factor behind this study. It is realized that maximization of the success factors and minimization of failure factors will ensure the construction industry realizes its efficient goals. The study was seeking to assess challenges facing timely completion of road projects managed by Kenya national highways authority. More specifically, it aimed at examining how budgetary provision and procurement procedures affected timely completion of road projects by Kenya National Highway Authority. The study adopted a descriptive survey design. The target population of the study was all the road engineers, middle managers and a few staffs (361) in the construction department of KeNHA. The sample size was 190 respondents. The researcher used stratified random sampling method to select the desired sample size of 150 respondents from the project Managers, top managers and few staffs. Data collected was cleaned, pretested, validated, and coded, summarized and analyzed using statistical package of SPSS V 23. The study findings were presented using graphs, histograms, bar charts and pie charts. Conclusions were derived based on the P. value and the coefficient of determination. The findings indicated that majority of the respondents agree that budgetary provision and Procurement procedures strongly affected timely completion of road projects in KENHA implemented road projects in Kenya. 1212 - \| The Strategic Journal of Business & Change Management. ISSN 2312-9492(Online) 2414-8970(Print). www.str Kenya National Highway Authority (KeNHA) Kenya National Highway Authority (KeNHA) Kenya National Highway Authority (KeNHA) The Kenya National Highway Authority (KeNHA) is an autonomous agency for the management, development, rehabilitation and maintenance of truck roads linking centers of international importance and crossing international boundaries or terminating at international ports (class A). In addition it is also in charge some national truck roads links internationally important centers (class B), and primary roads linking provincial centers or two higher class roads (class C). Kenya National Highway Authority core functions are to construct, upgrade, rehabilitate and maintain Class A, B, C roads, implement road policies on National roads and ensure adherence to guidelines on axle load control in the traffic Act. Its other roles are to ensure that the quality of roads is in accordance with defined standards and to collect data on use of national roads (KeNHA, 2011). The Authority is 100% owned by the Government as a state corporation established under Kenya Roads Act, 2007. The organization operations are also governed by relevant legislations and regulations such as the Finance Act, the Public Procurement Regulations, and Performance Contracting. The road transport industry includes large companies and individual owner operators; it is highly competitive and rates are determined by the market; the industry responds quickly to changes in demand, and road conditions and regulations. Even so, weak legal and regulatory framework has impacted negatively on the quality and reliability of services and on safety to the users (Kenya Transport Sector Support Project (KTSSP) Project Appraisal Document PAD, (2011). International development funds have had a big role in improving major routes. The road transport industry includes large companies and individual owner operators; it is highly competitive and rates are determined by the market; the industry responds quickly to changes in demand, and road conditions and regulations. Even so, weak legal and regulatory framework has impacted negatively on the quality and reliability of services and on safety to the users (Kenya Transport Sector Support Project (KTSSP) Project Appraisal Document PAD, (2011). International development funds have had a big role in improving major routes. But these improvements are not long lasting, and soon after a road project is completed, its condition deteriorates fast resulting in huge potholes, broken bridges and culverts and muddy sections. Kenya National Highway Authority (KeNHA) Industrialists and other observers have attributed the poor state of Kenyan roads and road transport to the government’s failure to efficiently allocate financial resources and to raise additional funds for road extensions and maintenance (IEA 2008; Watts 2000). INTRODUCTION Research shows that 10% - 30% of projects are delayed in Saudi Arabia (Assaf& Al-Hejji, ) and 70% projects suffered delays during their execution in Nigeria (odeyinka&Ysuf, 2007 Cited in sambassvan& soon, 2007). Ganaway, () Key contributors to project failures are poor project management practices and lack of skills/experience of management personnel. The consequences may rather be in terms of loss in productivity, additional expenditures by way of rework and repair, re-inspection and retest in the short term. In the long term, poor efficient can hurt reputation, and if the company continues in the same way it might have to close its shop for want of new projects. If a number of construction companies of a country start neglecting the efficient aspects in their projects, this also starts reflecting on the reputation of the country (Hyvari, ). Helping the construction companies to identify the critical attributes responsible for achieving the desired efficient level (success factors) and also to find the attributes adversely affecting the project efficient (failure factors) has been the motivating factor behind this study. It is realized that maximization of the success factors and minimization of failure Journal of Business & Change Management. ISSN 2312-9492(Online) 2414-8970(Print). www.strategicjournals.com In Kenya, road is the predominant mode of transport accounting for 93 percent of all freight and passenger traffic in Kenya, but costs are high (KACC, 2007). The road sub-sector is relatively large, with a total classified network of 160,886 km (of which 11,197 km are paved and 149,689 km are gravel or earth) and over 60,000 km of unclassified community roads (with corridors typically less than nine meters wide). This provides a reasonable network of roads in the densely populated parts of the country and some access throughout the rest. Unlike the neighbors, which have major areas without all-weather roads, the key challenge for Kenya is to bring the network in poor condition (56 percent) to good condition (currently just 11 percent), while ensuring that adequate maintenance is carried out on the rest. This has seen this Government initiate several road constructions projects which includes Thika road, expansion of outering road, southern by pass and many others. Statement of the problem Globally, transport infrastructure is an important factor in the development of a nation through its direct and indirect contribution to economic growth. It is in view of this that the Kenya Government has invested heavily in the road construction project and according to the Road Sector investment plan (2010-2024) the Government has allocated and continues to allocate significant resources toward improvement of transport infrastructure. The information held by Kenya Roads board Website indicates that in the financial years 2009/10, 2010/2011 and 2011/12 the government of Kenya allocated Ksh 19 billion, Ksh 20 billion and 1213 - \| The Strategic Journal of Business & Change Management. ISSN 2312-9492(Online) 2414-8970(Print). www.str Journal of Business & Change Management. ISSN 2312-9492(Online) 2414-8970(Print). www.strategicjournals.com Ksh 23.3 billion respectively for construction of road projects( KeNHA, 2010). In the road sub-sector, the extent of cost and time overruns in the overall portfolio is high. As at February 2007, 35 on-going projects out of a total of 207 showed cost overrun, translating into a cost overrun of Kshs. 7 billion. With regard to time overruns 184 projects exceeded their original completion time agreed at the tendering stage. On average, the actual time for completion was more than two times that at the tender stage (World Bank, 2007). In view of the above, the study, therefore, intends to establish the challenges facing timely completion of road projects in Kenya. Objectives of the study The main objective of the study is to establish challenges facing timely completion of road projects managed by Kenya National Highways Authority. The specific objectives were: The specific objectives were:  To establish how budgetary provision affect timely completion of road projects managed by Kenya national highway authority. The inability to complete projects on timecontinues to be a problem worldwide. According to (Ahamedet. al 2002) Overruns on Construction Projects are a Universal Phenomenon. Azhar (2008) states that, trend of time overruns is common worldwide and that is more severe to developing countries. In Kenya information held by KeNHA of a few sample of road construction in Kenya have showed delay in completion for instance Rehabilitation and Construction of Londian – Fortenan Muhoroni Road (KeNHA 2010) was awarded 27April 2010 and received order to commence on 22 June, 2010. The initial completion period for works was 24 months with completion date of 19July, 2012. The work was completed after additional 8 months. Similarly the Construction of KCC (Sotik) – Ndanai – Gorgor Road (15) (KeNHA 2011). The contract was awarded with commencement date of 7th September, 2011 to 6th September, 2013 but completion date revised to 7th February, 2014. A time overrun of six months (Al- Momani, 2010)  To establish how procurement procedures affect timely completion of road projects managed by Kenya national highway authority. Theory of Constraints The basic premises of the Theory of Constraints assume that people can think, they are good and systems are simple (The Choice, Eliyhau M. Goldratt, North River Press, 2009). Yet, there must be something missing. Why do good, thinking people have so much trouble with projects? After all, projects are simply a set of tasks which must all be done within some precedence order before the project is complete. What is missing? It must be something that is a hidden understanding of how project systems perform. Or, it must be something acting upon the project management system: good, thinking people that do things to actually make the problems worse. Homabay – Mbita road located in Homa bay and suba Districts of Nyanza in Western Kenya. Date of commencement of construction was 5-02-2010 for a period of 30 months with a completion date of 03- 08-2012 but the completion was first revised to 23- 10-2013 and a gain revised to 13-01-2014. (KeNHA, 2013) Theory of Constraints argues that an organization facing challenges in cost management, poor performance and chronic conflicts is as a result of poor management practices and lack of necessary intervention. Eliyahu developed the theory of Journal of Business & Change Management. ISSN 2312-9492(Online) 2414-8970(Print). www.strategicjournals.com 312-9492(Online) 2414-8970(Print). www.strategicjournals.c constraints in the early 1980s to help organizations decide what to change, identify a desirable new condition and how to trigger the change. He recommended first identifying the main factors affecting budget estimates in an organisation. He then suggested that the managers figure out how to handle the constraints or barrier to success within prescribed budget. By focusing on fixing the main problem, overall performance could be improved (Eliyahu, 2004). Additionally, Baloi& Price observed that most organizations fail to examine their operations as a whole when developing cost estimates (Baloi& Price, 2003). By focusing only on short-term goals, long-term success becomes jeopardized so he suggested establishing a long- term view. According to this theory, all systems operate in an environment of cause and effect. One event causes another to happen thus prompting for factors analysis as a measure. Adherence to cost estimates is either a constraint or has the potential to become a constraint. This cause-and- effect relationship can be very complex, especially in complex systems such as those of construction projects. Theory of Constraints Capturing the essence of cause and effect within the system and identifying factors that emulate these relationships are the keys to system performance and excellent adherence to cost estimates. Projects involve a high level of uncertainty and depend heavily on the contributions of individuals. Project manager needs to work with different departments involved in the project to estimate lead times so that they meet the needs of the critical chain. The critical chain concept starts with a set of talented and driven project managers and assumes that the resource constraints are within the scope of the project but not in its leadership. Budgetary Provision g y Budgeting is the process of allocating finite resources to the prioritized needs of an organization. In most cases, for a governmental entity, the budget represents the legal authority to spend money. Adoption of a budget in the public sector implies that a set of decisions has been made by the governing board and administrators that culminates in matching a government's resources with the entity's needs. As such, the budget is a product of the planning process. The budget also provides an important tool for the control and evaluation of sources and the uses of resources. Using the accounting system to enact the will of the governing body, administrators are able to execute and control activities that have been authorized by the budget and to evaluate financial performance on the basis of comparisons between budgeted and actual operations. Thus, the budget is implicitly linked to financial accountability and relates directly to the financial reporting objectives (Chan, 2006). It should be noted that complexity-based factors related to project dynamic are often abound. This is due to the difficulties in well understanding the nature of dynamic in construction project in order to identify relative complex factors. In other words, planning for a dynamic system is difficult due to changes in environment and circumstances. It is even more difficult to estimate when considering dependent on environmental conditions and other unknowns. There is an ongoing research in this regard and more details need to be revealed and findings explored. Project management systems are considered dynamic systems, similar to those in nature, which means they change over time and are hard to predict. This increasingly fast-paced system is creating a complexity explosion, which is affecting the way project managers need to govern. Although they are changing, there is usually an underlying predictability that can be identified. Kaliba, Muya and Mumba (2009) found that the major causes of delays in construction projects in road construction projects in Zambia were delayed payments, financial deficiencies on the part of the client or the contractor. One of the major sources of funding for road works in the country is the fuel levy fund which was introduced in 1993 and is mainly used for the maintenance of roads. Complexity Theory The theory of classifying problems based on how difficult they are to solve. A problem is assigned to the P-problem (polynomial-time) class if the number of steps needed to solve it is bounded by some power of the problem's size. A problem is assigned to the NP-problem (nondeterministic polynomial-time) class if it permits a nondeterministic solution and the number of steps to verify the solution is bounded by some power of the problem's size. The class of P-problems is a subset of the class of NP-problems, but there also exist problems which are not NP. A prominent author in the field of complexity is Terry Williams who shares the view of other scholars on complexity but extends it by one additional dimension of time estimates. In addition to the two components of complexity, vis-à-vis the number of factors and the interdependency of these factors, he introduces the third factor which is uncertainty. Since uncertainty adds to the complexity of a project, time estimates therefore can be viewed as a constituent dimension of project complexity that can be as a result of various factors (Williams, 2008). Projects occasionally demand for more additional funds as there is an increasing desire to reduce time to market thus affecting the cost estimates of the project (Williams, 2008). Kahane on the other hand puts a lot of emphasis on talking and listening to each other when solving tough problems when developing estimate costs. His approach to The Theory of Constraints (TOC) approach focuses on successful on-time completion of the entire project. According to TOC, the main constraint in any project is the time taken for completion of the critical chain. Therefore emphasis is laid on completing activities in the critical chain without wasting any time. Hence, cutting safety time from individual activities eliminates the major cause of time wastage, thereby removing the constraint. However, this does not mean that the project is to be left unprotected against any unforeseen delays in any individual activity. The project is to be guarded against delays by providing time buffers. Figure 1: Conceptual Framework. complexity is deeply rooted in a social environment. He distinguishes complexity in three ways. These are; Dynamic Complexity which means that the cause and effect are far apart and it is hard to grasp from firsthand experience. Budgetary Provision The fuel levy fund is collected by KRA and administered by KRB, which was established by an Act of Parliament in the year 2000, with the responsibility of presiding over planning, development and maintenance of roads. The KRB has three main agencies through which funds for roads rehabilitation, maintenance and repair are disbursed. These are: The Department of Roads at the Ministry of Roads and Public Works, which deals with Class A, B & C roads. These are international highways, the national - 1216 - \| The Strategic Journal of Business & Change Management. ISSN 2312-9492(Online) 2414-8970(Print). www.strategicjournals.com Conceptual Framework Independent Variables Dependent Variable , y ( ) major causes of delays in construction projects in road construction projects in Zambia were delayed payments, financial deficiencies on the part of the client or the contractor. One of the major sources of funding for road works in the country is the fuel levy fund which was introduced in 1993 and is mainly used for the maintenance of roads. The fuel levy fund is collected by KRA and administered by KRB, which was established by an Act of Parliament in the year 2000, with the responsibility of presiding over planning, development and maintenance of roads. The KRB has three main agencies through which funds for roads rehabilitation, maintenance and repair are disbursed. These are: The Department of Roads at the Ministry of Roads and Public Works, which deals with Class A, B & C roads. Budgetary Provision These are international highways, the national Budgetary provision  Disbursement procedure  Cash flow  Budgetary cycle  Budget implementation  Monitoring and evaluation Procurement procedures  Procurement policies  Tendering process  Standards determination  Price negotiation Timely completion of road projects  Identifying specific milestone  Documentary relationship of proper activities  Estimate activity resources  Develop schedule  Control schedule Conceptual Framework Budgetary provision  Disbursement procedure  Cash flow  Budgetary cycle  Budget implementation  Monitoring and evaluation Procurement procedures  Procurement policies  Tendering process  Standards determination  Price negotiation Timely completion of road projects  Identifying specific milestone  Documentary relationship of proper activities  Estimate activity resources  Develop schedule  Control schedule Budgetary provision  Disbursement procedure  Cash flow  Budgetary cycle  Budget implementation  Monitoring and evaluation Timely completion of road projects  Identifying specific milestone  Documentary relationship of proper activities  Estimate activity resources  Develop schedule  Control schedule Independent Variables Dependent Variable Independent Variables Dependent Variable highways and trunk roads; the District Roads Committees (DRC), which deals with Class D, E, and other roads. These are rural access roads and feeder roads and the Kenya Wildlife Service (KWS), which deals with all the construction and maintenance of roads in the national parks and game reserves (KeNHA, 2011). structures, unaccountable political class, weak legislative regimes on the devolved funds, apathy among the citizenry, and corruption among others as Kimenyi (2005) explains. Kenya Roads Board (KRB) manages road maintenance funds from the Fuel Levy Fund by disbursements in tranches to appointed agents for roads maintenance. Disbursement of funds to districts by the Ministry of Roads and Public Works is done after receiving funds from KRB. Upon receiving the funds, the Ministerial allocation committee sits to deliberate on its distribution (KeNHA, 2011). The fuel levy fund that the KRB administers is distributed amongst these agencies, in accordance with a formula that is spelt out in the Act: 57% goes to the Department of Roads of the Ministry of Roads and Public Works; 24% goes to the District Roads Committees, Roads Department, Local Authorities and Kenya Wildlife Service; 16% goes to Constituencies through District Roads Committees and 3% goes towards the overhead costs of the KRB. The other major source of funds for road works is budgetary allocation by the Exchequer. Budgetary Provision This is directly allocated to the Ministry of Roads and Public Works. The Government also receives substantial amounts of funding for road works from development partners. In addition to disbursements from KRB, KWS also occasionally receives funds from development partners for specific road projects and allocates part of the internally 22 generated funds for road works. Local authorities allocate part of their LATF and internally generated funds to road works. Other players in the roads sub- sector such as KTDA and tea factories, KSB and sugar companies do not receive any government funding (Mbeche 2000). Procurement Procedures The understanding of dynamics of procurement management has been predominantly seen through experience as dissatisfying where the procurement unit was viewed from other entities in the organization as an insignificant, reactive and an administrative part of the business. The potential, however, for the procurement organization to be significant in the company was argued to be vast (Ammer2012, Ellram& Car 2012, Van Weele 2005). In order to change the situation of the procurement organization, procurement management was informed that they should in gradual steps develop the procurement organization towards more sophisticated and significance (Reck& Long 2008) producing strategies that were aligned with overall company strategy including the development of policies, procedures, systems, tools and processes( cousins 2002, cousins et al 2008). This process changed the perspective of the procurement organization which among other things, allowed the procurement entity to contribute to implementation of supply chain management (freeman &Cavinato 1990). Effective participation of the Kenyan people in budgetary allocations remains an elusive mirage owing to a number of factors including: inadequate information on the devolved funds, exclusion of citizenry in decision making processes regarding the funds, poor coordination resulting in projects duplication, the culture of political patronage, wanting citizenry capacity to demand accountability from the ruling elite, unresponsive government Gesuka and Namusonge (2013) conducted a study on the factors affecting compliance of PP in Kenya Journal of Business & Change Management. ISSN 2312-9492(Online) 2414-8970(Print). www.strategicjournals.com with 70 respondents that were purposively selected from the Butere district commissioner’s office, procurement committees, procurement unit, user departments and suppliers. Primary and secondary data was collected through the administration of a structured questionnaire, interviews and records analysis respectively. The findings indicated some level of compliance to the legal requirements, but also revealed weak familiarity of procurement rules of all the stakeholders. Eyaa and Oluka (2011) also conducted a cross sectional study on the causes of non- compliance in public procurement in Uganda that targeted was 120 Central Government Procuring and Disposing Entities (PDEs).The authors’ model indicates a 52.4% variation in compliance with the regulations. including; coordinating, organizing, leading and controlling. This study dwelt on planning only and did not take cognizance of other roles. Further study fell short of explanation on the sample. It did not highlight whether the sample was representative of the local government agencies to enable generalization of findings. Procurement Procedures Kenya through the Public Procurement and Disposal Acts 2005, created the Public Procurement Oversight Authority (PPOA), the Public Procurement Advisory Board (PPAB) and the continuance of the Public Procurement Complaints, Review and Appeal Board as the Public Procurement Administration Review Board (PPARB). The PPOA is mandated with the responsibility of ensuring that procurement procedures established under the Act are complied with, monitoring the procurement system and reporting on its overall functioning, initiating Public Procurement Policy and assisting in the implementation and Operation of the public procurement system. In the context of road construction, procurement is very critical in the sense that for the projects to be completed at the right time, procurement of the project requirements should be done at the right time and right quality, according to Kagiri and Wainaina (2007), donors require the recipient to follow specific rules (i.e procurement guidelines) for identifying the contractor who constructs the road and to set up specific financial right systems to oversee the use of donor funds. Basheka (2008) undertook a study among 99 local government stakeholders selected from 11 Districts of Uganda, using a correlation research design. The data was analyzed using principal component factor analysis that aimed at identifying the critical components of procurement planning and accountable local governments systems in Uganda. Results of the study indicated a significant positive relationship between procurement planning and accountable local government procurement systems. He critically analyzed the contribution of the roles of a manager one of which is planning. - 1218 - \| The Strategic Journal of Business & Change Management. ISSN 2312-9492(Online) 2414-8970(Print). www Budgetary Provision Various studies have been conducted on organization resources and projects. Weiss, Hoegl and Gibbert (2014) conducted a study on the perceptions of Material Resources in Innovation Projects: What Shapes Them and How Do They Matter? This paper focused on team members' perceptions of the provided material resources' adequacy to address this gap. Various studies have been conducted on organization resources and projects. Weiss, Hoegl and Gibbert (2014) conducted a study on the perceptions of Material Resources in Innovation Projects: What Shapes Them and How Do They Matter? Various studies have been conducted on organization resources and projects. Weiss, Hoegl and Gibbert (2014) conducted a study on the perceptions of Material Resources in Innovation However, the scholar failed to acknowledge that while planning is key in the roles and responsibilities of managers, there are other roles Journal of Business & Change Management. ISSN 2312-9492(Online) 2414-8970(Print). www.strategicjournals.com Projects: What Shapes Them and How Do They Matter? This paper focused on team members' perceptions of the provided material resources' adequacy to address this gap. municipalities could not comply to the regulations because they were not familiar with them. Gelderman et al.(2006) confirmed this position in a survey on compliance with EU procurement directives. Given that the procurement profession is still relatively new in Kenya, with the regulations having come into force in 2006, it is possible that the level of familiarity with the regulations is still low. On the other hand, it is possible that those who are familiar with the regulations know it so well that they know how to beat the loopholes to their advantage. It is worth noting that the ambiguity in the private procurement procedures may provide a chance for dubious acts including opaque tendering and discriminate supplier selection which may progress into poor compliance levels. Some theorists have noted that deficient familiarity of the procurement procedure by all the internal stakeholders may affect compliance. Procurement procedures According to Huberts (2010), compliance with the formal elements gives an indication of knowledge of the rules. Gelderman et al. (2006) maintained that private purchasers will comply with the rules if they perceive them as clear. They added that the simple fact that the management of a private agency is familiar with the essence of the EU rules could function as an organizational incentive to comply. It is further argued that Lack of clarity is believed to increase the possibilities for (un)deliberate non- compliance. Educating and training private purchasers will be an effective tool for increasing the compliance with the directives (Gelderman et al., 2006). Eyaa and Oluka, (2011) stated that lack of familiarity with procurement rules results into poor compliance levels. They also found out that in the Kenyan context, familiarity with procurement regulations significantly predicted compliance with procurement regulations. A study by Heneghan and O’Donnell (2007) indicated that the high levels of non-compliance were partly attributable to the complex legislative requirements of the procurement policy and legal framework. Lazarides (2011) also adds that compulsory compliance is the result of among other factors clarity or lack of vagueness of provisions. Thus increasing knowledge of the law can possibly improve compliance. RESEARCH METHODOLOGY The study embraced descriptive research designin order to provide a framework to examine current conditions, trends and status of events.Descriptive research design was more investigative and focused on a particular variable factor. Kenya National Highway Authority had a total of 188 Projects being undertaken . The study population therefore was project managers (total=188) in charge of the projects in KeNHA. A questionnaire was designed to collect information from engineers which contained open ended and closed ended questions. This provided a basis to identify the critical success factors affecting timely completion of road projects. This research study used self-structured questionnaires to collect primary data from respondents. Open and closed ended questions were included in the questionnaires so that each respondent was capable of receiving the same set of questions in exactly the same way. According to De Boer and Telgen (1998) as quoted by Gelderman et al. (2006),one of the factors causes of non – compliance with procurement regulations is the level of familiarity with the procurement regulations. De Boer and Telgen (1998) assert that during the early days of the inception of private procurement regulations in The Netherlands, many Journal of Business & Change Management. ISSN 2312-9492(Online) 2414-8970(Print). www.strategicjournals.com validity of the instrument was considered reasonable (Brown, 2006) The study sought to establish the age of the respondents in order to determine if the age corresponded with Timely completion of road projects in Kenya. Majority (57.6%) of the respondents were in the age category of 25-35 years, 26.9% both were in the age category of 36 and above years 15.38% were in the age category of below 25 years. validity of the instrument was considered reasonable (Brown, 2006) Questionnaires may therefore yield data more comparable than information obtained through an interview. The questionnaire allowed respondents to express their opinions hence collection of objective data. The study sought to establish the age of the respondents in order to determine if the age corresponded with Timely completion of road projects in Kenya. Majority (57.6%) of the respondents were in the age category of 25-35 years, 26.9% both were in the age category of 36 and above years 15.38% were in the age category of below 25 years. Data was analysed using both descriptive and inferential statistics. RESEARCH METHODOLOGY This is because descriptive statistics helps to describe the data collected and aim to summarize a sample while inferential statistics are used to interpret the meaning of descriptive statistics besides making propositions about populations and so helps in drawing conclusions. The filled in questionnaires were collected, cleaned, coded and fed in the computer for analysis by SPSS V22 for both descriptive and inferential statistics. The study sought to establish the education level held by the respondents in order to ascertain if they were equipped with relevant knowledge and skills to understand the study. From the study findings, majority (64%) of the respondents were post graduate students followed by 21% Graduate education level and 15% who were diploma education level. FINDINGS A total number of 188 questionnaires were administered to the sample selected. 130 questionnaires were collected back The response of 69% facilitated towards gathering sufficient data that was generalized to reflect the opinions of respondents. This was in tandem with Graham (2002) that a response rate above 60% of the total sample size contributes towards gathering of sufficient data that could be generalized to represent the opinions of respondents in the target population on the sought study problem. The study sought to establish the length of service the respondents worked in order to establish the familiarity with the sector. 38.89% had worked less than five years, 25.5% had worked between 5 to 10 years, 11.11% had worked 11 years and 15 years, 6.35% had worked 16 years and 20 years, 7.94% had worked between 21 years and 25 years and 10.32% are not employed. Timely completion of road projects in Kenya This research sought to get from the respondents the status on Timely completion of road projects in Kenya. The findings indicate that 52.9% strongly agree that Timely completion of road projects in Kenya is a major issue in Kenya. 35.5% agree, 5.8% disagree and 5.0% strongly disagree. These findings are supported by ROK (2014) that Timely completion of road projects in Kenya is an issue that needs to be addressed with urgency. Procurement Procedures Procurement Procedures The study sought to investigate the influence of Procurement Procedures on Timely completion of road projects. The results in table 2 below, 8.1 % strongly agree that Procurement Procedures affects disagree. These findings tally with AGC (2011) that Procurement Procedures is a critical aspect of Timely completion of road projects in Kenya. Table 2: Procurement Procedures Frequency Valid Percent Valid SD 17 13.7 D 32 25.8 N 34 27.4 A 31 25.0 SA 10 8.1 Total 130 100.0 These findings tally with AGC (2011) that Procurement Procedures is a critical aspect of Timely completion of road projects in Kenya. NAP was an operational document – a methodology to steer country action on Timely completion of road projects in Kenya on the basis of a common agreement reached by all interested parties. All priority programmes were alignment to Kenya vision 2030 flagship projects and programmes. Basically the development plan was currently being implemented in a systematic manner under overall national macro policy framework of the sector Medium Term Plans (MTPs) for the spans of five year planning period. Within the macro context, empowerment was critical for the nation both in the short and long run. NAP was an operational document – a methodology to steer country action on Timely completion of road projects in Kenya on the basis of a common agreement reached by all interested parties. All priority programmes were alignment to Kenya vision 2030 flagship projects and programmes. Basically the development plan was currently being implemented in a systematic manner under overall national macro policy framework of the sector Medium Term Plans (MTPs) for the spans of five year planning period. Within the macro context, empowerment was critical for the nation both in the short and long run. Budgetary Allocation NAP was an operational document – a methodology to steer country action on Timely completion of road projects in Kenya on the basis of a common agreement reached by all interested parties. All priority programmes were alignment to Kenya vision 2030 flagship projects and programmes. Basically the development plan was currently being implemented in a systematic manner under overall national macro policy framework of the sector Medium Term Plans (MTPs) for the spans of five year planning period. Within the macro context, empowerment was critical for the nation both in the short and long run. Conclusion From the finding, the study concludes that Timely completion of road projects in Kenya is affected by Budgetary provision and Procurement procedures. Budgetary provision was a major factor in Timely A 42 32.3 SA 19 14.6 Total 130 100.0 Procurement Procedures The study sought to investigate the influence of Procurement Procedures on Timely completion of road projects. The results in table 2 below, 8.1 % strongly agree that Procurement Procedures affects Timely completion of road projects in Kenya, 25% agree while 25.8% disagree and 13.7% strongly disagree. These findings tally with AGC (2011) that Procurement Procedures is a critical aspect of Timely completion of road projects in Kenya. Table 2: Procurement Procedures Frequency Valid Percent Valid SD 17 13.7 D 32 25.8 N 34 27.4 A 31 25.0 SA 10 8.1 Total 130 100.0 NAP was an operational document a methodology Budgetary Allocation The study sought to investigate the influence budgetary allocation on Timely completion of road projects in Kenya. 14.6 % strongly agree that budgetary allocation timely completion of road projects in Kenya, 32.2% agree while 21.5% disagree and 4.6% strongly disagree. These findings are in line with Ryder (2016) that budgetary allocation timely completion of road projects in Kenya. The pilot study involved questionnaires from 8 respondents. Cronbach’s alpha of well above 0.7 and most of it above 0.8 implying that the instruments were sufficiently reliable for measurement. The study accepted a Cronbach alpha of 0.7 and above. Since most items total correlations were reasonably high, the construct Table 1: Budgetary Allocation - 1220 - \| The Strategic Journal of Business & Change Management. ISSN 2312-9492(Online) 2414-8970(Print). www.strategicjournals.com Table 1: Budgetary Allocation Frequency Percent Valid SD 6 4.6 D 28 21.5 N 30 23.1 - 1220 - \| The Strategic Journal of Business & Change Management. ISSN 2312-9492(Online) 2414-8970(Print). www.strategicjournals.com A 42 32.3 SA 19 14.6 Total 130 100.0 Procurement Procedures The study sought to investigate the influence of Procurement Procedures on Timely completion of road projects. The results in table 2 below, 8.1 % strongly agree that Procurement Procedures affects Timely completion of road projects in Kenya, 25% agree while 25.8% disagree and 13.7% strongly disagree. These findings tally with AGC (2011) that Procurement Procedures is a critical aspect of Timely completion of road projects in Kenya. Table 2: Procurement Procedures Frequency Valid Percent Valid SD 17 13.7 D 32 25.8 N 34 27.4 A 31 25.0 SA 10 8.1 Total 130 100.0 Timely completion of road projects in Kenya This research sought to get from the respondents the status on Timely completion of road projects in Kenya. The findings indicate that 52.9% strongly agree that Timely completion of road projects in Kenya is a major issue in Kenya. 35.5% agree, 5.8% disagree and 5.0% strongly disagree. These findings are supported by ROK (2014) that Timely completion of road projects in Kenya is an issue that needs to be addressed with urgency. SUMMARY OF FINDINGS, CONCLUSIONS AND RECOMMENDATIONS Summary of the findings The study established that Budgetary provision and Procurement procedures affected Timely completion of road projects in Kenya in Kenya. Suggestions for Further Research The results of this study can be further utilized to suggest several directions for future research. Finally, more research on this area is needed because this study has investigated a subset of the variables found to be important determinants. Other variables that may affect private label projects need be investigated. Further research can examine these possibilities and the extent of their influence. Summary of the findings From the finding, the study concludes that Timely completion of road projects in Kenya is affected by Budgetary provision and Procurement procedures. Budgetary provision was a major factor in Timely completion of road projects in Kenya and that policy priorities for tackling timely completion of road projects in Kenya is essential. These policy priorities are not presented in any order of precedence. From the finding, the study concludes that Timely completion of road projects in Kenya is affected by Budgetary provision and Procurement procedures. The study established that Budgetary provision and Procurement procedures affected Timely completion of road projects in Kenya in Kenya. Rather, they require sustained and concerted action – by several actors over an array of policy areas. The Kenya National Highways Authority (KENHA) had developed the National Action Plan on Timely completion of road projects in Kenya (NAP). The The study established that Budgetary provision and Procurement procedures affected Timely completion of road projects in Kenya in Kenya. Rather, they require sustained and concerted action – by several actors over an array of policy areas. Budgetary provision was a major factor in Timely completion of road projects in Kenya and that policy priorities for tackling timely completion of road projects in Kenya is essential. These policy priorities are not presented in any order of precedence. The Kenya National Highways Authority (KENHA) had developed the National Action Plan on Timely completion of road projects in Kenya (NAP). The Journal of Business & Change Management. ISSN 2312-9492(Online) 2414-8970(Print). www.strategicjournals.com An integrated approach (e.g. national action plans on Timely completion of road projects in Kenya), where not just the Ministry of of transport, but also other key Ministries, address the issue of Timely completion of road projects in Kenya, could be helpful in this regard. programs should comply with nationally recognized quality assurance standards to ensure training is relevant and appropriate for development. Life-long career paths must be brought to the fore of Timely completion of road projects in Kenya rather than focusing on “short-term” isolated job interventions. There need to prioritize the development of an Integrated system that the country meets the ever- increasing demands for timely and accurate completion of road projects. 1222 - \| The Strategic Journal of Business & Change Management. ISSN 2312-9492(Online) 2414-8970(Print). www.str gic Journal of Business & Change Management. ISSN 2312-9492(Online) 2414-8970(Print). www.strategicjournals.com Recommendations The study established that Budgetary provision and procurement procedure need to be checked in a more appropriate for change. Provision of accessible and practical skills training that has been developed with input, either additionally or independently of formal education. Training REFERENCES Chan, A.P.C. (2006), Determinants of project success in the construction industry of Hong Kong, University of South Australia, Armidale, AGC (2011).Partnering A Concept for Success, Associated General Contractors, Washington, DC. 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W4389694962.txt	https://ojs.academicon.pl/np/article/download/7550/6986	pl		Bożogrobcy w Miechowie (w 800-lecie sprowadzenia zakonu do Polski)	Nasza Przeszłość	1,963	cc-by-sa	28,447	BOŻOGROBCY W MIECHOWIE * (w 800-lecie sprowadzenia zakonu do Polski) 1. STRÓŻE GROBU CHRYSTUSOW EGO W JEROZOLIM IE Zdobycie Jerozolim y przez europejskie rycerstwo podczas pierwsze wyprawy krzyżow ej w roku 1099 oddało z pow rotem w ręce świata chrześcijańskiego m iejsce Grobu Chrystusowego . Pow stałe królestw o jerozolim skie, z B aldw m em z B om B on n , ukoro nowanym w bazylice betlejem skiej w roku 1 1 1 0 , m iało byc zaczął kiem idei zjednoczenia całego chrześcijaństwa Baldw in m e mial tych .w “ p ,.ló w c ...... . jeg©Gotfryd * B '»u ll.n , k .o r , M a c - , . k , do; bywca Jerozolim y wybranym prtez rycerstwo władcą Ziem i Świętej przyjął jedynie skrom ny tytuł „o b ro ń cy Grol.u Świę tego" s Żył jeszcze zaledwie prze* kilka m iesięcy, ale pozostawił p o sobie idee służenia pobożnym pielgrzym kom ściągającym do Jerozolim y ze wszystkich zakątków średniowiecznego chrzesci^a" Osadzony w Jerozolim ie patriarcha obrządku łacińskiego — pośród wewnętrznych sporów rycerstwa rządzącego ^ feudalnym i wśród walk z muzułmanami — musiał oprzeć się o zakony oddane służbie samarytańskiej i gotow e jednocześnie d zbrojne" obrony. Tak powstał zakon rycerski obron cow Grobu Chrystusow ego \ R ycerze, Czyli kawalerowie Grol.u Pańskiego, wolni byli od ślubu bezżeństwa, nosili suknie białe po kostki długie, a na płaszczu krzyz pojedynczy czerw onego koloru, w którego czterech kątach były umieszczone „te ry małe krzyżyki tegoż k oloru , na kształt herbu miasta Jerozol.m y. * Opracowanie powyższe jest szkicow e, ja k o czSSĆ badań naukow ych i wyko paliskow ych podjętych przez autora, które wymagają dłuzszego okresu 1 J. D ą b r o w s k i , D zieje E uropy od X w ieku. W ielka H istoria Powsz. 1. IV. s. 176. * K»m Zp . P ę k a l s k i , O początku rozkrzetcw niu i upadku zakonu W K anoników Stróżów iw. Grobu J erozolim sk,ego, Kraków 86- .. 17. Z b roja ich była: szyszak, pancerz, tarcza, m iecz obosieczny, koncerz, łuk i strzały. Obow iązani byli odprow adzać pątników przybyw ających d o Je rozolim y dla zwiedzenia m iejsc świętych, b ron ić Grobu Chrystusowego, iść z w ojskiem do boju przeciw n ieprzyjacielow i wiary św iętej” 4. B ożogrob cy zwali się m iędzy sobą „b ra ćm i” — Fratres cruciferi donunici sepulcri Hierosolymitani 5. W roku 1114 otrzym ali od patriarchy jerozolim skiego regułę św. Augustyna. W m iędzy czasie charakter rycerski zakonu zm ienił się na klasztorny i dlatego nosili nazwę Canonici regulares Sanctissimi Sepulchri Hieroso- limitani 6. Biały płaszcz z czerw onym krzyżem nosili też templariusze, tak zwani od Salom onow ej świątyni ( templum), na m iejscu k tórej miał pow stać ich klasztor w Jerozolim ie. Zakon templariuszy został założony przez dziew ięciu rycerzy francuskich w roku 1118. Z ubioru byli w ięc p odob n i do b ożog rob ców , związanych z kapi tułą patriarchy jerozolim skiego, która miała prawo do jeg o w y boru. Stąd przypuszczalnie w yw odziła się nazwa kanoników Grobu Chrystusowego i pow ierzona nad nim piecza strażnicza 1. Ludność m iejscowa, obserwująca element rom ański dom inujący wśród krzyżow ców pierwszej w ypraw y i dw ór króla Baldwina, nazywała europejskie rycerstw o F ra n k a m i8. Tym należy też sobie tłum aczyć przybycie do Polski w roku 1163 pierwszego b o żo grobca, zwanego M arcinem Gallem. W roku 1122 zakon b ożog rob ców został zatw ierdzony przez papieża Kaliksta II; w krótce potem , bo w roku 1144, dom y klasz torne w ym ienione były w siedmiu m iejscow ościach Palestyny . Po zdobyciu Jerozolim y przez Saracenów w roku 1187 siedziba zakonu ustaliła się w Ptolem aidzie. W tym właśnie czasie rozwinął się klasztor w M iechow ie, a po w ypędzeniu krzyżow ców z Ziem i Świętej w roku 1291, nabrał on specjalnego znaczenia dla pa triarchy jerozolim skiego. Zakon utraciwszy podstawę dla swej działalności na Bliskim W schodzie, mógł ją skutecznie rozw ijać w takim właśnie kraju jak Polska, gdzie posypały się bogate fundacje i istniała m ożliw ość pracy na ugorach, gdzie zakładano parafie. P o roku 1291 rozszerzyli się b ożog rob cy także na terenie W łoch, Hiszpanii, F rancji i N iem iec; nie odgryw ali tam jednak 4 tamże. M. H e i m b u c h e r , D ie Orden u. K ongregation en d er katholisclien K irehe, Paderborn 1934 t. I. s. 411. 6 tamże. Ks. Fr. B o g d a n, Z e studiów nad egzem pcją klasztorów w śred n iow iecznej P olsce. Polonia Sacra 1957 s. 233— 52. H J. D ą b r o w s k i , D zieje E uropy, s. 176. 9 H e i m b u c h e r , D ie O rden , s. 24. większej roli i ograniczali się jedyn ie do działalności szpitalnej 10. Rów nocześnie rycerze szpitala św. Jana w Jerozolim ie, zwani joannitam i, przenieśli się po roku 1291 na Cypr, gdzie rów nież osiedlili się templariusze. R ok 1291 był dla patriarchy jerozolim skiego i kapituły datą zasadniczą, gdyż katedra została im osta tecznie odebrana. N ajw idoczniej w Rzym ie uważano bożogrobców nadal za zakon krzyżow y, skoro papież Innocenty V III w roku 1484 połączył ich z kawaleram i maltańskimi osiadłym i jeszcze wówczas na wyspie R odos u. W kilka lat późn iej, w roku 1496, papież A leksander VI dokonał ponow nego rozdziału obu zakonów. W m iędzyczasie, od roku 1304, zezw olono zakonnikom św. Fran ciszka na o b jęcie pieczy tak nad pielgrzym am i udającym i się do Pa lestyny jak i nad G robem Chrystusowym, z zachowaniem dla gwar diana zakonu bernardynów w Jerozolim ie prawa do pasowania ry cerzy tegoż Grobu 1_. W^ ten sposób rozwiązano zagadnienie uczcze nia pobożn ych fundatorów , odbyw ających w bardzo ciężkich a na wet wręcz niebezpiecznych warunkach pielbrzym ki do Ziem i Świę tej, względnie w spierających kościoły i klasztory tam się znajdujące. Jakkolw iek nie było to rów noznaczne z udziałem krzyżow ców w wyprawach w ojennych przeciw muzułmanom, niem niej groziło wielu niebezpieczeństwam i rycerzom udającym się do Jerozolim y przez kraje opanowane przez A rabów . Inaczej rozwijał się w krajach europejskich zakon b ożog rob ców , liczący w roku 1471 ponad dwa tysiące kanoników rozsiedlo nych po różnych klasztorach 1S. Patriarchow ie jerozolim scy, zmu szeni do opuszczenia nie tylko Jerozolim y ale i Palestyny, zm ie niając zależnie od warunków politycznych i w yboru następcy swą siedzibę, zatwierdzali do roku 1428 proboszczy m iechowskich w Polsce H. R zecz naturalna, że polscy b ożogrob cy uważali się za kontynuatorów idei zakonu i dlatego — mimo wygaśnięcia zakonu kanoników G robu Chrystusowego na Bliskim W schodzie — w dal szym ciągu uznawali zw ierzchnictw o patriarchów jerozolim skich, ch ociaż ci zajm owali ju ż tylko tytularne stanowiska, a obrządek łaciński usunięty został z B liskiego W schodu. Uważano jednak u m iechow itów , iż nadal pozostała łączność z G robem Chrystuso wym i z władzą zwierzchnią w osobie patriarchy jerozolim skiego, usuniętą przez wojska muzułmańskie. P raw dopodobn ie w Polsce w wieku X IV widziano w m iechow itach zakon krzyżow y tej rangi 10 tamże. 11 P ę k a l s k i , jw ., s. 18. 12 tamże. 13 S. N a k i e 1 s k i, M iech otia sive prom ptuarium antiąuitatum M onasterii M iechoviensis, Cracoviae 1634 s. 305. 14 Ks. M. B u 1 i ń s k i. W iadom ość historyczna o zakon ie kanoników re gularnych Stróżów Grobu Chrystusow ego. Pam iętnik R eligijn o — Moralny 1852 s. 466. 15 Ks. B o g d a n , jw . s. 262, przypis 166. Is P ę k a l s k i , jw ., s. 71. obronną z wieżą w Miechowie W roku 1163 przybyli pierwsi przedstawiciele zakonu Stróżów Grobu Chrystusowego z Jerozolim y do Polski. Nikt wtedy jeszcze nie przewidywał późniejszego biegu w ypadków tj. utraty przez krzyżow ców Palestyny, zaniku działalności zakonu m ającego swe źródło w pozbawieniu kanoników celu w ytyczonego im przez regułę, em igracji kapituły patriarchy jerozolim skiego i zaniku w pływ ów związanych z tym stanowiskiem. N ic też dziw nego, że pow yżej opisane w ypadki odcięły wszystkie klasztory b ożog rob ców od podstaw działalności na terenie Ziem i Świętej; rów nocześnie wypadki te wysunęły na plan pierwszy najsilniejszą enklawę z pośród wszystkich klasztorów w E uropie, jaką była baza kano ników G robu Chrystusowego w M iechow ie. Ponieważ w M iech o wie, działając szeroko na teren całej Polski, um ocnili się m iech o wici bardzo silnie w wieku X II i X III i w ybili się w tym czasie na czoło polskich zakonów, tym samym zyskali sobie podstawę dla dalszej egzystencji. Tym też sobie należy tłum aczyć ich siłę gospodarczą i w pływy polityczne, które niefortunnie użyte przeciw Ł okietkow i z początkiem wieku X IV zadecydow ały o zawężeniu ich ekspansji na ziemiach polskich. Od całkow itej likw idacji urato wało icłi pokonanie przez A rabów chrześcijaństwa na Bliskim W schodzie oraz nimb otaczający ich jako zakonu jerozolim skich strażników G robu Pańskiego. Pozostając wielką siłą gospodarczą nie m ogli w późniejszych wiekach odegrać większej ro li; musieli ustąpić miejsca zakonom lepiej politycznie ustosunkowanym i tym samym przez w ładców polskich usilnie popieranym . T o jednak, czego dokonali w wiekach X II i X III, a później kontynuow ali przez z górą pięćset lat, jest w ystarczające by ich działalność duszpasterską i samarytańską prow adzoną poprzez bezpośrednie bożogrobców 2. SPR O W AD ZE N IE BOŻOGRO BCÓW DO M IECH OW A W ROKU 1163 1 Kościół co templariusze czy joannici, gdyż w dokum encie z roku 1375 nazwano ich „c ru cife ri” I5. Nie bez znaczenia było także, że w p o bliskim M iechow ow i — Zagościli, znajdow ali się joan n ici, spro wadzeni tam przez Henryka Sandom ierskiego w roku 1153. Patriarchow ie jerozolim scy doceniali p ozycję zakonu w Polsce i stawali się obrońcam i M iechow a, gdy np. w roku 1365 przeor klasztoru b ożogrob ców w Peruggi (W ioch y ) zażądał uiszczenia przez polskich bożogrob ców danin i opłat zwierzchnich pod grozą kar kościelnych. Filip, patriarcha jerozolim ski, rozstrzygnął wtedy spór na korzyść polskich b ożogrob ców , zakazując klasztorow i w Peruggi roszczenia sobie pretensji do danin i zniósł nałożone kary kościelne. —rw .ię lrze k o ś c io l. l.o io g ro b có w .n iecl.ow .k icl. odbudow ane p o p o ia r e w r. 1 . 1.) kontakty z Ziem ią Świętą i Jerozolim ą, ocen ić pozytyw nie w osiemsetletnią roczn icę sprowadzenia tego zakonu do Polski. Jaksa z M iechowa w ioząc z Z iem i Świętej — w drodze p o w rotnej trw ającej ok oło p ół roku — kilka w orków ziem i z góry G olgoty, zapoczątkow ał ważne i bezpośrednie kontakty z Bliskim W schodem . Należy bow iem pam iętać o ówczesnej pen etracji ce sarstwa rzym skiego narodu niem ieckiego na ziem iach w schodnich prow adzonej za bezpośrednictw em łacińskiego kleru, ksiąg, bu dow nictw a i organizacji gospodarczej. Utrzymanie kontaktu ów czesnej Polski z Rzym em b yło m ożliw e jedynie za pośrednictw em elementu germ ańskiego, który pilnow ał krajów E uropy w schodniej, w tym i Polski, uważanej za sferę w pływ ów cesarstwa. H istoria Polski wykazuje dobitnie jak trudno przychodziło w ładcom pias towskim wyłam ywać się spod twardej i ciążącej na każdym k ro ku obłudnie chrześcijańskiej ręki niem ieckiej. Stąd też d ecyzje, podjęte przez Henryka Sandom ierskiego i Jaksę z M iechow a, spro wadzenia do P olski zakonników reprezentujących świat rom ański oraz w pływ y w rogie cesarstwu należy uznać z perspektyw y dzie jow ej za rzecz bardzo doniosłą. P odporządkow anie się w zakresie w pływ ów religijnych zw ierzchnictw u patriarchy jerozolim skiego nie odcinało Polski od zawsze żyw ych kontaktów z Rzym em ani też nie paliło m ostów z zachodnim sąsiadem, ale w prow adzało nowy i ożyw iony nurt związany z ideą wypraw krzyżow ych. W okresie, w którym przybyw ali b ożog rob cy do M iechow a, ich zakon związany bezpośrednio z kapitułą katedry jerozolim sk iej i patriarchą był u szczytu potęgi. Zarów no H enryk Sandom ierski sprow adzający joannitów do Zagościa, jak i Jaksa z M iechowa sprow adzający b ożog rob ców , uniknęli błędu p opełn ion ego przez swych następców w wieku X III, którzy ściągnęli krzyżaków . Jeśli prawrdą było książęce p och odzen ie Jaksy, w yw odzące się z p le m ion słowiańskich w ytępionych przez N iem ców, to myśl jeg o osa dzenia na ziem iach polskich zakonu założonego przez francuskie rycerstw o miało swoją wym owę. R oczn ik m iechowski, spisany rękam i osiemnastu osób, z k tó rych pierwsza zakończyła wpisy kronikarskie w roku 1388, zano tow ał: Jaxa udał się do Jerozolim y pod rokiem 1162 i w rok późn iej przybyli do M iechow a bracia Grobu Pańskiego z jednym kanonikiem przysłanym przez patriarchę M onacha 11. Cronica Polonorum M iechow ity, wyd. w r. 1572, zapodaje rok fun dacji Jaksy w M iechow ie w r. 1162 (s. X C I III). H istoryk i kanonik zakonu b ożog rob ców , Samuel Nakielski, k tóry wydał swoje dzieło oparte na materiałach źródłow ych zachow anych w archiwum m iechow skim w pierwszej p ołow ie wieku X V II, często p ow ołu je się na 11 A. B i e 1 o w 8 k i, Monum entu P olon ia e H istoriea, Warszawa 1961 t. II. R ocznik M iechowski, s. 882. Ołtarz głów ny w kościele m iechowskim z drugiej połow y X V III wieku R ocznik M iechow ski jako jedną z najstarszych kronik. D zieło N akielskiego pt. Miechovia sive Promptuarium Monasterii Miechoviensis zostało wydane w K rakow ie w roku 1634 i stanowi p o d stawowe źród ło w iadom ości o zakonie Stróżów G robu Chrystuso wego od początków jeg o istnienia. Nie trzeba podkreślać jak ważne jest to źródło dla historii zakonu b ożog rob ców w P olsce; zawiera ono sumiennie zebrane i w nikliw ie opracow ane w iado m ości lecz m im o to nie daje właściwego poglądu na szereg za gadnień dotyczących M iechowa w okresie X II i X III wieku. Nie ulega w ątpliw ości, że b ożogrob cy m iechow scy do czasu buntu w ójta A lberta w roku 1411 odgryw ali na terenie Krakowa, ówczesnej stolicy Polski, nie mniejszą rolą niż benedyktyni w w ie ku X I 18. Dlatego wiązanie bożog rob ców z kościołem św. Jadwigi oraz klasztorem i szpitalem na Stradomiu d opiero w wieku X IV jest zgodne z w yw odam i Nakielskiego, lecz niewystarczające. O d kryte na Kazim ierzu w K rakow ie fundam enty rom ańskiego koś cioła św. W aw rzyńca o rzucie charakterystycznie w ydłużonego prezbiterium z absydą (przypom ina rzut kaplicy G robu Chrystuso wego w Jerozolim ie) wskazują na wczesne usadowienie się m ie chow itów pod bokiem w ładców P o ls k i10. Jest bow iem w iadom e, że wieś Bawół na krakowskim Kazim ierzu znajdowała się w rę kach b ożog rob ców m iechow skich; później d opiero przeszła ona na własność kapituły krakow skiej, od której odkupił ją Kazim ierz W ielki i przekazał miastu K azim ierzow i przy nadaniu lokacji w roku 1335 20. Legenda o powstaniu kościoła św. W awrzyńca drogą dwunastowiecznej fundacji Piotra W łostow icza, teścia Jaksy z M iechow a, jest rzeczą praw dopodobną, jak i osadzenie b o żo grobców w tyin kościele jest m ożliw e. Należy zw rócić uwagę na w iadom ość o m iechowitach w przyw ileju lokacyjnym miasta K ra kowa z roku 1257, w którym Bolesław W stydliwy nadaje: „n a tejże rzece (Prądnik) ustępujem y dwa nasze młyny i trzeci, który należał niegdyś do braci z M iechow a” “ . Ustalenie dawnego k o ryta rzeki Prądnik do połow y wieku X V II, uchodzącego do nie istniejącego dzisiaj koryta W isły w pobliżu klasztoru św. Jadwigi b ożog rob ców na Stradomiu, wskazuje na dawne ich związki z tą 18 M. T o b i a s z , K raków b en ed yk tyń sk i w w ieku X I. Nasza Przeszłość t. X I V s. 6— 8. 19 O dkrycie przez Mieczysława Tobiasza fundam entów kościoła św. "Wa w rzyńca w r. 1961, w piw nicach dom ów narożnych przy ul. Bartosza G łow ac kiego 4 i W aw rzyńca 20 na Kazim ierzu, stanowi zapoczątkow anie badań nad architekturą m iechow itów w przedlokacyjnym K rakow ie. Prowadzone w są siedztw ie na Placu Baw ół archeologiczne sondaże przez D yr. Radwańskiego •w okresie dw óch lat w ilości 60, nie przyniosły pozytyw nego rezultatu. 20 W . K o n i e c z n a, P oczątki K azim ierza. B ibl. Krak. nr 94 s. 14. 21 KI. B ą k o w s k i , K raków przed lokacją w r. 1257, K raków 1935 s. 1T0. częścią przedlokacyjnego Krakowa ” . Tak więc rola m iechow itów w ówczesnej stolicy P olski w okresie X II i X III wieku jest dotąd nieprzebadana i wymaga gruntownej rew izji dotychczasow ego przekonania o uplasowaniu się ich tu d opiero za czasów Kazi mierza W ielkiego. Za podstawę osadzenia b ożog rob ców w M iechow ie przyjm uje się dokum ent patriarchy jerozolim skiego Monachusa z roku 1198, w którym napisano: „g d y pan Jaksa, mąż świętej pam ięci, odw ie dził G rób Pański i oddawał cześć Panu w m iejscu, gdzie stąpały J ego nogi, wśród innych odznak swej p obożn ości, prosił aby k ościół G robu Św. wysłał z nim razem do Polski jedn ego ze swych kanoników , którem uby Jaksa spełniając ślub, m ógł nadać dobra. P oniew aż był on mężem prawdziwie chrześcijańskim i pełnym szczególnego nabożeństwa do G robu Pańskiego, chciał obdarzyć nasz kościół m ajątkiem i pom n ożyć w łości Chrystusa wTśród dale kich ludów tamtejszego kraju, aby Chrystus, k tórego dziedzictw o na ziem i powiększył, zapisał go w niebie jako dziedzica w iecznego miasta. Przyw iózł w ięc do Polski jedn ego z kanoników i wyzna czył mu i jeg o następcom za wolą i zgodą znakom itego księcia Bolesława trzy wsie, to jest M iechów , Zagórzyce i K om orow o jako wieczystą jałm użnę” 2S. Jaksa z M iechowa herbu G ryf, dumnie w yzierającego z tarcz rycerskich w dawnym opactw ie m iechow skim : „b y ł — według Kroniki W ielkopolskiej — księciem Sorabów, plem ienia słowiań skiego z nad Łaby; wygnany z ojczyzny i osiadły w Polsce, gdzie dał początek możnemu rod ow i G ryfitów ” S4. Liczne swe włości zawdzięczał posagow i żony B eatryczy, córki Piotra W łostow icza z rodu Łabędziów , m ożnow ładcy oślepionego przez Bolesława K rzyw oustego; W łostow icz oprom ieniony był legendą założenia 70 czy 77 kościołów . Tak piszą o kom esie P iotrze źródła nie m ieckie. Za przykładem swego teścia spieszył Jaksa z M iechowa. Dokum ent partriarchy jerozolim skiego, zatw ierdzający ugrunto wany już w Polsce zakon Stróżów Grobu Chrystusowego w trzy dzieści sześć lat po ich sprowadzeniu do M iechow a, wymienia hojne donacje jakie posypały się po roku 1163; don acji tych była pokaźna ilość bo aż trzydzieści p ięć *. W iesci o wyprawach do Ziem i Świętej, o walkach z Saracenami. a także m ożność uzyskania odpustów — bez konieczności odbywania pielgrzym ki do G robu Chrystusowego — za pośred 22 M. T o b i a s z, H istoryczny rozw ój sieci w odn ej K rakow a i je j w pływ na u rbanistykę miastu. Zesz. Naukowe W ydziału Architektury P olitech niki K ra ków . nr 2. 1958 s. 23— 28. 23 Z. K o z ł o w s k a - B u d k o w a , Z przeszłości pow iatu m iech ow skiego, 1929 s. 20. 24 tamże. 25 tamże. nictwem przedstawiciela kapituły jerozolim sk iej osiadłego w M ie chow ie, inspirowała hojne przyw ileje książęce i nadania rycerskie. Przypuszczać należy, że w ślad za M arcinem Gallem przybyli następni b ożog rob cy do M iechow a, gdyż liczne darowizny wym a gały w prow adzenia we w łościach d obrej organizacji adm inistracyj no-gospodarczej, duszpasterskiej, a także utrzymywania stałego kontaktu m iędzy ofiarodaw cam i a Jerozolim ą. Dokum ent pa triarchy Monachusa wymieniał w śród ow ych 35 posiadłości przy należnych i podległych klasztorow i w M iechow ie: 2 wsie tar gowe, 3 kościoły (Chełm no, Skaryszew, G niezn o), liczne dziesięci ny nadane przez biskupa krakow skiego Gedkę, 3 źródła solne, uw olnienie od ciężarów prawa książęcego, podatków , świadczeń, robocizn na rzecz księcia, jakich nie posiadał żaden ówczesny klasztor w P olsce” 25. Szybki rozw ój klasztoru m iechow skiego w Polsce znajdowrał odpow iednik i w innych krajach; dlatego należy tu p rzytoczyć bullę papieża H onoriusza III z roku 1220, w której w ym ienione są klasztory bożog rob ców w Akrze, Tyrze, A ntioch ii, K onstanty n opolu, Tessalonice, w Lom bardii, Toskanii, N eapolu, Sycylii, Hiszpanii, Czechach, na W ęgrzech, w Slawinii, N iem czech, F rancji, A n g lii27. Tak w ięc szeroko rozprzestrzenił się ten zakon w kra jach i na w yspach opanow anych przez chrześcijaństwo — m im o utraty Jerozolim y i G robu Chrystusowego, będących podstawą działalności stróżowania, duszpasterstwa i opieki. Z upływ em lat klasztory bożog rob ców na terenie innych państw stopniow o zani kały mimo oddania się sprawom duszpasterstwa i szpitalnictwa. O dw rotnie rzecz się miała w Polsce, gdzie zakres działalności M iechowa stale wzrastał, a wybudow anie w kościele m iechowskim kaplicy G robu Chrystusowego, będącego kopią jerozolim skiej -— zwłaszcza po opanowaniu Jerozolim y przez mahometan — p o d niosło znaczenie prawa o udzielaniu zastępczych odpustów. Przez cały wiek X III m nożyły się fun dacje p obożn ego rycerstwa p o l skiego, podkreślającego w ten sposób w olę odbycia pielgrzym ki d o Ziem i Św iętej; w obec niem ożności jej zrealizowania — wzamian za udzielone odpusty — ofiarow yw ało bogate jałmużny. 3. P IE R W O T N Y KOŚCIÓŁ I K A P L IC A G RO BU CHRYSTUSOW EGO W M IECH OW IE Pierw szy kościół murowany — w edług opisu N akielskiego — przypuszczalnie posiadał założenie centralne na planie kwadratu, na w zór k ościołów w sch od n ich !8. K anonicy jerozolim scy, osiedli 26 tamże. 27 Ks. J. G a c k i , B ożogrobcy. Pam iętnik R eligijn o — Moralny. 1853 s. 111 28 N a k i e 1 s k i, jw . s. 63/64. w M iechow ie, w zorow ali się p raw dop od obn ie na typie rozsze rzonej rotundy tzw\ Anastazis, zbudow anej przy bazylice je r o z o lim skiej. Budowla ta miała stać w miejscu dzisiejszej kaplicy G robu Chrystusowego, połączon ego później z krużgankami klasz toru. Ten bardzo interesujący typ budow li sakralnej b yć m oże przeniesiony przez b ożog rob ców na ziem ie polskie w prost z Pa lestyny jest dotąd sprawą otwartą z p ow odu niem ożności p rzep ro wadzenia badań w ykopaliskow ych 2#. W krypcie tego pierw otn ego i niewątpliwie rom ańskiego k o ścioła m iechow skiego pierw szych bożog rob ców w Polsce, spoczęły zw łoki ich fundatora Jaksy z M iechow a, a w wieku X V I I Samuela N akielskiego. Pierwszy k ościół nosił przypuszczalnie wezwanie B o żego G robu i ozdob ion y był — ówczesnym zw yczajem — herbami rycerstwa, które było d obrodziejam i klasztoru. N akielski w wieku X V I I widział jeszcze zniszczone epitafium Jaksy-założyciela; we dług tradycji Jaksa miał przyw ieźć z Jerozolim y kilka w orków ziem i p o d budow ę pierw szego kościola-kaplicy 30. N akielski pisze, że sami zakonnicy w ym urow ali pierwszy kościół m iechowski, co w ydaje się p raw dopodobn e i rów nocześnie utwierdza w przekona niu, iż czerpiąc w zory bezpośrednio z Ziem i Świętej w prow adzili tamtejszą architekturę do swych k ościołów w P olsce 31. Na miejscu owego pierwszego kościoła m iechow skiego stoi obecn ie kaplica kryta kopułą na pendentyw ach z latarnią. Kaplica ta jest fundacją prepozyta m iechow skiego Tom asza z Olkusza z pierwszej połow y wieku X V I, odrestaurowana zaś została w roku 1880. W je j wnętrzu znajduje się kopia G robu Chrystusowego wykonana w dolnej partii z ciosu, górą z d rz e w a 32. Z tego względu kaplica ma wezwanie G robu Pańskiego. Spodem , przez niew ielki otw ór, w W ielk i Piątek wsuwa się do wnętrza figurę zm arłego Chrystusa przyniesioną w p rocesji przez dziewczęta. W edług panującego zw yczaju w W ielki Piątek i W ielką Sobotę przy grobie czuwali na zmianę czterej zakonnicy b o ż o g r o b c y 3S. P obożn i, udający się do m iechow skiej kaplicy G robu Pańskiego, otrzym ywali takie odpusty jak pielgrzym i jerozolim scy. Należy podkreślić, że w innych krajach nie ma zwyczaju — począw szy od W ielkiego Piątku — dekorow anie w kościołach jed n ej z ka plic i urządzania w niej G robu Chrystusowego. Zw yczaj ten roz 29 A u tor jest w trakcie uzyskania zezwolenia na badania zespołow e w ka plicy G robu Chrystusowego w M iechow ie. Jak do tej pory badań w tym m iejscu nie prow adzono. Badania mają na celu odszukanie fundam entów pierw szego kościoła rom ańskiego z X I I wieku. 30 N a k i e l s k i , jw ., s. 81. 31 T o b i a s z , K raków b en ed yk tyń sk i, s. 6. 32 Ks. J. W i ś n i e w s k i , D ekanat M iechow ski, M iechów 1917 s. 119— 120; Ks. M. N o w o d w o r s k i , Encykl. K ośc. t. IX . Warszawa 1876 s. 464. Stróże G robu ] erozolim skiego. 33 tamże. powszechni! się w P olsce w ciągu w ieków dzięki m iechowskim b ożogrob com , którzy zapoczątkow ali w ielkopostne nabożeństwo, nauki i odw iedzanie p o kościołach grobów z figurą zm arłego Chrystusa Nabożeństwo przy G robie jerozolim skim i w Święta W ielka nocne znane było tylko b ożogrob com w Palestynie (nie od pra wiano go nawet w Rzym ie) i zostało przez nich przeniesione do M iechow a; po utracie bazyliki jerozolim sk iej rozpow szechnione zostało przez kanoników tego zakonu na terenie całej Polski . „O p rócz tego w niedzielę drugą po Zm artwychwstaniu Pańskim odprawiali b ożogrob cy uroczystość Grobu Chrystusowego, jako święto tytularne swego zakonu z takim obrzędem , z jakim w samą wielkanocną niedzielę kościół polski odprawia. Na szczególną tę uroczystość ułożyli w przepisanym porządku pacierze kapłańskie z pięknym hymnem zaczynającym się Sancti Sepulchri gloria etc.” 83. Na prośbę generała kanoników m iechow skich, ks. Jakuba R adlińskiego, nabożeństw o pow yższe zatwierdził w roku 1744 papież Benedykt X IV i nakazał je w prow adzić na terenie całej p rzedrozbiorow ej Polski łącznie z Litwą 37. Od tego czasu b o ż o grobcy odprawiali uroczystą procesję — p odobn ie jak w nie dzielę wielkanocną — w ostatnią niedzielę po uroczystości Z ie lonych Świątek z obnoszeniem godeł Zmartwychwstania. B o ż o grobcy mieli przyw ilej papieski na używanie rokiety i m antoletów takich, jakie noszono w Jerozolim ie i jakie w olno b yło używać biskupom i rzymskim p ra ła tom 38. Papież Leon \ w breve z roku 1520 zezwalając na noszenie rokiety, a Grzegorz X III — na prośbę generała m iechowskiego A ndrzeja B atorego — zezwalając na m antolety, podkreślili znaczenie M iechowa jako spadkobiercy zw yczajów jerozolim skich 39. R ów nież — jak w Jerozolim ie patriarcha przyjm ow ał do zakonu bractwa rycerzy zasłużonych dla G robu Chrystusowego — tak też i m iechow itom prawo to nadał patriarcha Monachus w roku 1198: „przyjm ow an ie świeckich osób do konfraterni Grobu Świę tego w M iechow ie z zezw oleniem noszenia krzyża p od w ójn ego” 40. Tą drogą w owym czasie zostali p rzyjęci z rodzin książęcych w P olsce: B olesław Kędzierzawy, M ieszko Stary z żonam i Elż bietą i Eudoksją oraz z synami z obu tych małżeństw, dalej K azi mierz II z żoną Heleną i synami Leszkiem Białym i K onradem 34 35 3# 37 38 tamże. P ę k a l s k i , jw., s. 43— 48. tamże. tam że. M. N o w o d w o r s k i , jw ., s. 465. P ę k a l s k i , jw. tamże. 40 tamże. oraz książęta opolscy i r a cib o rs cy 4ł. Ponadto — jak p odaje Nakielski według dokum entu patriarchy Monachusa — do zgrom a dzenia weszli dostojn icy duchow ni: arcybiskupi gnieźnieńscy Piotr i Jan oraz biskupi krakowscy G edeon i Pełka. Spośród rycerstwa na pierwszym miejscu w ym ieniony był Jaczo czyli Jaksa z M ie chowa z wielu ziemianami, ofiarodaw cam i licznych wsi uposaże niow ych J2. Fundatorow ie m ieli praw o noszenia na szatach p o d w ó j nego czerw onego krzyża, będącego godłem stróżów G robu Chry stusowego. N akielski podkreślił, że biskup krakowski G edeon pośw ięcił pierwszy kościół m iechow ski na chwałę G robu Świętego, a będąc już członkiem bractwa b ożog rob ców pośw ięcił także kościół w Skaryszew ie43. Poniew aż w innym m iejscu N akielski podał, że k ościół m iechowski nosił wezwanie św. Katarzyny A leksandryj skiej, przypuszczać należy, iż miał na myśli wezwanie drugiego k ościoła; kościół m iechow ski bow iem na w zór bazyliki Grobu Pańskiego w Jerozolim ie od pierwszej chw ili p raw dop od obn ie był pod tym właśnie wezwaniem. Stąd też kaplica, która przetr wała na jego przypuszczalnym miejscu i nazwana kaplicą Grobu Pańskiego, wezwanie to przejęła. Ponieważ w Jerozolim ie przy G robie Chrystusowym paliły się światła w ciągu dnia i nocy, dlatego Leszek Biały w roku 1 2 1 7 przeznaczył na ten cel fundusze dla kościoła m iechow skiego; fundusze te zawarowane były doku mentem wydanym w obecn ości W incentego Kadłubka, biskupa krakow skiego oraz prepozyta i archidiakona krakow skiej kate dry 4\ Oczywiście pierwszy kościół okazał się w krótce zbyt mały dla potrzeb pielgrzym ek korzystających w warunkach X II i X III wieku z odpustów związanych z Ziem ią Świętą. Przystąpiono więc do wznoszenia now ego k ościoła (fundam enty i mury tej budow li znajdują się częściow o w dolnych kondygnacjach dzisiejszego k o ścioła). K aplica Grobu Chrystusowego została połączona z kruż gankami, w których ustawiono stacje M ęki Pańskiej. Jednocześnie postępow ała budowa klasztoru i budynków gospodarczych, o czym będzie m owa poniżej. 4. UPO SAŻENIE KO ŚCIO ŁA I K LA SZTO R U M IECH OW SKIEGO Dokum ent patriarchy Monachusa z roku 1 1 9 8 w yliczał dokład nie donacje na rzecz m iechow skich b ożogrob ców — poza w ym ie nionym i już trzema wsiami ofiarow anym i przez Jaksę 4‘ . W roku 1 2 0 8 papież Innocenty III przyjął konwent m iechow ski pod 41 N a k i e l s k i , jw ., s. 81— 83. 42 tamże. 43 tamże. 44 tam że , 8. 135. 45 tam że, 8. 81— 83. „N ie znam y ich rozm ia rów , d ok u m en t nazyw a k a żd y z n ich p łu g ie m , ale oniekę Stolicy A postolskiej i w dw óch bullach zatw ierdził wszystkie d arow izn y48. Majątek m iechow skich b o zo g ro b co w stale wzrastał, gdyż prowadzili oni racjonalną gospodarkę rolną i umiel i n i e pomnażali swe dochody. Ta gospodarność przebija się przez wszystkie karty historii pośw ięconej klasztorow i m iechowskiem u. B ożogrobcy przeprow adzili „reform ę gospodarczą i prawną, p o łączoną z akcją osadniczą tzw. k olon izacji na prawie niem ieckim . Powstają nowe wsie założone na tym p r a w i e , j a k W ola Bukow ska, W ola Podm iejska, W ola Podleśna, W ola Kalińska. Ą\ dawniej istniejących przeprowadzają kom asację i podział gruntów d ostoso wany do trójpolow ego systemu uprawy a różn orodn e świadczenia chłopskie w naturze zostają zamienione na czynsz pieniężny W połow ie wieku X IV dobra m iechowskie były zreform ow ane oraz oczynszowane i dlatego w kilka lat późn iej przynosiły pokaźn e sumy; dochody te budziły niestety wiele zazdrości i przyczyniły się do zmiany dawnego charakteru zakonu i właściwego jeg o prze znaczenia. Zebrane kapitały szły oczyw iście na budow le, b ib lio tekę, aparaty kościelne, na zakup zegara i organów, ale ró w n o cześnie wprowadzały wśród duchowieństwa niezdrow e am bicje do zawładnięcia administracją M iechow a, co później spow odow ało niewłaściwe przeznaczenie fundacji. Z dokumentu Kazimierza W ielkiego z roku 1354 wynika, że klasztor w M iechowie posiadał wsie: L n iejów , Zagórzyce, Strzeżów, W olę Bukowską, Kalinę Małą, Szczepanowice, Przeslaw ice, Jaksice, Kam ieńczyce, Falniów, Przedm iejską W olę (nazwaną W olą za Stawem), M ich a łow ice48. Były to wsie p ołożon e w naj bliższej okolicy M iechow a; oprócz w ym ienionych uprzednio jeszcze około dwudziestu wsi w dalszych i bliższych stronach stanowiło ogromny majątek rolny bożogrobców . D o połow y wieku X V , czyli do czasów Długosza, doszły jeszcze dalsze wsie p ołożon e w pobliżu opactwa: Chodów, Orłów, Podleśna W ola, Brzuchanie, Parkoszowice, Pstroszyce, Siedliska W ola, Kalińska czyli Kalina Mała ". Szereg wsi powstało przed rokiem 1339 na zasadzie osadnictwa lokacyjnego, polegającego na zrzeczeniu się przez klasztor d o ch o dów przez okres lat 20 względnie na czas dłuższy w postaci tzw. wolnizny; do takich wsi należały: M ichałow ice, Bukowska W ola. Falniów, Smroków, Przeslawice, Kalina Mała i Podm iejska W o l a 50. Folwarki klasztorne w roku 1349 były w następujących m iej scowościach: M iechowie, Kalinie, Szczepanowicach, Jaksicach, Strzeżowie. 46 tamże, s. 128— 129. d o c h o d y p od a je n ie je d n a k o w e od 1.5 d o 3 w łó k licz y ły k a żd y , ty lk o f o l wark » M iech ow ie nazyw any 2 płu ga m i, m usiał o b e jm o w a ć 6 w łó k zie m i o rn ej. W raz z p ow oln ą d ew a lu a cją tem zboża, p rzyb yw a ło fo lw a r k ó w klasztoru. Za D ługosza p r zy b y ły czynszów i p o w ię k s z a ją cy m zu ży tk o w u ją cy ch je s z cze r o b o c iz n ę fo lw a r k i k la szto rn e K om orow ie, M ich a ło w ica ch , U n ie jo w ie , C h o d o w ie , Siedliskach, P a rk o szo w ica ch , F a ln io w ie , S m ro k o w ie , O rło w ie , się zby p o d d a n y ch we w sia ch : B rz u ch a n i, P s tro s z y ca ch , Z ago- rzy ca ch , w k tóry ch w ro k u 1456 zeb ra n o 7 tysięcy s n o p k ó w p s z e n icy , rze cz niesłychana i nie d o w iary dla ty ch , k tórzy teg o nie w id z ie li 51. O p actw o w M iech ow ie uw ażało, że spotyk a g o „k o s z t o w n e w y różn ien ie” przez w ysok ie ota k sow a n ie d o c h o d ó w z d ó b r , p r z e w yższających d o ch o d y w szystkich in n y ch za k o n ó w w P o ls ce i d la tego w roku 1349 p rz e p ro w a d z o n o — na p ro ś b ę z a k o n n ik ó w — p on ow n e otaksow anie icli m ajątku , k tó re b y ło m n iej słuszne w stosunku d o d ok on a n ego w rok u 1325 T o w ie lk ie g r o m a d z e nie d ó b r ziem skich przez zak on b o żo g ro b có w n ie szło w p arze z przeznaczen iam i d o ch o d ó w z nich na a k cję ch a ry taty w n ą czy samarytańską. Długosz w ręcz zarzucał z a k o n o w i w w iek u X V , że „s ła b o przestrzegał regu ły św. A u gu styn a” i3. R ów n olegle ze sku pyw an iem p rzez klasztor m ie ch o w sk i wsi i folw a rk ów szło w yk u p yw a n ie z rąk sołtysów lo k a cy jn y c h b o g a tych sołectw i obsadzanie ich lu d źm i zależn ym i o d za k on u . P r z y kładem tego b yło w yk u p yw a n ie częściam i wsi B rzu ch a n ie z rąk 14-tu „s o b ie p a n ó w ” tj. szlachty ch o d a c z k o w e j. W ro k u 1394 większa część tej wsi zn ajd ow a ła się ju ż w ręk a ch k la sztoru i o b e j mowała i łanów k m iecych , z k tó ry ch czynsz w y n o sił: 6 s k o jcó w . od n iek tóry cli 10 s k o jcó w , ja j 30, k a p ło n ó w 4, serów 2 oraz r o b o cizna w ynosząca sprzężajem 1 d zień w tyg od n iu p rzez ca ły ro k oraz pow aby na w iosnę i w zim ie Na każdą sprzeda ż w y sta w io ny był od ręb n y d ok u m en t: sprzedaż z rok u 1392 (je d n a ż w ie lu ) dokon an a przez 1 om asza z B rzu ch au ia na rę ce Stanisław a, p r o boszcza m iech ow sk iego, została zatw ierdzon a przez sę d zie g o D ragom ira i podsędka ziem i k ra k ow sk iej K rzesław a w K siążu Jak bardzo p ow ik łan e b yły spraw y g o s p o d a rcze fo lw a rk ó w L W" m“ w , i IIIOŻna za o l,se rw o " a ć na p rzy k ła d zie Sm rokov%a . W roku 1236 p ro b o sz cz k ra k ow sk i W it sprzeda ł H en ry■p re P“ zy t? w i , m iech ow sk iem u , w ieś swą S m r o co w za 60 wien. ^ roku 1339 Jakub de S m rocew , sołtys k lasztoru m ie52 53 54 55 56 ta m że. tam że. G eo g ra ,icz n y K r “ lestu a P o lsk ieg o , t. I, g. 426. jw ., t. X , s. 924. 2 — Nasza P rzeszłość chowskiego, występuje na jednym z (lokiinientów. W roku 1319 w związku z otaksowaniem dochodów opactwa, prokurator klasz torny Mateusz Krzeczkowicz oznaczył dochód jaki wieś ta przy nosiła na 6 grzywien i 1 ferton. W roku 1379 — Jarosław ze Skotnik, arcybiskup gnieźnieński, polecił kanonikowi Albertowi i altarzyście katedry krakowskiej Mikołajowi Skałce rozstrzygnie cie sporu między klasztorami miechowskim i mogilskim o dzie sięcinę ze wsi Smroków. W roku 1380. Halla z Proszowa sprzedał pól łanu Markowi, sołtysowi tamecznemu, za 12 grzywien. W roku 1385. w obecności wójta i sędziego najwyższego sądu niemieckiego we dworze klasztoru miechowskiego, sprzedał Dominik Krzeczak '/u łanu wolnego za 11 grzywien. roku 1385. sołtys Marek z żo ną Hanką. Marcinem bratem Hanki i Dominikiem Krzeczakiem, sprzedali klasztorowi swoje łany. Wieś o takiej przeszłości, będąca w połowei wieku XV własnością klasztoru miechowskiego, miała 18 łanów kmiecych, karczmę z rolą i 3 zagrodników z rolą. Z tych łanów płacono dziesięcinę snopową i konopną wartości 10 grzy wien biskupowi krakowskiemu. Folwark klasztorny płacił dziesię cinę wartości -1 grzywien klasztorowi mogilskiemu. Prócz tego z czterech łanów specjalnych płacono dziesięcinę wartości 4 grzy wien klasztorowi cystersów w Mogile. Z łanu dawano meszne ko ściołowi parafialnemu w Wielkich Czaplach: 4 korce jęczmienia 1 2 korce owsa. W roku 1581 własność prepozytury miechowskiej w Smrokowie wynosiła: 7.5 łanów kmiecych. 3 zagrodników z rolą, 2 komorników z bydłem. 2 komorników bez bydła. 5. M IASTO MIECHÓW l stóji klasztoru prędko powstało miasto; czerpało ono zvski nie tylko z targów licznie uczęszczanych przez ludność okoliczną ale także z pielgrzymek spieszących do Grobu Chrystusowego w kościele bożogrobców, udzielających odpustów. Trudno ustalić początkową datę odbywania się tego targu: mówi o nim dopiero dokument Henryka Brodatego z roku 1232, wspominający jednak, że już dawno targ ten is t n ie je D o k u m e n t Monachusa z roku 1198. wspominający targi w Chełmie nad Rabą i w Skaryszewie, milczy o M iechow ie3. Bolesław Wstydliwy przywilejem z roku 1259 ,,... zakazał swemu myncerzowi i poborcy ceł urzędować na targu miechowskim (nie wolno było ściągać kary, pozywać przed sąd. wymieniać pieniędzy i pobierać cła od handlujących). W wy padku nieusłuchania proboszcz miechowski mógł bezkarnie wyrzu cić urzędników książęcych z Miechowa. Bożogrobcy pilnowali porządku wolnego targu, ale transakcje musiały być dokonywane i>: K o z ł o w s k a - B u ii k o w a, ju\, s. 35. 58 tum ie. w monecie obowiązującej w dzielnicy krakowskiej. Klasztor tą drogą nie dzielił się dochodami z urzędnikami panującego księcia’ 59W roku 1290 istniejące juz miasto Miechów otrzymało przy wilej lokacyjny księcia Przemysława II, który zezwalał obywate lowi miasta Krakowa, Gerardowi, na założenie miasta na prawie n i e m i e c k i m W imieniu klasztoru prepozyt Piotr, kustosz Henryk i przeor Bogufał — oddali osadźcy, wójtowi Gerardowi, 600 łanów wielkich (każdy liczący około 48 morgów) z prawem założenia na nich miasta. Każdy z działów otrzymywał role podzielone na trzy pola i plan na dom przy Rynku lub jednej z ulic. Sołectwo i jurysdykcja przysługiwało Gerardowi na takim prawie, jakim rządziło się miasto Środa na Śląsku (rządziło się ono prawem wzo rowanym na miastach niemieckich — Halla i Magdeburg). P o nieważ Gerard traktował wójtowstwo miechowskie jako transakcję handlową, z której czerpał zyski w zamian za zapłaconą klaszto rowi sumę, dlatego wyręczał się sędzią miechowskim Adamem z Komorowa Kazimierz Wielki w roku 1365 zatwierdził w Krakowie wy kupiło wójtostwa miechowskiego z rąk następców Gerarda, braci Mikołaja i Spytka, w zamian za odstąpienie przez klasztor trzech wsi: Karniowa wsi przynależnej do parafii Czulice, INieszkowic w parafii Pogwizdów w powiecie bocheńskim i Udorza w parafii Cłilina w powiecie olkuskim Tą drogą bożogrobcy uzyskali prawo do obsadzania wójtostwa miechowskiego ludźmi sobie od danymi. co miało zasadnicze znaczenie dla wszystkich spraw gospo darczych i sądowniczych. Klasztor rządził miasteczkiem poprzez radę miejską i lawę przysięgłych, do której wchodzili wójtowie z wsi zależnych od bożogrobców. W roku 1373 istniał sąd prawa niemieckiego dla sołtysów dóbr klasztornych tzw. sąd leński, w którym zasiadali sołtysi ze wsi Szczepanowie i Smrokow'a ',3. Za czasów Długosza Miechów liczył 70 domów miejskich z pra wem sprzedawania napiwków, 16 zagród, 2 młyny, cech rzeźniczy liczący członków 26, a szewski 20. W wieku X V I istniały tam nadto następujące cechy: stolarski, bednarski, sukienników. Z do kumentów królewskich wynika, że istniały inne cechy: rymarski w roku 1604, krawiecki w roku 1641, i kowalski w roku 1708 M. Bogactwo klasztoru i ściągające do Miechowa rzesze pielgrzymek o0 l a m i e . 60 K odeks D yplom atyczn y M ałopolski, K raków 1886, t. II, s. 179. 61 K o z l o w s k a - B u d k o w a , dz. cyt., s. 37. 62 K od. Dypl. Malop. II, s. 187. 63 K o z l o w s k a - B u d k o w a . jw., 9. 23 64 Ks. Fr. N i w i r r a . M onografia kościołów d iecezji k ieleck iej. Warszawa 1911, t. II, 8. 274. przyczyniały się do dobrobytu miasta. Znakom ite gleby i związane z nim i świetne urodzaje ściągały na targi m iechowskie okoliczną ludność, zaopatrującą się tam także w w yroby przem ysłowe. Liczne pożary i klęski w ojenne kilkakrotnie obracały miasto w perzynę, po których odbudow yw ało się ono mniej lub w ięcej szczęśliwie. Y^alki o tron krakowski w X III wieku odbijały się niekorzystnie na M iechow ie i narażały m ieszkańców na poważne straty. Tak musiało być w okresie walk dzielnicow ych, podobnie jak w okresie buntu wójta A lberta; we wcześniejszym okresie nastąpiło dwukrotne zniszczenie miasta, uważanego za własność b ożog rob ców : w roku 1294 przez wojska księcia p łockiego B o lesława. a w roku 1300 przez Kum anów i W ęgrów '"'. Vt roku 1342 miasto płacąc za lojalność w obec Kazimierza W ielkiego zostało splądrowane przez wojska Jana czeskiego. W zamian za to król otoczył M iechów opieką; chąc uzyskać hojniejsze wspom ożenie dla filii klasztoru b ożogrobców ze szpitalem św. Jadwigi na Stradom iu — obsadził w roku 1360 probostw o z zarządcą Strzeszein z Pstroszyc, którv nakładał duże kontrybuje i bvł dla .,braci uciążliwy” 6C. M iechów ulegał już wcześniej zniszczeniom : m iało to miejsce w czasie najazdu Tatarów w roku 1241; podobn ie zrujnow ali oni miasto i klasztor z kościołem w roku 1289. Te klęski były właśnie pow odem nadania M iechow ow i wr roku 1290 prawa now ej lokacji przez Przemysława łl. ^ roku 1344 wybuchł w klasztorze pożar, który pow tórzył się także w roku 1506; w roku 1595 spaliło się zaś całe m ia s to b7. N iedobrze obeszli się z M iechow em także Szwedzi w roku 1655. D ocierały tam także głód i zarazy; okres największego głodu przeżyli mieszkańcy miasta w roku 1736. W reszcie w roku 1745 spalił się kościół i miasto. B ożogrob cy od budowali wprawdzie budynki kościelne, ale miasto nie podniosło się już właściwie z ruiny i przez długie lata prow adziło żywot wegetatywny. 6. O R G A N IZA C JA Z A K O N l BOŻOGROBCÓW MIECHOW SKICH W wewnętrznej organizacji polskich bożogrob ców stali p rep o zyci i plebani na czele podległych k ościołów i b en eficjów parafial nych wTzględnie szpitalnych; zwierzchnikam i szpitali byli czasem mianowani m istrzowie szp italn i" . Na wakujące stanowiska byli wysuwani kandydaci z pośród braci-kanoników , typowani przez proboszcza m iechowskiego względnie — w wypadku nieobecności tegoż przez jego zastępcę. W wryborze brali udział członkow ie 63 60 67 •8 Koztowsk B i e Io w sk N a w a r r a, Ks. G a c k i. a - B u d k o w a , jw ., s. 24— 26. i, jw . t. II s. 886. jw ., s. 274. jw .. s. 221— 2. kapituły m iechow skiej, w zorującej się w tym względzie na zw y czajach panujących w kapitule jerozolim skiej. Następnie prezento wano wybranego kandydata diecezji, w której wakowało p robos two i w tedy biskup ordynariusz instytuował kanonika-hożogrohca, rozciągając nad nim władzę taką, jaką posiadał nad duchow ień stwem świeckim lecz bez praw a j u r y s d y k c j i K a ż d y z p robosz czów — z wiedzą i zgodą p rzełożon ego klasztoru w M iechow ie — przybierał sobie do pom ocy drugiego członka zakonu, którego bez ob edien cji opata nie m ógł ani przyjąć ani zw olnić. Przepisy zakonne wymagały bowiem na probostw ie oprócz proboszcza także obecności wikariusza z tegoż zakonu celem wspólnego przestrze gania reguły. Stąd też obu obowiązywał tryb życia taki. jaki przestrzegany był w klasztorze m iechowskim . Tam. gdzie zacho dziła potrzeba, wysyłano z M iechowa trzeciego zakonnika, który poświęcał się wyłącznie obow iązkom kaznodziejskim i ci należeli do w ysoko wykształconych w zakresie filozofii i teologii, stąd piastowali rów nież godności p rofesorów . W kilku wypadkach rezy dowali w macierzystym klasztorze m iechowskim , oddając się w y łącznie pracy pisarskiej i studiom . P repozyci pełnili swoje obow iązki dożyw otnio, natomiast wi kariusze winni byli zm ieniać się co trzy lata. ale to w wrieku X V III przestało być stosowane. P roboszczów obow iązyw ało co ro cz ne wysyłanie do Miechowa rozrachunków z przychodów i rozch o dów : na każdym zebraniu kapituły składali oni sprawozdania ze stanu spraw poruczonego ich opiece probostw a C hodziło tu nie tylko o odprowadzanie d och od ów — częściow o także i do skarbca m iechowskiego — ale głównie o uzm ysłowienie członkom zakonu, że są tylko administratorami zakonnym i. Zależność od kurii biskupiej proboszczów -bożogrobców osadzonych na beneficjum w danej diecezji była identyczną z zależnością duchowieństwa świeckiego. Prawo do pełnienia obow iązków , przysługujących w zasadzie duchowieństwu świeckiemu, posiadał zakon m iechow itów na m ocy przyw ilejów papieskich Urbana IV (1261— 1264) oraz B on ifacego IX (1389— 1404). który to prawo p o tw ie rd ził71. W Jerozolim ie, po patriarsze jerozolim skim , trzecie stanowisko w hierarchii k o ścielnej zajmował przełożony zakonu bożogrobców . Dlatego w M ie chow ie najwyższe stanowisko zajmował w zakonie bożogrob ców prepozyt kościoła m iechowskiego, później nazwany generałem i opatem m iechowskim. Tutaj odbyw ały się kapituły zakonu, k tó rym przew odniczył tenże proboszcz-prepozyt, obok k tórego — jeszcze w X III wieku — występował w dokum entach przeor 89 tam ie. 70 tamże. 71 Ks. 6 u I i ń s k i. art. cy t., s. 467. miechowski później zas ta godność znika. Proboszcz-prepozyt zajmował pierwsze miejsce wśród braci zakonnej i wybór jego był zatwierdzany przez patriarchę jerozolimskiego do roku 1428: po tym okresie nastąpiła zmiana i Wacław ze Zdziechowa, jako proboszcz pierwszy otrzymał zatwierdzenie od biskupa krakow skiego Zbigniewa Oleśnickiego Przez Oleśnickiego został rów nież zatwierdzony następny prepozyt miechowski, Jan Oczko, który uczestniczył w soborze bazylejskim. Odtąd wszyscy opaci miechowscy byli zatwierdzani przez biskupów krakowskich. Stąd wielu dygnitarzy kościelnych w X V II i XVIII wieku, posiadają cych tytuł prepozyta miechowskiego, zostawszy biskupami krakow skimi musiało zrezygnować z Miechowa. W roku 1383 — prepozyt miechowski Stanisław Stoikoń — otrzymał od patriarchy jerozolimskiego prawo używania tytułu jenerała zakonu bożogrobców na terenie Słowiańszczyzny, gdyż jego poprzednik Marcin Czczikus uzyskał ten tytuł, lecz tylko wy łącznie dla bożogrobców miechowskich Znaczenie stanowiska proboszcza miechowskiego znacznie wzrosło, gdy papież Jan \ \ I I I zezwolił w roku 1412 ówczesnemu prepozytowi miechowskiemu, Michałowi z Radomska, używać insygni biskupich, tj. infuły, pierścienia, pastorału, dalmatyki i in nych części stroju pontyfikalnego, z prawem udzielania błogo sławieństwa w czasie uroczystych nabożeństw w razie nieobecności hisku pa czy legata papieskiego 7’. Drugie po prepozycie miechowskim stanowisko w zakonie zaj mował kustosz, zwany także starszym mistrzem klasztoru, który w hierarchii klasztoru stał w wieku X III wyżej od przeora 7®. Według uchwały kapituły z roku 1470 kustoszem musiał być naj zdolniejszy z pośród braci zakonnej. Jego obowiązkiem był nadzór nad wykonywaniem przez wszystkich obowiązującej reguły zakon nej. Z tego tytułu korzystał on z pom ocy kantora, dbającego znów o śpiewy w chórze. Przez ręce kustosza przechodziły do skarbca zakonnego dochody ze wszystkich probostw. Kustoszowi podlegał, niższy od niego rangą, skarbnik, sprawujący pieczę nad wszyst kimi kosztownymi aparatami kościelnymi i srebrami. Do zakresu czynności skarbnika należała też inwentaryzacja i pieczołowite przechowywanie przywilejów zakonu. Po nim następował hrat-zakrystian. czuwający nad porządkiem nabożeństw, kolejnością odprawianych mszy, spowiedzi, przepisanych modlitw i uczestnic twem w uroczystościach. Zależny znów od niego młodszy zakry stian prowadził księgi chrztów, małżeństw, zgonów i pogrzebów; '2 Ks. G a c k i , jw ., s. 224. ' Ks. U u 1 i ń s k i, jw ., s. 467. 4 Ks. N a w a r r a, jw., s. 412. Ks. B u l i ń s k i, jw., s. 467. ,B Ks. G a c k i , jw., s. 224— 5. pilnował nadto porządku i czystości oraz miał pod sobą wszelkie klucze. Czynności gospodarczych w klasztorze dozorował szafarz, któremu podlegali ekonomowie osadzeni na folwarkach “ . Konserwatorowie dokonywali poboru dochodów, sprawowali czynności zaopatrzeniowe, prowadzili księgi inwentarzowe biblio teki, zakupywali i wypożyczali księgi, zapisywane w dwóch rejes trach. z których jeden był w przechowaniu u proboszcza mie chowskiego. drugi zaś służył do ich podręcznego użytku. Ponie waż w owych czasach księgi szły na wagę złota, a więc były rzeczą bardzo kosztowną, dlatego nadzór nad biblioteką klasztorną liczącą do dziewięciu tysięcy tomów — był zajęciem ważnym Oprócz powyższych godności klasztornych — pieczę nad szpi talem i apteką sprawował kanonik-przełożony, zwany też mistrzem szpitalnym. Odrębnie prowadzony nowicjat miał profesora teologii i filozofii, tzw. magistra, któremu podlegali klerycy; mieszkali oni osobno, a tylko odbywali posługi kościelne i zasiadali do stołu razem z kanonikami-bożogrobcami. Od roku 1585, prepozyta miechowskiego i kustosza wspierała rada, złożona z czterech kanoników, z których jeden musiał stale mieszkać w Miechowie 79. Mieli oni pod opieką klucze od archi wum z przywilejami, lustracjami, korespondencją oraz większą pieczęć, używaną do aktów uroczystych. Wyrażali również zgodę na wydzierżawianie folwarków klasztornych i na obsadzanie pro bostw, zarządzanych przez bożogrobców. Ponieważ od roku 1620 ustanowiono, że proboszcz miechowski nie może sam dokonywać wyboru kustosza, a jedynie może nie wyrazić zgody na jego obiór, rada czterech zbierała się dla obsadzenia wolnych beneficjatów i wyboru kandydata na ten drugi w hierarchii zakonu urząd. W roku 1791 dokonano tej zmiany, że członków rady i pro wincjałów wybierano na kapitule, a ci kustoszem wybierali na okres lat trzech kanoników na poszeczególne stanowiska w klasz torze. Strój bożogrobców miechowskich był czarny z podwójnym krzy żem czerwonym na lewym boku. Od roku 1520 — na mocy pozwo lenia papieskiego — używali mantoletu z czarnego sukna, względ nie z jedwabiu: był to ,,... płaszczyk kolisty z otworami przy ramionach do wyłożenia rąk z pod niego . Również na mocy tego pozwolenia używali: „... mucetii czyli peleryny kolistej z ramion po łokcie spuszczonej. Młodzi księża i klerycy profesi wdziewali na rokietę nuicet; przełożeni zaś i proltoszczowie wkładali na rokietę mantolet” m. Do stal w kościele dopuszczano kleryków 77 tamże. 78 tamże. Ks. P ę k a l s k i , dz. cyt., s. 45, 80 tamże. zaraz po złożeniu ślubów zakonnych na równi z kanonikami, także w refektarzu podawano posiłki wszystkim jednakowe dla pod kreślenia równości panującej w zakonie. 7. Z A M E K M IECH O W SK I — K LASZTO R BO ŻOGROBCÓW Pierwszy klasztor wystawiony był prawdopodobnie w drugiej połowie wieku XII. W wieku X IV był on częściowo murowany, częściowo zaś drewniany81. Z roku 1389 wiadome jest nazwisko muratora; był nim Mikołaj rodem z Prus. Następnie odbudowy wano budynki zniszczone pożarami, które poczyniły większe szkody w latach: 1506, 1732 i 1745 w. Klasztor — wzniesiony na planie prostokąta z wirydarzem. w pośrodku którego pozostawio no kaplicę Bożego Grobu i krużganki w dolnej kondygnacji — graniczył z kościołem po stronie zachodniej. Na zewnątrz ma on charakter budowli renesansowej z obramieniami okien profilo wanych z ciosu i przedstawia się jako okazały pałac obronny. Po stronie południowej przylegał do klasztoru budynek gospodarczy, oddzielony dziedzińcem od zachodniej fasady kościoła. Po stronie wschodniej, równolegle do drogi Miechów-Jędrzejów, stoi po dzień dzisiejszy okazały dwukomlygiiacjowy budynek, będący siedzibą generałów i opatów zakonu i mający na piętrze sale reprezenta cyjne, połączone ze skarbcem i zakrystią kościoła. Tędy można było zawsze wejść bezpośrednio do stall w prezbiterium. Klasztor miechowski, niegdyś dwukondygnacjowy, po przerób kach dokonywanych przez znajdujące się w nim urzędy w wieku X IX — podniesiony został do trzech kondygnacji, z wyjątkiem skrzydła północnego ,.Na parterze dookoła wirydarza prostokątne krużganki gotycko-renesansowe, ze sklepieniam i krzyżow o-żebrow ym i ze zwornikam i w kształcie tarcz z herbam i: T op ór, Odrowąż, Łodzią, Łabędź. Dołęga i z wspornikami przeważnie w form ie kapiteli toskańskich; w skrzydle południow ym kilka piw n ic; w narożniku południow ow schodnim od r. 1”>90 kaplica Chrystusa Ukrzyżow anego; w skrzydle pn. trzy duże sale oraz w narożniku półn ocn ozachodnim t. zw. karcer, nakryty czterema sklepieniami krzyżowym i wspartymi na filarze. W kondygnacji środkow ej w skrzydle w schodnim i zachodnim wzdłuż korytarzy cele, w skrzydle południow ym m. in. refek tarz. w skrzydle półn ocn ym częściow o szkarpam i” 84. biblioteka. Na zewnątrz budowla opięta 81 Z. B o c z k o w s k a. Powiat M iechow ski, K atalog Zahytk. Szt. w Polsce, t. I, z. 8, s. 24— 25; K ościół bożogrob ców w M iechowie. Przyjaciel Ludu 1837. I I I ; S o b i e s z c z a ń s k i . F. M. Tygodn. i 11. 1861. IV ; K łosy. t. IX. 82 B o c z k o w s k a, jw. 83 tamże. 84 tamże. K rucyfiks z wieku X V I w lewej nawie kościoła b ożogrobców m iechow skich Sklepienie k ościoła m iechow skiego odbu dow anego " drugiej połow ie wieku W Iii-g o Fragment dekoracji nawy głów nej w kościele m iechow skim i XXI I I wieku W Roczniku Miechowskim pod rokiem 1311 zachowała się notatka o inkastelacji klasztoru przez Jana, syna Budivogia, wy słanego przez Władysława Łokietka. Być może, że z tych czasów zachowała się baszta „na narożniku muru otaczającego zabudo wania klasztorne od wschodu i północy: z dzikiego kamienia, wieloboczna, przysadkowata, nakryta dachem namiotowym” 85. Biblioteka, znajdująca się w klasztorze, posiadała w swych zbiorach kilkaset rękopisów i około dziewięciu tysięcy różnych ksiąg: uległa ona pożarowi w roku 1736 86, z którego niewiele uratowano. Do zachowanych najcenniejszych rękopisów należy wspomniany już Rocznik Miechowski, który był wszyty do ko deksu pergaminowego i wywieziony został — przypuszczalnie z biblioteką biskupa Załuskiego — z Warszawy do publicznej bi blioteki w Petersburgu Pisany w wieku XIV, zawiera bardzo cenne zapiski — początkowo rekonstruowane — od roku 1389 i kończy się opisem koronacji Władysława Warneńczyka i omó wieniem niedoli klasztoru za rządów opata Lisowskiego, który żyjąc rozpustnie marnotrawił majątek opactwa i zadłużył wiele dóbr klasztornych88. W wieku XV II — ze zbiorów biblioteki i z archiwum czerpał cenne wiadomości kanonik Samuel Nakiel ski. wydając swe bezcenne dzieło o historii miechowitów w roku 1634. Biblioteka miechowska szczególnie bogatą była w dzieła teologiczne, filozoficzne itd. Nakielski podaje, że Miechowici zaj mowali się przepisywaniem ksiąg i wymienia Mikołaja z W ol borza, zmarłego w roku 1406 '". Wśród rękopisów zwracało uwagę dzieło Mikołaja, archidiakona krakowskiego, z roku 1286 pt. Summa decretalium per dominum N. .4. C. collecta. przepisane w roku 1467 przez W ojciecha de Perusio i nabyte w roku 1534 przez kanonika sandomierskiego Tomasza z Oświęcimia. Znany bibliofil, biskup Załuski, widział to dzieło w klasztorze mie chowskim. ,.Kustosz klasztoru P iotr B oroviusz przeniósł bibliotekę do sali skle p ion ej nad zakrystią dzięki temu mial ocalić ją w czasie pożaru w roku 174.r>, gdy pożar zniszczył klasztor. Po kasacie zakonu, dyrek tor generalny bibliotek Samuel Bogum ił Linde, z polecenia władz Królewstwa Polskiego zabrał z M iechowa 17 pak książek do Biblioteki G łów nej w Warszawie w roku 1818; dostarczone najpierw do Brzeska, a następnie przew iozione W isłą wraz z księgozbioram i innycli skasowanych klasztorów . Tą drogą znalazło się w dziejskiej” 90. Ściana romańska drugiego kościoła b ożogrobców w M iechowie, odkryta w r. 1948 Warszawie 787 tom ów 85 tamże. H6 Ks. B u 1 i ń s k i, dz. cy t., s. 479. 88 tam że. 87 B i e l o w s k i , dz. cyt., t. II s. 881. 89 Ks. W i ś n i e w s k i , dz. cyt., s. 129. 90 tamże. treści teologiczn ej i kazno 8. EGZEM PCJA M IECH O W ITÓW Ponieważ bożogrobcy w innych krajach podlegali władzy bi skupiej, dlatego i klasztor miechowski wraz ze wszystkimi za konnikami początkowo musiał podlegać biskupom polskim. Ka pituła jerozolimska otrzymała w roku 1 168 od papieża Alek sandra Ił władzę jurysdykcji, co ją uniezależniało od pa triarchy91. W roku 1170 tenże papież udzielił prepozytowi bożo grobców w Jerozolimie prawa pierwszeństwa przed wszystkimi Opatami i przeorami patriarchatu s!. Mimo to w dokumentach z lat 1182 i 1205 wyraźnie była zaznaczona jurysdykcja patriarchy jero zolimskiego nad bożogrobcami i trwała ona do momentu połącze nia bożogrobców z joannitami w roku 1489. W roku 1261 papież Urban IV nadał egzempcję bożogrobcom zamieszkałym w Szderasz w Czechach, umotywowaną przynależ nością ich do jerozolimskiego ośrodka kanonickiego w. Klasztor miechowski nie mógł się od razu uwolnić od władzy biskupiej; świadczą o tym dokumenty z lat 1239. 1243. 1262. ..Biskup w rocławski Tomasz ... oddając bożogrobcom szpital pod Nysą. d o łączył zastrzeżenie zobow iązujące obdarow anych kanoników nie tylko do za sięgania rady w sprawach ważniejszych, ale do wFyraźnego posłuszeństwa w o b ec ordynariusza. P od obnie książę Przemysław przy przekazaniu m iechow i tom gnieźnieńskiego szpitala w roku 1243. że na wypadek, gdyby przełożony szpitala okazał się nieodpow iednim będzie na żądanie darczyńcy i arcybi skupa zw olniony. W 12 lat później arcybiskup Pełka przywłaszczył sobie praw o patronatu. M iecłlowici znosili to aż do otrzymania egzem pcji, bo za raz p o je j otrzym aniu, w piśm ie z 2 X 1262 r. zaświadcza Urban IV . że na prośbę m iechow skiego prepozyta zatwierdza mu (a nie arcybiskupow i) praw'o patronatu nad wspomnianym szpitalem i kościołem z nim połączonym . Wrreszcie trzecim dokumentem jest bulla papieża datowana dnia 9 czerwca 1202 r., w której papież przyznaje m iechow itom różne sw obody, zabrania ich karać. W trzy miesiące po w spom nianej bulli otrzym ali nową bullę, która zapoczątkowała cały szereg przyw ilejów przyznających im zasadniczą egzem pcję” B1. Tą drogą uzyskali miechowici wyjęcie spod władzy karnej biskupów i swobodę w sprawach świadczeń materialnych. Papież wysuwał klasztor miechowski na czoło wszystkich klasztorów bożo grobców na terenie Polski. Rusi, Czech. Węgier, i Niemiec i nadał mu pełną egzempcję, którą transumował arcybiskup Świnka w ro ku 1302. a miechowici uważali bullę tę za tak ważną, że przed* 91 85 9,1 94 Ks. B o g d a n, <lz. cyt., s. 233— 262. tamże. tamże. tamże. łożyli ją do zatwierdzenia soborowi hazylejskiemu w roku 1434 ’ 5. W międzyczasie Indię papieża Urbana IV potwierdzili jego następ cy: w roku 1281 — papież Marcin IV, w roku 1372 i 1374 — Grzegorz XI, w roku 1520 — Leon X. Na tej podstawie Długosz, oglądnąwszy oryginały w klasztorze miechowskim, pisał z sza cunkiem o Miechowie, że tam znajdują się różne immunitety, swo body, egzempcje zakonu bożogrobców. W oparciu o bulle papieskie patriarcha jerozolimski uznał w roku 1365 drugiem miejsce — po kapitule jerozolimskiej — zakonu bożogrobców w Miechowie, jako siedzibie bożogrobców w północno-wschodniej Europie i na terenie Słowiańszczyzny. Za braniał jednocześnie swej kapitule mieszać się w sprawy Miecho wa zaznaczając, że polscy bożogrobcy podlegają tylko papieżowi i jemu Na tej zasadzie patriarcha jerozolimski interweniował kilkakrotnie w sprawach miechowitów, a także wysyłał do Polski swego przedstawiciela w osobie wikariusza generalnego w roku 1302. Gdy w roku 1344 zakwestionowano niemiecką narodowość wizytatora, wyznaczonego przez patriarchę Henryka, w wyniku interwencji Kazimierza Wielkiego przyszła zgoda na objęcie tej funkcji przez prepozyta miechowskiego W roku 1371 patriarcha Wilhelm nadał prepozytowi miechowskiemu, Marcinowi Czczikonowi. tytuł generalnego wikariusza dla miechowitów polskich i wę gierskich. Potwierdził to następca Wilhelma — patriarcha Fernandus. Z końcem wieku XIV, w związku z coraz silniejszą pozycją klasztoru w Miechowie i słabnącą władzą patriarchów, przyszedł czas na wyjęcie bożogrobców spod ich władzy. W roku 1395 papież Bonifacy IX podporządkował bezpośrednio swej osobie obsadę stanowiska opata miechowskiego H. Pierwszym nominatem papieskim stał się Michał z Radomska i odtąd każdego prepozyta miechowskiego uważano za generalnego przełożouego bożogrob ców. Odtąd też i Kuria rzymska obdarzała icli zaszczytnymi funk cjami. uchylając równocześnie wszelkie pretensje klasztoru w Peruggi do zwierzchnictwa nad Miechowem, jako nieuzasadnione (r. 1414). W wieku X IV miechowici zwracali się ze skargami bez pośrednio do Rzynui. 1 tak papież Benedykt X II w roku 1339 nakazał arcybiskupowi, jako swemu delegatowi, zaprowadzić po rządek w klasztorze, gdy władzę objął samozwańczo prepozyt Henryk. Także Grzegorz XI nakazał klasztorowi nyskiemu pod porządkować się miechowitom, gdy ten samowolnie poddał się pod jurysdykcję prepozyta w Szderasz M. 95 98 97 98 99 tamże. tamże. tamże. tamże. tamże. M iechow ici — obok norbertanów — byli jedynym i w Polsce kanonikam i regularnym i w yjętym i spod jurysdykcji biskupów. Mi mo to — od wieku X V — w ybór na stanowisko opata m iechow skiego podlegał zatwierdzeniu przez biskupa krakowskiego. M ie chow ici brali także udział w synodzie w roku 1456, jak to wynika z aktów kapituły gnieźnieńskiej. Z egzem pcją bożogrob ców w P o l sce wiązały się następujące upraw nienia: Pow yższe nadania wiązały się p raw dopodobn ie tylko z okresem panowania wystawiającego go w ładcy; d opiero patriarcha Monachus — zgodnie z polityką duchowieństwa nadał im charakter „na w ieczność” . N iem niej „...pierw sza wzmianka o obow iązku szanowania nadań przez na stępcę znajduje się w dokum encie Bolesława z roku 1264. O następcach o b darowanego wspomina Przemysław w r. 1290. .....niezależna władza spowiadania swoich podw ładnych, prawo wykonywania nadaniach Łokietka z r. w obec nich władzy karnej, w olność od prokuracyj, prawo sw obodnej se- 1325 i 1331 brak jest wzmianek o obow iązyw aniu dokum entu w czasie, przy czym król zatwierdził w olności nadane przez poprzedników . Nie wspomniał pultury nawet w czasie interdyktu, praw o przenośnego ołtarza, prawo odprawiania nabożeństw w czasie interdyktu. pod czterema jednak zw yczaj przyw ilej Bolesława nymi warunkami” ,lw. ności” 105. 9. K SIĄ ŻĘ C Y I MMI M T E T SĄ D O W Y W DOBRACH BOŻOGROBCÓW M IECH O W SK ICH Od m om entu założenia siedziby b ożog rob ców polskich w M ie chow ie panujący Piastowie bardzo hojnie uposażyli ich licznym i przyw ilejam i, w ym ienionym i przez N akielskiego: nadali immu nitet skarbowy dla m ieszkańców wsi klasztornych, według którego byli oni zw olnieni od następujących świadczeń: od obowiązku udziału w wyprawacli w ojennych, od uczestnictwa w budow ie gro dów książęcych, nie dawali poradlnego i stróży. nie dostarczali pow ozu i podw ód oraz nie podlegali m incerzow i panującego księcia 101. Ten ostatni przyw ilej był uwolnieniem od jurysdykcji sądowej m incerzy, sprawowanej przez nich w imieniu księcia na targu m iechowskim 10!. W ykaz patriarchy Monacha poświadczał przyw ileje: Bolesława K ędzierzaw ego z roku 1174 dla ludności wsi klasztornych m iechow itów ; potw ierdzone powyższe w olności przed rokiem 1178 przez Mieszka Starego, a także przez K azi mierza Sprawiedliwego przed rokiem 1 1 8 7 103. Drogą tych przyw i lejów klasztor uzyskiwał egzem pcję dla wszystkich wsi pierw otnie nadanych i później uzyskanych dóbr. Tą drogą sądowa władza książęca przechodziła na klasztor, który sprawował ją za pośred nictwem prałata klasztornego względnie katedralnego. „Sądow y immunitet m iechowski był pełny, bo poza teoretyczną od pow ie dzialnością przed Bogiem , a praktycznie przed prałatem klasztor nym nie przewidywał innej instancji ’04. Przyw ilej ten był za twierdzony przez Henryka B rodatego w roku 1232. Konrada w roku 1243 i Bolesława W stydliw ego w roku 1259. 100 101 102 10:ł 104 tamże. R. G r o cl e c k i. Początki im m unitetu w P olsce, Lwów 1930 s. 42. 61. tamże. tamże. tam że. też o nadawaniu na w ieczność Kazim ierz Wiel ki w r. 1354, zatwierdzając W stydliw ego z r. 1256 i dodając sam pewne w ol Pierwszy immunitet książęcy obejm ow ał tylko mieszkańców M iechowa wraz z trzema wsiami nadanymi przez Jaksę, a rozciąg nięcie przyw ileju egzem pcyjnego na wszystkie dobra bożogrobców przez patriarchę Monacha w' dokum encie z roku 1198 — należy uznać za zgodne z życzeniem i dążeniami m iechowskiego klasztoru. Z tego względu bulla Innocentego III z roku 1208. zatwierdzająca immunitet i nadania Henryka B rodatego z roku 1232, wspominała przede wszystkim o M iechow ie 10,,. W roku 1243 M iechów otrzy mał od Konrada imm unitet na wszystkie wsie nie tylko nadane lecz i zakupione przez b ożogrob ców . Szereg późniejszych przyw i lejów nie wnosiło pow ażniejszych zmian i dotyczyły one wsi, które przybyły bożogrobcom ju ż po wydaniu przywileju przez Konrada. D opiero dokum entem z roku 1295 została objęta całość dóbr klasztornych; zawierał on generalne zezwolenie na lokację na prawie niem ieckim w posiadłościach m iechow itów 10'. Przyw ilej ten był bardzo ważny dla M iechowa i stanowił ukoronow anie wy siłków zakonu: „zaw ierał immunitet sądowy dla wszystkich wsi lokow anych, a raczej m ających być lokow anym i, w którym król W acław uwolnił je od sądownictwa wszystkich urzędników kra kowskich i sandom ierskich. Dla króla i starosty zastrzeżono tylko te sprawy, które proboszcz czy sołtysi zaniedbali rozsądzić. Spra wy te miały być wywrołane pozw em króla lub starosty, a sądzone według prawa niem ieckiego, tego, jakim mieszkańcy byli lo k o wani" 10". W roku 1354 nastąpiło zrównanie wsi na prawie polskim i niem ieckim , zam ykające trzy etapy w procesie odstępywania przez księcia jurysdykcji na rzecz klasztoru m iechow skiego. Osią gając już w początkow ym stadium tego procesu uniezależnienie 105 Z. K a c z m a r c z y k , Im m unitet sądotcy w dobrach kościoła iv P olsce, Poznań 1936 s. 172— 182. 10,i tamże. 107 tamże. 108 tamże. się od ju rysdykcji biskupiej i książęcej — zakon b ożogrobców w M iechow ie zapewnił sobie pełną sam odzielność i stał się samo istną władzą, rządzącą się podług wewnętrznych praw i reguł panujących w zakonie. U zależniając tą drogą od hierarchii ka pituły m iechow skiej wszystkich poddanych swych wsi i miasteczek, w tym szczególnie M iechowa i Skaryszewa, rządząc nim i przez podw ładnych sobie w ójtów i sołtysów — stawał się wyłącznym panem w swych posiadłościach. JO. W A L K A O Z A G A R N IĘ C IE W Ł A D Z Y PREPO ZYTA M IECH OW SKIEGO I W PRO W AD ZEN IE O P A TÓ W- KOMEND A T A RIUSZ Y Powyższa pełnia władzy i sam odzielność bożogrob ców w M ie chow ie stworzyła bardzo silną enklawę gospodarczą, m ogącą być przykładem — jak to podkreślił Długosz — dla wszystkich zain teresowanych racjonalnie prow adzonym i gospodarstwam i rolnym i. I właśnie te wielkie zasoby materialne m iechow itów i ich gosp o darność stały się przyczyną walki — prow adzonej z zewnątrz — o opanow anie stanowiska proboszcza m iechow skiego. Nie p om ogło uchylenie się w średniowieczu od ju rysdykcji biskupiej, gdyż najpierwsi dygnitarze kościelni ubiegali się o bogate probostw o m ie chowskie ze względu na jego w ielkie doch ody. W alka ta toczyła się ze zmiennym szczęściem i przez długie dziesiątki lat zakon p o trafił zw ycięsko bronić swej niezawisłości, późn iej jednak uległ. Nie wszyscy jednak opaci m iechowscy przychodzący z zewnątrz przyczynili się do rozkładu w ew nętrznego i zaprzepaszczenia głów nego celu, jakiem u bożogrobcy m ieli służyć społeczeństwu p o l skiemu. W ielu z nich przyczyniło się do okresow ego podniesienia świetności zakonu; ostatecznie jednak byli właściwą przyczyną w ew nętrznego rozkładu i spow odow ali kasatę zakonu. Opanowanie władzy przez tytularnych dygnitarzy kościelnych — którzy przez swą n ieobecność w M iechow ie w yw oływ ali brak wewnętrznej spo istości i dyscypliny, a także ich nastawienie niemal wyłącznie mające na celu czerpanie korzyści materialnych — spow odow ało zanik działalności zakonu w myśl założeń jego reguły. Walka powyższa toczyła się niemal przez okres trzystu lat, a więc przez czas dostatecznie długi dla wprowadzenia rozkładu w najbardziej nawet organizacyjnie i ideow o silnej społeczności kościelnej. Przebieg tej walki był następujący. O bowiązkiem pre pozyta m iechow skiego było zwoływanie co trzy lata kapituły ge neralnej do M iechow a; w kapitule tej uczestniczyli wszyscy b ożo grobcy osadzeni na probostw ach, w szpitalach i szkołach Obra dy kapituły trwały trzy dni i na niej wybierano hierarchię zakonu; nieprzybycie groziło utratą beneficju m , a dla wikariuszy klątwą. >°» Ks. G a c k i , Jz. cyt., s. 138— 140. Kapituła podejm ow ała uchwały mające służyć dobru zakonu, dla poży tku wszystkich braci. K apituły z r. 1620 i 1625 określiły obow iązki każdego zakonnika, w słusznej dążności do zreform ow a nia tradycji, która — w okresie zakończenia walk z różnow iercami — musiała iść z postępem czasu. Wybrany przez kapitułę generalną p roboszcz m iechow ski miał obow iązek zwizytowania przynajm niej raz w roku — wszyst kich placów ek podległych klasztorowi w M iechow ie. Ponieważ brak czasu i duże odległości utrudniały wykonyw anie tych czyn ności dlatego prepozyt m iechowski rządził tymi placów kam i przez czterech prow incjałów , których ustanow iono w roku 1585 <1la : W ielkopolski, Małopolski zachodniej i w schodniej oraz M azow sza uo. W szczególnych wypadkach proboszcz m iechowski m iano wał w izytatorów , którzy potem składali sprawozdania. Każdy z prow incjałów był przełożon ym któregoś z kościołów w swej p ro wincji i składał sprawozdanie cztery razy do roku proboszczow i miechowskiem u, radząc się go w ważniejszych sprawach. Z początkiem wieku X V I w pływ ow e duchow ieństw o p od jęło pierwsze starania o obsadzenie probostw a m iechow skiego osobami z zewnątrz, z uwagi na w ielkie dochody jakie ono dawało. W roku 1505 na sejm ie lubelskim — kanclerz królewski Jan Łaski, mając na względzie słabe uposażenie diecezji w łocław skiej, w k tó rej był dziekanem, postawił wniosek o przyłączenie do diecezji w łocław skiej probostw a w Gostyninie, będącego w ręku b ożo grobców Przeszło to łatwo i biskup płocki. Erazm, wniosek ten zatw ierdził. W odpow iedzi na to prepozyt m iechowski, T o masz, odwołał się do Rzymu, gdzie uzyskał w roku 1513 hreve Leona X , w którym papież — pod groźbą kary kościelnej — za bronił uszczuplania dóbr m iechow skich. Fakt ten nie powstrzymał jednak biskupa przem yskiego. Piotra T om ickiego (popartego przez króla Zygmunta I u tegoż papieża Leona X ), od starań o przy łączenie probostwa m iechow skiego do biskupstwa przem yskiego ,1S. Papież dokonał tego przyłączenia lecz w obec stanowczego sprze ciwu opata m iechow itów , Tom asza, doszło do procesu w Kurii Rzymskiej. Sprawa toczyła się przez dziew ięć lat; rów nocześnie Miechów interweniował na sejmach, a danie dygnitarzom kra kowskim sporych sum (kasztelanowi krakowskiemu Szydłow ieckieniii 40 dukatów, A n drzejow i Tęczyńskiem u, w ojew odzie kra kowskiemu, 130 dukatów, a M ieleckiem u 200 złoty ch ), a dalej łączne wydatki w Rzym ie 200 dukatów i na sejm ach 5.665 z ło tych — zadecydow ały o odw ołaniu tej decyzji. Papież Leon X wydał w roku 1520 breve, w yłączające zgrom adzenie m iechowskie 110 tamże. 111 Ks. P ę k a l s k i , dz. cy t., s. 89. 118 tam że; ks. Wł . S a r n a , E piskopat przem yski, Przem yśl 1902 s. 87. spod władzy diecezjalnych biskupów lls. Papież Klemeus VII bul lami z r. 1522 i 1524 unieważnił przyłączenie dóbr miechowskich do biskupstwa przemyskiego i nakazał ks. Tomaszowi Rożnow skiemu, oficjałow i krakowskiego konsystorza, przeprowadzić od łączenie dóbr miechowskich przyłączonych kiedykolwiek do któ regoś z biskupstw 1U. Również Zygmunt Stary podpisał uchwały sejmową w Piotrkowie wr roku 1524, dotyczącą nietykalności fun duszów z dóbr bożogrobców polskich oraz ich dalsze prawo do wyboru na kapitułach miechowskich generała zakonu z zastrzeże niem zatwierdzania go przez biskupa krakowskiego. Zygmunt Au gust w roku 1562 potwierdził wolny obiór prepozyta generalnego miechowskiego i wyraził słowa uznania dla działalności zakonu. Kiedy jednak ówczesny przełożony Miechowa Jan z Lwówka z powodu podeszłego wieku chciał zrezygnować z piastowanej godności, postanowił skorzystać z tego ulubieniec królewski ks. Szymon Ługowski, kanonik katedry krakowskiej i wysunął swoją kandydaturę na generała bożogrobców ni>. Kapituła miechowska w roku 1566 — mimo przysłania na nią wysokiego urzędnika kró lewskiego, mającego poprzeć starania ks. Ługowskiego — nie wybrała go swym przełożonym. Królowi przedstawiono jednak, że powodem odmowy był brak jednomyślności kapituły; wtedy to Zygmunt August zażądał od biskupa krakowskiego Padniewskiego wprowadzenia ks. Ługowskiego na prepozyturę miechowską. W odpowiedzi na to żądanie królewskie biskup Padniewski, łącz nie z legatem papieskim, zwołali synod biskupów do Łowicza, na którym — w' roku 1566 — uchwalono „... by tylko zakonni ków według ustaw kościelnych na opactwa i probostwa wolno było wybierać” . Nie wiele to pomogło. Po śmierci dziewięćdziesięcioletniego pro boszcza Jana — król Zygmunt August — nie zważając na uchwały synodu łowickiego polecił instytuować na opactwo miechowskie kanonika Ługowskiego, co biskup krakowski wykonał. Odtąd we szło w zwyczaj obsadzanie opactwa miechowskiego przez dostoj ników kościelnych diecezjalnych — ze względu na wielkie do chody związane z Miechowem n\ Ks. Ługowski z dochodów tych zakupił dla swojej rodziny dobra Korzkiew, a bożogrobcy tyle zyskali na jego szesnastoletnich rządach, że odtąd opat mie chowski otrzymał w stallach kanoniczych katedry krakowskiej miejsce, jakie już dawno posiadali opaci — tyniecki i mogilski. Ks. Ługowski został biskupem przemyskim, ale — przez naruszenie 113 N a k i e l s k i , ilz. cyt., s. 599. 114 tam że, s. 604— 606. 115 P ę k a l s k i , rfi. cyt., s. 94— 102; ks. L. Ł ę t o w s k i , K atalog bisku pów , prałatów i kanoników krakow skich, Kraków 1852 t. III s. 320; H. B ar y c z , H istoria Uniw. Jag., K raków 1935 s. 567. 118 P ę k a l s k i , ,lz. cyt. Boży G rób w kaplicy w ybu dow anej w X V I wieku na m iejscu pierwszego kościoła rom ańskiego w Miechowie G ryf Jaksy z Miechowa fundatora zakonu bożogrobców p ol skich w r. 1163. uchwal synodu łowickiego i hull papieskich — nie mógł uzyskać zatwierdzenia Rzymu. Po dwóch latach zmarł u bożogrobców na Stradomiu w Krakowie. Zanim kapituła miechowska zdążyła wybrać z pośród siebie nowego generała — przyjechał sekretarz królewski, kanonik Piotr Tylicki; przedłożył on list wysuwający kandydaturę Andrzeja Ba torego, bratanka króla Stefana Batorego W związku z tym w roku 1583 przyjechał do Miechowa biskup krakowski, Piotr Myszkowski, w sprawie wyboru Andrzeja Batorego, popieranego również przez legata papieskiego. Jakkolwiek legat papieski stwierdzał na piśmie prawo bożogrobców do wyboru prepozyta spośród nich samych — zgodnie z ustawami zakonnymi, ale równo cześnie wyliczał korzyści mogące spłynąć na opactwo po obraniu bliskiego krewnego króla. Batory, liczący wówczas zaledwie dwa dzieścia dwa lata, został wybrany proboszczem miechowskim i re zydował w Miechowie. Udawszy się do Rzymu został mianowany kardynałem przez papieża Grzegorza X III. Tak na czele bożo grobców stanął najbliższy krewny królewski i kardynał, który rzeczywiście przysłużył się dla podniesienia znaczenia zakonu i uporządkował wiele spraw wymagających reformy. Częściowo roztrwonione przez ks. Ługowskiego wsie miechowskie Batory odebrał i mimo administrowania diecezją warmińską okazywał bo żogrobcom wiele życzliwości aż do swej śmierci w czasie wyprawy wołowskiej w roku 1599. Zygmunt III Waza uważając, że przyjął się zwyczaj obsadza nia probostwa miechowskiego kandydatami z pośród protegowa nych króla, wysunął na to stanowisko kanonika krakowskiego Fogelwedera, którego wyborowi nikt się już w zakonie nie sprze ciwiał “ 8. Fogelweder okazał się dla miechowitów pożytecznym, lecz jako sekretarz króla nie wiele w Miechowie przebywał. Po jego śmierci objął tamtejszą prepozyturę wpływowy dygnitarz królewski i biskup warmiński Piotr Tylicki. Odtąd utarł się zwy czaj, że biskup czy inny dygnitarz kościelny obdarzany był przez króla dochodami z probostwa miechowskiego i w wypadku wakansu ubiegał się o wybór na opata w zakonie. Tylicki był dobrze znany w Kurii Rzymskiej i dlatego wybór jego został potwierdzony przez papieża Klemensa V III w roku 1603; z probostwa miechow skiego zrezygnował, gdy został biskupem krakowskim w roku 1606, ponieważ nie mógł być własnym zwierzchnikiem. Uprzed nio nie przeszkadzało mu w utrzymaniu tej godności przejście z biskupstwa warmińskiego na włocławskie. Za jego następcy, biskupa przemyskiego Macieja Pstrokońskiego, wszedł w życie zwyczaj mianowania przez każdego tytularnego proboszcza mie7 tamże. 118 P olsk i Sloienik B iograficzny, t. V II s. 44— 46. chowskiego jego zastępcy z pośród bożogrobców; w rzeczywistości spełniał on wszystkie obowiązki lecz nie korzystał z dochodów. W ten sposób koadjutorem tytidarnego proboszcza miechowskiego został w imieniu biskupa przemyskiego — Szymon Turski, kano nik Grobu Chrystusowego i przełożony klasztoru św. Jadwigi na Stradomiu Zatwierdzenie go przez Zygmunta III Wazę utoro wało mu drogę do następstwa po śmierci biskupa Pstrokońskiego; po dwóch latacłi samodzielnych rządów proboszcz miechowski-koadiutor Turski zmarł w roku 1611. Odtąd, po śmierci któregokolwiek z proboszczów miechow skich, król przedkładał zgromadzeniu miechowitów — na komendatariusza któregoś z wpływowych członków rodzin inożnowładczych czy też biskupów, piastujących w'ysokie stanowisko na dworze. Przestrzegano jednak, by nowomianowani biskupi kra kowscy — w wypadku, gdy byli prepozytami miechowskimi — godność tę składali. Za rządów opatów-komendatariuszy dobra miechowskie szły w dzierżawę i gospodarka upadała, gdyż tytu larni proboszczowie przebywali poza Miechowem i zależało im tylko na wyciągnięciu jak największych dochodów z fundacji za konnych. W roku 1642, przy nominacji na proboszcza miechowskiego Jana Gembickiego, kapituła bożogrobców usiłowała bronić się i przy wyborze postawiła nominatowi warunki: stałego zamieszka nia w Miechowie, przyjęcia ślubów zakonnych, odebrania włości miechowskich zastawionych przez jego poprzedników i oddania dochodów zgodnie z intencją fundacji, a nie traktowania ich jako funduszy świeckiego uposażenia 120. Gembiski odrzucił kategorycz nie słuszne żądania bożogrobców i nie chciał słyszeć o potrzebach zakonu. I tak już pozostało aż do kasaty zakonu miechowitów w roku 1818, choć niektórzy z rzeczywistych generałów usiło wali ratować zakon przed upadkiem materialnym i czynili wysiłki dla podniesienia spoistości reguły zakonnej bożogrobców. W międzyczasie, w roku 1768, Stanisław August Poniatowski przyłączył dobra miechowskie do Akademii Krakowskiej i od roku 1784 uczelnia ta miała sprawować pieczę nad opactwem w Miechowie. Ponieważ administratorem z ramienia Akademii chciał zostać Hugo Kołłątaj — król odmówił mu i w wyniku tego tylko cztery wsie przypadły Uniwersytetowi: Michałowice, Parkoszowice, Smroków, i Orłów, zamiast projektowanych je denastu m. 119 Ks. P ę k a l s k i , dz. cyt., s. 103. 120 ta m ie, s. 110. 111 tam że, s. 115— 119. 11. P R Ó B A R A T O W A N IA ZA K O N U BO ŻOG RO BC ÓW PRZED RO ZKŁAD EM PO D JĘTA W X V III W. Trwające przez dwa wieki rządy komendatariuszy nad zako nem miechowitów, ograniczające się tylko do ściągania dochodów z ich dóbr, bardzo rzadkich ich przyjazdów do Miechowa względ nie całkowite zaniedbywanie spraw opactwa sprawiły, że z po czątkiem wieku X V III zakonnicy postanowili ratować się przed ostatecznym rozkładem. Wiedząc, że komendatariuszaini są bisku pi, arcybiskupi i protegowani królów, którym niepodobna się oprzeć i przeciwstawić, uradzili wybrać z pośród siebie general nego przełożonego z wyznaczeniem kilku wiosek miechowskich na uposażenie związane ze stanowiskiem122. Faktycznie stało się to w roku 1709, ale zatwierdzenie trwało przez pewien czas. Prośba, wniesiona przez miechowitów do Augusta Mocnego, uzyskała aprobatę w roku 1725 i król „wydał przywilej, że kano nikom Grobu Chrystusowego wolno wybierać generalnego przeło żonego ze swego Zgromadzenia” 123. Wybór ten miał być tajny — w oparciu o zezwolenie papieża Jana X X III z roku 1412; wy brany opat miał używać w czasie uroczystości infuły i pastorału. Kustosz zakonu, ks. Stępkowski, zwołał kapitułę generalna na 28 sierpnia 1725 roku tj. na dzień św. Augustyna, uroczyście ob chodzony przez kanoników tejże reguły. Na kapitule wybrano ks. Stępkowskiego generalnym administratorem do czasu uzyskania zatwierdzenia przywileju przez papieża; ze względu na to, że bożogrobcy wyjęci zostali spod jurysdykcji biskupiej, chcieli unik nąć zatwierdzania go przez biskupa, obawiając się jego sprzeciwu w związku z komendatorią. Papież Benedykt X III zatwierdził przywilej Augusta Mocnego i ponowił wszystkie poprzednie przywileje dla bożogrobców w Miechowie. W międzyczasie komisarze — wyznaczeni przez kapitułę — przeznaczyli osiem wsi na uposażenie opata mie chowskiego: Pstroszyce, Kalinkę Małą, Bukowską W olę i pięć mniejszych wsi tj. Podmiejską Wolę, Podleśną Wolę, Brzuchanię, Biskupice, Strzeszów oraz czynsz z miasta Miechowa. Gdy król za twierdził powyższe w roku 1726, zebrana kapituła wybrała ponow nie ks. Stępkowskiego generalnym przełożonym, a biskup krakowski bez sprzeciwu wprowadził go na opactwo 124. Od tego czasu, aż do kasaty w roku 1819, rządzili zgromadzeniem bożogrobców opaci w Miechowie, którzy położyli duże zasługi dla utrzymania swego zakonu w dawnej świetności. Będąc bożogrobcami dbali o regułę i ratowanie powierzonych ich pieczy kościołów, szpitali i resztek dóbr klasztornych. 122 tamże. 123 tamże. 124 tamże. Z tych czasów dochow ały się w dawnym opactw ie m iechowskim księgi kapituły generalnej, zbierającej się przepisow o co trzy lata oraz inne dokum enty świadczące o przyw róceniu uporządkow a nych stosunków w zakonie. Księgi te są następujące: 1. Notata varia, A. D. 1746, 2. Acta seu Gęsta Regni Poloniae itern Conventus Miechoviensis, A. D. 1757, 3. Acta Conventus Generalis Miecltoi-iensis ordinis Canonicorum Regularium St. Sepulcri, A. D. 1774, 4. Visitatio generalis Ecclesiarum Ordinis Nostri Canonici in Regno Galiciae et Lodomeriae e.ristentis, A. D. 1777. Księgi te zawierają wszystkie rozporządzenia dotyczące d óbr zawiadywawanych przez bożogrobców , w izytacje kościołów , dokum enty od noszące się do sprzedaży oraz dzierżaw, porządkujące poprzedni stan rabunkowej gospodarki prow adzonej przez kom endatariuszy itd. Oczywiście rozkład polityczn y R zeczypospolitej i rozbiory, które podzieliły dobra m iechowskie m iędzy Prusy, Austrię i oca lałą jeszcze część państwowości polskiej — nie sprzyjały całko witemu opanowaniu sytuacji. Znamienne jednak było to, że próba ratowania zakonu przed całkow itym upadkiem wyszła spośród samych m iechow itów i rzeczyw iście dokonali oni w wieku X V III — w okresie najm niej sprzyjającym przeprow adzeniu tego zadania — dzieła uporządkowania prawie wszystkich spraw k o ścielnych i m ajątkow ych. Znamienną jest karta tytułowa Księgi kapituły generalnej m iechow skiej z roku 1774; u samej góry um ieszczony jest kape lusz kardynalski, stanowiący nawiązanie do osoby proboszcza m ie chow skiego A ndrzeja B atorego. Kapelusz kardynalski stanowi zwień czenie podw ójn ego krzyża Stróżów Grobu Chrystusowego, którego podstawą jest korona królestwa jerozolim sk iego; po lewej stronie kapelusza kardynalskiego infuła opata, po prawej zaś pastorał, będące od roku 1412 insygniami przełożonych m iechow skich, d o p iero na prawo od kartusza z napisem — wsparty o niego pazu rami — G ryf Jaksy z M iechow a, fundatora i dobrodzieja zakonu. Począwszy od opata Stępkowskiego zakonem w M iechow ie rządzili generalni przełożeni, którzy w yprow adzili b ożog rob ców z upadku lecz ukończeniu tego ich dzieła przeszkodziły warunki polityczne i rozbiory. Zadecydow ały one w końcu o kasacie za konu. P o Stępkowskim wybrany został na kapitule generalnej w roku 1744 proboszczem m iechow skim ks. Jakub Radliński, p eł niący p oprzed n io obow iązki proboszcza w Leżajsku 1‘ °. Celem ra towania swego zgrom adzenia dał on w roku 1733 ze swego pryw at nego majątku czterdzieści tysięcy zł. p oi. na rzecz K ościoła św. Jadwigi na Stradomiu. Gdy zaś k ościół m iechowski spłonął w roku 1745 — przystąpił natychmiast d o jego odbudow y i tak stanął now y kościół dochow any p o dzień dzisiejszy. Za jego rządów do125 tamże. konano gruntownej w izytacji wszystkicłi k ościołów , szpitali i wsi będących pod zarządem b ożog rob ców , co przyczyniło się do napra wienia wielu błędów i w ypaczeń z okresu zaniedbań p opełn ion ych przez komendatariuszy. Tą drogą opactw o w M iechow ie stało się znowu generalną siedzibą dla wszystkich czterecłi p row in cji za konu i — bez względu na odległość — koncentrow ało wszystkie ich sprawy. Zarów no doprow adzenie do porządku wewnętrznej dyscypliny jak i zewnętrzne uporządkow anie społeczności zgro madzenia zawdzięczano rządom opata Radlińskiego (1760— 1762), doktora teologii, człowieka w ykształconego i roztropn ego w p o dejm owaniu niełatwych decyzji. P o Radlińskim generalnym przełożonym został wybrany w ro kit 1762 ks. Florian B ujdecki, d ok tór teologii i protonotariusz apostolski, przełożony gnieźnieńskiego klasztoru 1 . Jemu zawdzię cza kościół m iechowski kontynuowanie budow y, a zakon dalsze uporządkowanie swych wszystkich spraw. Był on autorem b io grafii biskupa Macieja Łubieńskiego który stworzył wiele fundacji dla b o żog rob ców ; biografia ta została wydana w roku 1752 127. Po śm ierci B u jdeckiego w roku 1765 opatem m iechowskim w y brano jeg o rodzonego brata, Mateusza B ujdeckiego, który rządził do roku 1788. Na kapitule w roku 1788 obrano generalnym przełożonym za konu Tomasza N owińskiego, który zmarł w roku 1830; za jego rządów — w roku 1819 — nastąpiła kasata z a k o n u 128. Nowiński, rzymski doktor teologii, był kustoszem m iechow itów w roku 1786, a na kapitule w roku 1788 wybrany został opatem m iechow skim ; w roku 1816 biskup W oron icz konsekrował N owińskiego na bisku pa biblejskiego. Po nim proboszczem m iechowskim był jeszcze w latach 1830— 1852 człon ek zakonu, kanonik ks. Baltazar Chwalbiński; od jego następcy rozpoczyna się działalność proboszczy, rekrutujących się z duchow ych diecezjalnych 128. 12. KOŚCIÓŁ M IECH OW SKI W W IE K U X V III O R AZ JEGO Z A B Y T K I W wieku X III zaistniała potrzeba wzniesienia kościoła w ięk szego rozm iaram i od dotychczasow ego, który już został p oprzed nio opisany. Pod jego budow ę wybrano m iejsce zlokalizow ane w- kierunku południow ym od kaplicy B ożego Grobu, która znalazła się w pośrodku wirydarza i krużganków klasztornych. Budowa tego drugiego kościoła objęła lata 1233— 1293; w roku 1947/48 odkryto ścianę zachodnią, rom ańską, u dołu mającą gzyms z wałkiem 1S0, 128 127 128 129 tamże. Ks. G a c k i , dz. cyt., s. 233. P ę k a 1 s k i, dz. cyt., s. 120— 121. Ks. N a w a r r a, dz. cy t., s. 522. z oknem na zewnątrz rozglifionym , z przeźroczem czterolistnym . Przypuszczalnie jest to ściana drugiego kościoła zbudow anego w stylu rom ańskim . Fasada ta, zbudowana z dużych kamieni i kostek ciosow ych, podzielona jest na dwie kondygnacje od stro ny wewnętrznej kościoła; we wnęce fresk z przedstawieniem Ukrzyżowania z roku 1380. W późniejszym czasie kościół ulegał kilkakrotnie przeróbkom w latach 1394— 1410. Rocznik M ie chowski pod datą 1394 wspom ina o zburzeniu poprzedn iego k o ścioła i rozp oczęciu budow y now ej świątyni; odkryta zachodnia fasada romańska zdaje się jednak przeczyć całkow item u zburzeniu drugiego kościoła m iechow skiego. P ożar w roku 1506 miał znisz czyć kościół, a przynajm niej dach kryty ołow iem i wiązania zgo rzały, na w idok czego zwariował ówczesny proboszcz m iechowski M arcin z R adom ia; kościół odbudow ał jego następca Tomasz z Olkusza i pokrył go d a ch ó w k ą m. P o pożarze w roku 1745, w otaksowaniu kosztów odbu dow y brali udział w roku 1749 mi strzowie z Krakowa — murarze D om inik i W ojciech Puckow ie oraz M aciej Cepigowski i cieśle Jakub N ow erkow icz i Jan N ow a kow ski 1J'. Odbudowa kościoła w stylu p óźn obarokow ym trwała do roku 1771. K ościół niewątpliwie zawiera mury rom ańskie, go tyckie, które d o 't e j pory nie zostały przebadane. K ościół m iechowski, zgodnie z założeniem budow y średnio w iecznej. jest orientowany, m urowany i zgodnie z tradycją zako nu m iechow itów nosi wezwanie B ożego G robu. Po stronie południow o-zachodniej stoi luźno — w stosunku do korpusu k o ścioła — wieża o charakterze obronnym , znamienna dla zna czniejszych kościołów b ożog rob ców w Polsce. W doln ej kon dyg nacji wieży kościoła m iechowskiego można się doszukać śladów budowy drugiego kościoła rom ańskiego l33. W ieża ma sześć k on dygnacji i wykazuje nawarstwienia budow y kamiennej z lat 1233— 1293, gotyckiej z w ieków X IV i X V o cegłacli z wzoram i zendrów ki: u szczytu nadbudówka z początku wieku X IX i hełm w kształcie kuli, pokryty blachą miedzianą. Zw ieńczenie wieży sym bolizuje kulę ziemską. Przypuszczalnie ten typ wieży w prow adzili bożogrobcy do swej architektury sakralnej po przy byciu do Polski, przeszczepiając w ten sposób z Ziem i Świętej bezpośrednie w pływy budownictwa spełniającego podw ójną rolę — sakralną i obronną. W ieża kościoła m iechow skiego stała się nie wątpliwie w zorem przy budowaniu wież w kościołach b o żo g ro b ców na Spiszu, w Przeworsku, Leżajsku i Gostyniu. B ożogrob cy przypuszczalnie przejęli zarów no architekturę Palestyny jak 131 Ks. N a w a t r a , dz. cyt., s. 412. 132 Z. B o c z k o w s k a, dz. cyt., s. 21— 25. 133 T. S z y d ł o w s k i , P om nik a rch itek tu ry Piastow skiej. K raków s. 31. 1928 i wpływy architektury rom ańskiej przeszczepionej do Ziem i Świę tej przez krzyżow ców . Stąd luźno stojące wieże obronne przy kościołach m iechow itów wykazują w pływ y architektury w łoskich cainpanilli. Korpus kościoła m iechow skiego posiada prezbiterium zam knię te w ieloboczn ie, trójprzęsłow e, p ołączon e z trzynawową bazyliką rów nież trójprzęsłow ą 1S4. Nawy boczne zamknięte są kaplicam i: po lew ej — kaplica Ukrzyżowania, po prawej — M. B. Często chow skiej. D o p ółn ocn ej ściany prezbiterium dobudow ana została dw ukondygnacjow a budowla w roku 1410; m ieści ona na d ole zakrystię i na piętrze — z lat 1611— 1616 — skarbiec i klatkę schodową. P o stronie połu dniow ej, kruchta i rów nież dw ukon dygnacjow a budowla z biblioteką na piętrze. Od strony p ółn ocn ej przylega do kościoła tzw. Zam ek czyli pałac prepozytów m iechow skich połączon y dobudow anym przejściem z zakrystią. Od strony za chodniej lewa nawa połączona jest z krużgankami klasztoru, a chór z dawnymi celam i zakonników . „W ew nątrz bazyliki ściany prezbiterium podzielon e gzymsem na dwie kondygnacje: w dolnej podział ram owy, w górnej za pom ocą pilastrów i belkowania z attyką, w polach m iędzy pilastrami balkoniki. Nawa główna otwarta do naw bocznych arka dami filarow ym i; podział w form ie parzystych pilastrów, b elk o wania i attyki. Tęcza w yodrębniona opilastrowanym i filaram i przy ściennymi. Sklepienia kolebkow e z lunetami, na gurtach p o jedynczych i parzystych, lub krzyżow e w nawach boczn ych (urato wane od pożaru dachu nawy głów nej w roku 1506). Chór m uzycz ny przechodzący przez całą szerokość korpusu, na pięciu arkadach filarow ych opilastrow anych, o linii falistej” 135. K lerycy m iechow scy tw orzyli ch ór w zorow any na chórach katedralnych; ch ór ten stal na wysokim poziom ie i cieszył się dużą sławą. W edług N akielskiego — przed pożaram i k ościół m iechow ski i jego krużganki ozdobion e były herbami fundatorów i d ostojn i ków, których pam ięć chciał w ten sposób uczcić zakon m iechow i tów 136. Miał tam znajdow ać się herb francuski patriarchy jerozo lim skiego A lm eryka, z czasów pobytu Jaksy w Ziem i Świętej i wysłania do Polski pierw szego kanonika-bożogrobcy. W pierw szym rom ańskim kościele miały być przypuszczalnie jeszcze herby dostojników kościelnych z drugiej połow y wieku X I I : Piotra — arcybiskupa gnieźnieńskiego, Mateusza — biskupa krakow skiego, W altera i Żyrosława — biskupów w rocławskich, Gudnera — biskupa w łocław skiego, Świętosława, Gerarda, A rnolda, M okroty — biskupów poznańskich, W ilka, W ita — biskupów płockich 1:14 Z. B o c z k o w s k i, dz. cyt. 8. 21— 25. 135 tam ie. , 136 Ks. W i ś n i e w s k i . dz. c\t.. a. 120— 122. oraz innych dostojników . Nie zapom niano też o Łabędziu Piotra W łasta, teścia Jaksy. Z k olei szły herby rycerstwa, które uposa żyło m iech ow itów : Odrow ąż — Radosława z K ońskich, N ałęcz — Gniewom ira z Czarnkowa (Ł ętk ow ice), Starykoń — Szafrańców , Habdank — Kagnim ira (B ieganów ), Ośłagłowa — Jakuba (o fia r o dawcy wsi K arcze), B ogorja — M ikołaja (Jaksice i R zep lice), Strzemię — Zem enty (U n iejów ), Radwan — Pakosława (U darz), N ieczuja — W szebora (G olczew o), Sasor — H inczów (Szczepanowice, Pstroszyce, C bod ów ), D em bno — Żyry (Sam ogost, Stawiany), Tarnawa — B udziw oja (G oresław ice), Nowina — Świętosława (C hycza), Doliwa — Jana K rzyżow ca, drugiego prepozyta niiecbow skiego, Lubicz — Budzisława spod Łęczycy. Z tych herbów kilka pozostało w krużgankach, a to: T op ór, Odrowąż, Łodzią, Łabędź i Dołęga. W nętrze kościoła posiada figuralną i ornamentalną d eko rację stiukową w stylu rokokow ym , wykonaną przez W ojciecha R ojow sk iego w latach 1765— 1771 13‘. N ajw idoczniej rob oty restau racyjne trwały bardzo długo, skoro odbudow any kościół został p o nownie konsekrow any przez prepozyta N ow ińskiego w roku 1802 13s. Z dokum entów — w yjętych z puszek w czasie restauracji kościoła w roku 1882 — wynika, że ambona w stylu rok ok ow ym została ukończona w roku 1777, ołtarz zaś śś. Piotra i Pawła w roku 1781 ,39. „O łtarz głów ny ze stiukową płaskorzeźbą Zmartwychwstania Chrystusa, posągam i trzech Marii i anioła oraz aniołów adorujących, rokokow o-klasycystyczny, zapewne po r. 1771. Ołtarze boczne ze stiukowym i płasko rzeźbam i i posągam i, rokokow e: dwa przy tęczy po r. 1765, dwa przy fila ra ch ; dwa w nawach boczn ych , z obrazam i św. Anny i św. Józefa, ma lowanym i przez Franciszka Sm uglew icza; jed en w kaplicy pł. ok. 1771, w ykonany przez Franciszka R ojow skiego, w nim krucyfiks późnogotycki z pocz. X V w .; jeden w kaplicy połu dn iow ej z obrazem Matki B oskiej Częstochow skiej. Ołtarz w kaplicy św. Marii M agdaleny z obrazem św. Barbary, późnobarokow y. Ołtarz w kruchcie przy wieży z krucyfiksem i posągiem Matki B. Bolesnej późn ob a rokow y” 140. R ok ok ow e — ozdobion e krzyżem bożog rob ców i G ryfem Jaksy z M iechow a — są dębow e stalle z roku 1771, tron prepozytów i kon fesjon ały. Szafy i kom ody w zakrystii oraz lawaterz są w stylu klasycystycznyin. O pierw szych organach w m iechowskim kościele b ożog rob ców zachowała się wzmianka z roku 1376, poda na przez N akielskiego; były to organy 22-głosowe, a sprowadził je 137 138 139 141 Z. B o c z k o w 8 k a, dz. cyt., s. 23. Ks. W i ś n i e w s k i , dz. cyt., s. 115. tamże. Z. B o c z k o w s k a, dz. cyt., s. 23. , Portret ze zbiorów b. opactwa b ożogrobców w M iechowie z m s y g n i a m ce tem z podw ójn ym krzyżem czerw onym zakonu Strozow Bożego relikwiarzem zachowanym w skarbcu kościelnym i b is k n p im i^ m u Kardynał Andrzej Batory proboszcz m iechow ski, generał zakonu, zmarły w r. 1599. (Zbiory b. opactw a w M iechow ie). Portret Samuela N akielskiego historyka zakonu bożogrobców polskich w M iechowie (zbiory b. opactw a w M iechowie) Portret Piotra K orycińskiego. posła króla Michała K orybuta W iśniow ieckiego do pa pieża. generała bożogrobców m iechow skich, biskupa przem yskiego (zbiory b. opactwa w Miechów ie) M arcin Czczikus. Są także w zm ianki z wieku X V I I dotyczące restauracji organów kosztem biskupa Zadzika. O becne organy p o chodzą z czasów restauracji kościoła* Zegar na wieżę sprawili b o żogrobcy jeszcze w średniow ieczu; obecny p och od zi z roku 1872 i został ufundowany przez magistrat m ie ch o w sk iI41. Z zabytkow ych ksiąg liturgicznych kościoła m iechow itów za chow ały się: ilum inowany antyfonarz „op ra w ion y w deski obite białą skórą z wyciskam i, z drugiej połow y X V I wieku, w form ie ramek obiegających opraw ę, z roślinnym i figuralnym ornam en tem. Na okładce w idnieje łaciński napis majuskułami: A ntifonarius E cclesiae M iechoviensis A. D. M D L X X X . W narożnikach i na środku znajdow ały się niegdyś okucia, których obecnie brak. R ęk o pis składa się z 217 kart pergam inow ych. Tekst pisany gotycką majuskułą, nuty są w p ięć linii"’ 14\ Na w iniecie data 1562 i w y raźne w pływ y włoskie w dekoracji, jakby zaczerpniętej z m oty wów zdobn iczych architektury kaplicy Zygm untow skiej na W a welu, wskazują na czas powstania antyfonarza. Opis jed n ej z mi niatur, przedstawiającej Zbaw iciela, daje p ojęcie o je j wykonaniu: „Z b aw iciel przedstaw iony (jest) en face, twarz ma szeroką, p o krytą brodą, w łosy czarne spadają mu na ram iona, głowa w yko nana piórkiem i tuszem. G łowę otacza duży, żółty nim b. Suknia blado buraczkow ego koloru. U drapow anie szaty zaznaczone ciem nymi, rów noległym i liniam i w iśniow ej barwry. Przy rękawrach w i dać zieloną podszew kę sukni. Zbaw iciel podniósł praw'ą rękę jak do błogosławieństwa, w lewej trzyma kulę świata z krzyżem , op ie rając się o piersi” 143. Opis pow yższy jest ciekawy ze względu na kulę zakończoną krzyżem b ożog rob ców na hełm ie w ieży kościoła m iechow skiego; w obu w ypadkach można też doszukać się p o d o bieństwa z w yobrażeniem przedsionka z kulą świata w świątyni jerozolim sk iej w B iblii, w ydanej w Paryżu w roku 1563 144. Bardziej jeszcze charakterystyczny jest inny zabytek wr skarbcu dawnego opactwa m iechow skiego; jest to relikw iarz sprzed roku 1584, w yobrażający kaplicę G robu Chrystusowego w Jerozolim ie. Darowany on został przez św. Karola Borom eusza kardynałowi A n drzejow i Batorem u, przełożon em u bożog rob ców m iechow skich w czasie jego pobytu w Rzym ie. P od ob n y do m iechow skiego jest relikw iarz w kościele we W rocieryżu , należącym rów nież do b o żo grobców m iechow skich; p och od zi on z roku 1806 i przedstawia kopię Grobu Chrystusowego w Jerozolim ie. N iew ątpliw ie na re likwiarzu rzymskim , przyw iezionym przez B atorego do M iechow a, 141 Ks. W i ś n i e w s k i , s. 119— 131. 142 Ks. J. Z d a n o w s k i , Ilum inow ane ręk op isy k sięgozb iorów kapitularza k atedraln ego, Seminarium D uchow nego tv K ielcach i kościoła parafialnego w M iech ow ie, K ielce 1929 s. 30. 143 ta m ie, s. 35. 144 Ks. W i ś n i e w s k i , dz. cyt., s. 130. w zorow ane były p odobn e zabytki innych k ościołów b ożog rob ców i podkreślały w ten sposób kult G robu Chrystusowego, związany z regułą zakonu 14s. . 13. ZALEŻNOŚĆ OD M IECH OW A P A R A F II W CZTERECH PRO W IN CJACH I ROZW ÓJ D ZIAŁALN O ŚCI D U SZPASTERSKIEJ I SAM A RYTAŃ SK IEJ W kościele skaryszewskim, dochow ał się — z czasów jeg o przy należności do b ożog rob ców m iechow skich — kielich ufundow any przez W awrzyńca K oryck iego w roku 1763; na jeg o stopie znaj duje się w izerunek kaplicy B ożego G robu w Jerozolim ie 14e. W śród zabytków skarbca m iechow skiego zwracają uwagę trzy kielichy: p óźn og otyck i — bogato rzeźbiony i wysadzany perłam i — z ok oło roku 1500; rok ok ow y — z inicjałam i złotnika krakow skiego LM (M arcina Lekszyckiego) i datą 1773 — oraz rów nież rok ok ow y — z datą 1775. Cechami Lekszyckiego są jeszcze ozna czon e: rok ok ow y relikwiarz i tacka z roku 1774. Z zabytków baro kow ych znajdują się tam: pacyfikał z wieku X V II, krzyż ołta rzow y z postacią U krzyżow anego wykonaną z kości słoniow ej, am pułki cechow ane w Augsburgu oraz tacka z drugiej połow y wieku X V II ,47. P rócz tego jest wiele zabytkow ych tkanin, a to: kap, ornatów, dalm atyk z okresu X V I— X I X wieku oraz dwie infu ły opatów m iechow skich z p ołow y wieku X V II. Od pierwszej chwili przybycia do Polski m iechow ici — zgod nie z regułą zakonu — rozw ijali działalność duszpasterską i sa marytańską w czterech p row in cjach : M ałopolskiej ze Spiszem, W ielk opolsk iej, M azow ieckiej i Przem yskiej, zwanej czasem ruską. Bez om ówienia — ch oćby najbardziej szkicow ego — historii ro z w oju tych p row in cji m iechow itów (zawsze pozostawały w najściś lejszej zależności od prepozyta w M iechow ie i kapituły zakonu a także odbyw ały swe zjazdy w M iechow ie) nie da się w pełni zobrazow ać zadania, w ykonyw anego przez zgrom adzenie je ro z o limskie na ziemiach polskich. Zależność prow in cji od M iechowa była dlatego tak ścisła i ciągła, ponieważ bożogrobcy uzyskawszy uw olnienie papieskie od ju rysdykcji biskupów diecezjalnych od w oływ ali się do nich tylko w sprawach, o których rozstrzygnięciu nie mogła decydow ać hierarchia zakonu w M iechow ie. R ozpatrując kolejno historię bożog rob ców w tych czterech p ro w incjach należy podkreślić, że w spółzależność wizytacyjna i hie rarchiczna nie została rozluźniona nawet po rozbiorach Polski. M imo trudności w pokonyw aniu now ych granic w ramach admini stracji obcych rządów zachowane z tych czasów w izytacje wska zują, że bożogrobcy z innych p row in cji nie tylko utrzymywali kontakt z M iechow em ale byli mu tak posłuszni, jakby nie istniały podziały polityczne. Nawet p o kasacie zakonu, z niektórych parafii bożogrob ców znajdujących się pod zaborem austriackim czy pru skim, wysyłano do M iechowa cenne księgi i dokum enty rozum ie jąc. że tam jest ich główne przeznaczenie historyczne. W każdej z p row in cji rozróżniano w roku 1574 dwa rodzaje parafii t.j. zawiadywane przez p roboszczy b ożogrobców -prepozytów i bożogrobców -pleban ów (w hierarchii diecezjalnej tych ostatnich uważano za niższych 148). W roku 1574 proboszczy-prep ozytów miały parafie: w Gnieźnie, Przeworsku, K rakow ie, B y tomiu, Lendaku na Spiszu, W yszogrodzie, Leżajsku, Pyzdrach, Sie radzu, Łęgow icach, Żarnow cu, Sępolnie, G rodzisku, R ypinie, Górznie. Plebani rządzili na parafiach w Skaryszewie, Chełmie n. Rabą, W rocieryżu, U niejow ie, C hodow ie, R adołow icach , Giedlarowej, U rzewicach, Łapszach N iżnych i H anuszowcach na Spiszu. B iskupi nie chcieli zrezygnow ać z prawa ingerencji w w e wnętrzne sprawy b ożog rob ców na terenie swych d iecezji; przy kładem tego była wizytacja biskupa przem yskiego w Przeworsku w roku 1490, po k tórej zalecił: „b y przełożony dawał braciom dobre p iw o, trzymał dla nich służącego i miał pieczę nad chorym i, Reasumując powyższe należy stw ierdzić, że obecn y kościół m iechow ski znajduje się na fundam entach kościoła rom ańskiego z wieku X I I I ; odkryta fasada zachodnia wskazuje na to, że mury jeg o pow inny tkwić w późniejszych murach, poch od zących z ok re su przeróbek gotyckich, częściow o renesansowych i p óźn obarok ow ych ; te ostatnie wiążą się z okresem po pożarze w roku 1745. Do tej p ory brak było badań architektoniczno-archeologicznych, które nie są łatwymi do przeprow adzenia z uwagi na konieczność kontynuowania pracy duszpasterskiej. Prow adzenie badań wyma gałoby wyłączenia od pełnienia kultu głów nej nawy. W każdym razie konieczne jest przebadanie wieży, jako zabytku architektury sakralno-obronnej oraz krypt, których część m oże być dotąd zam urowa na i tym samym niedostępna. Nie należy rów nież lekcew ażyć p oda nia o połączeniu podziem nym piw nic kościoła z pobliskim rynkiem miasta, w pośrodku którego miała znajdow ać się studnia i z niej prow adziło tajne w yjście. B yć m oże, że naukowe badania ujawnią inne rozwiązanie tego podziem nego przejścia; m ogło się ono ciąg nąć spod kościoła czy klasztoru na dużo większą odległość — na wet poza teren miasta M iechow a. Liczne zbrojne napady na M ie chów i inkastelacja klasztoru w początkach wieku X III nasuwa przypuszczenie, że tego typu ukryte przejście było konieczne. 145 K atalog Zabytk. Szt. tv P olsce, t. III nr 10 s. 38— 10. 146 tam że, nr 3 s. 42. 147 Z. B o c z k o w s k a, dz. cyt., s. 24. 148 N a k i e l s k i , dz. cy t., s. 693. braciom natomiast przykazał: by się w mieście nie zabawiali, śpie wali godziny w kościele i nie m ieli żadnych rzeczy ponad osobistą kon ieczn ość” 149. R ów nież konsystorze biskupie rozpatryw ały spory m ajątkowe. Przykładem tego m oże być spór w kurii krakow skiej w roku 1486 m iędzy bożogrobcem M ikołajem ze Strojnow a, plebanem w Cheł mie, a Janem Pękosławskim , proboszczem m iechowskim , o za garnięcie dw orskich gruntów; spór zakończył odkopcow an iem ról zagarniętych przez plebana lso. W średniow ieczu biskupi dokonyw ali oceny działalności gospo darczej m iechow itów , dając im w nagrodę za karczowanie lasów — w olne rybołóstw o. N aogół wzajemne stosunki układały się harm onijnie; wyjątek stanowili ci biskupi, którzy działali w kierunku zawładnięcia d o brami b ożog rob ców wrzględnie stosowali nacisk przy obsadzaniu godności komendariusza osobam i kierującym i się względam i ma terialnym i; zakon z reguły m ocno się temu sprzeciwiał i tym sa mym popadał w otwarty lub ukryty k on flik t. Poniew aż stan taki trwał przez kilka w ieków, a pom im o to b ożogrob cy w sch od zili zawsze z tych spraw obronną ręką, należy docen ić ich zasługi w utrzymaniu reguły aż do kasaty zakonu w wieku X IX . Należy też podk reślić, że opactw o m iechowskie aż do schyłku R zeczyp o spolitej, a w ięc niemal do końca wieku X V III, m iało p odw ójn e zw ierzchnictw o w osobach: kom endatariuszy działających na szko dę podstaw materialnych zgrom adzenia i op atów -bożogrobców ra tujących wszelkim i siłami podstaw y istnienia zakonu, tak ideowre jak i finansowe. Przykładem takiej zlej w oli była postawa biskupa kam ienieckiego M ikołaja D em bow skiego, m iechow skiego proboszcza-komendatariusza w latach 1743— 1756, który po straszliwym pożarze kościoła m iechow skiego nie pozw olił na jakiekolw iek usz czuplanie swoich d och od ów na odbudow ę 151. Cały ciężar tej odbu dow y wzięli na siebie wyłącznie b ożog rob cy, zwłaszcza opaci m ie chow scy, m ając uszczuplone d och od y w stosunku do tego, czym dysponow ali m iechow ici w okresie średniowiecza. R ozpatrzenie historii przynależności parafii diecezjalnych i szpitali w czterech prow in cjach zakonu m iechow itów na zie miach polskich daje pełny obraz osiągnięć działalności klasztoru w M iechow ie. Osobną kartę stanowią szkoły prow adzone przez b ożog rob ców na marginesie duszpasterstwa; zagadnienie to w y maga osobnego om ówienia. 149 Ks. G a c k i , dz. cyt., #. 222. 150 tamże. 151 tam że, s. 247. A. Prow incja M ałopolska ze Spiszem P o M iechow ie najważniejszy ośrodek bożogrobców znajdow ał się w K ra kow ie, który — jako siedziba panujących — wymagał dość w czesnego obsa dzenia przez kapitułę miechowską. Przyw ilej lokacyjny Krakow a z roku 1257 wspom ina o młynie m iechow itów , zniszczonym przypuszczalnie w czasie najazdu Tatarów w roku 1241. Należy w ięc przyjąć, że przybycie b ożogrobców do sto licy nastąpiło przed tą datą; pewne jest posiadanie przez nich wsi B aw ół na Kazim ierzu i kościoła św. W aw rzyńca, pochodzących praw dopodobn ie z fundacji Jaksy z M iechow a; później wieś B aw ół przeszła w ręce kapituły (przypuszczalnie w okresie buntu w ójta A lb erta ). D opiero za Kazim ierza W ielkiego wypływa sprawa budow y k o ś c i o ł a ś w. J a d w i g i i s z p i t a l a d l a b o ż o g r o b ców n a S t r a d o m i u 152. Zniknięcie m iechow itów z terenu K rakow a za czasów Władysława Łokietka związane było z ukaraniem przez króla spiskow ców , do k tórych zaliczał się H enryk, będący prepozytem m iechow skim w okresie buntu w ójta A lberta 158. H enryk miał być spokrew nionym ze zbuntowanym w ójtem i w związku z tym miał mu w ydzierżaw ić jedną z w iększych wsi klasztornych Ł ętkow ice, przyno szącą rocznie 80 grzywien dziesięciny; dlatego właśnie wieś ta została na stałe skonfiskowana przez Łokietka w roku 1312. — Jest faktem , że bożogrobcy w roku 1311 zostali w ypędzeni z M iechow a, pozbaw ieni swych w łości i dop iero na interw encję papieża odzyskali M iechów i uposażenie w roku 1314. Papież K lemens V , zwracając się do arcybiskupa gnieźnieńskiego o w zięcie w obronę b ożogrobców m iechow skich, zalecał „u ży cie klątwy w ich obron ie” ,54. W pływ y posiadane przez b ożogrobców w kurii rzym skiej — zapewne interw eniow ali za pośrednictw em patriarchy jerozolim skiego — pow odow ały kilkakrotnie w y stąpienie papieża i szybkie w ycofanie się Łokietka. R ok 1314 był datą śmierci proboszcza m iechow skiego H enryka; dla ułagodzenia króla w ybrano jeg o na stępcą Polaka — Benedykta. Dla ścisłości należy zaznaczyć, że K azim ierz W ielki nie wykazywał najm niejszej n iechęci do zakonu; za je g o czasów otrzym ali szereg zatw ierdzających przyw ilejów , a nawet pom niejszone taksy przy opod a t kow aniu dóbr w roku 1349. Niem niej zniknięcie ich z Krakow a i to w chw ili uplasowania się w nim now ych zakonów (cystersi, duchacy, dom inikanie i fran ciszkanie) w wieku X III, musiało m ieć dla ich przyszłości pow ażne konsek wencje polityczne. Stąd wypływ a ich chęć do pow rotu na teren K rakow a i dla tego fundacja na Stradom iu stanowiła poniekąd m ożliw ość naprawienia błędów pop ełnion ych przez ich poprzedników . Długosz pod aje, że „K azim ierz W. chcąc przestrzeń zwaną Stradomiem zaludnić i zabudow ać — dla uczczenia Jadwigi obranej księżną wrocławską, z której krwi sam p och odził, założył tam klasztor b ożogrobców , reguły św. Augustyna, przeznaczając w r. 1360 m iejsce obszerne murem opasane na k ościół, klasztor i szpital dla ch orych , które też gmachy pod wezwaniem św. Jadwigi 152 St. T o m k o w i c z, K la sztor szpitalny św. Jadwigi. R oczn. Krak. X X II, 8. 59— 66. 153 E. D ł u g o p o 1 s k i, Bunt ivójta A lberta. R oczn. Krak. V II. s. 164— 167. 154 tam że, stawiać zaczął. A dalej, że siostra Kazim ierza W ., Elżbieta, królow a polska wzgórzu p o prawej stronie rzeki przy trakcie do Krakowa. Na m iejscu tym — i węgierska, dzieło przy śmierci brata n iedokoń czone i zaniedbane, na prośby prepozytów klasztornych: Benedykta gnieźnieńskiego i Marcina m iechow skiego mającym fu n dację uczynioną przez Kazim ierza wznow iła w r. 1375, a instytucję dała w posiadanie M iechow itów ” 155. Lokalizacja m iechow itów na Stradom iu pod względem gospodarczym była duże znaczenie obronne — skupiał się przypuszczalnie kult po gański w czasach przedchrześcijańskich. Z tego zdaje się w zględu osadzono tu b ożogrobców rów nocześnie z M iechow em , stwarzając filię klasztoru, która od roku 1417 podlegała k ościołow i św. Jadwigi na Stradom iu w K rakow ie. K rew ny Jaksy z M iechowa, M ichor, darował b ożogrobcom tę wieś p o pow rocie istniejące) oddzielające Stradom od Kazim ierza, na zapleczu zaś — w m iejscu Jaksy z Ziem i Świętej w roku 1163 159. K ościół, jaki tu pow stał, był drewniany; zachowana świątynia murowana została wybudowana wr latach 1738— 1749 dzisiejszych ogrodów misjonarskich — był staw św. Sebastiana, będący przysta i pośw ięcona w roku 1824 16°. Na je j fasadzie zachodniej um ieszczono kartusz znakomita. Od strony połu dn iow ej znajdow ało się koryto W isły (dziś nie nią dla szkutów i barek; nad stawem tym znajdow ały przeładunkow e żurawie 156. z herbem zakonu, a na w ieży i dachu kościoła pod w ójn y krzyż m iechow itów . Była też i słaba strona tego usytuowania budynków N ależałoby przebadać, czy na tym terenie nie ma fundam entów rom ańskiego k o ścioła czy kaplicy z wieku X II, gdyż istniejący w tym czasie bród na Rabie i dawne osadnictw o słowiańskie nasuwają przypuszczenie o dużej roli tej m iej m iechow itów na Stra dom iu, a m ianow icie niebezpieczeństw o zalew ów w czasie dość częstych pow od zi, 0 czym mamy szereg wzmianek. Długosz w X V wieku opisywał ten obiekt następująco: ..Klasztor św. Jad wigi położon y jest nad rzeką W isłą, w pobliżu mostu królew skiego (dzisiejsze skrzyżowanie Plant Dietla ze Stradom iem ), złączony po stronie połu dn iow ej scow ości przed napadem Tatarów w roku 1241. U n i e j ó w , wieś leżąca przy trakcie M iechów — Żarnow iec, została na dana klasztorow i miechow skiem u przez comesa Zem entę i je g o żonę w roku z kościołem św. Jadwigi częścią z kamienia, częścią z cegły przez króla K azi 1229 przed W iesławem, dziekanem katedry krakow skiej 161. Podczas badania mierza wzniesionym , inne budynki gospodarcze mając drewniane, piw nice zaś 1 mieszkania braci murowane. Ze względu na szczupłość doch odów prepozyt doch odów jedyn ie i czterech braci zw ykło tu m ieszkać, służbę Bożą sprawować i m ieć staranie o dusze przebyw ających w szpitalu. Szpital leży po drugiej, półn ocn ej klasztoru m iechow skiego w roku 1349 — prokurator Świętosław podał, że U niejów przynosi bożogrobcom 5 grzywien czynszu i 5 grzywien dzie sięciny. W roku 1373 prepozyt m iechow ski łącznie z braćm i zakonnym i nadał k ościołow i św. Wita w' U niejow ie, ja k o uposażenie, zarośla nieprzekraczające stronie; przylega i stanowi dalszy ciąg kościoła św. Jadwigi, w znoszącego się obszarem 1 łanu. Później nastąpiły dalsze fundacje b ożogrob ców dla kościoła w pośrodku, rów nież uniejowskiego. Od roku 1417 wieś stanowiła uposażenie klasztoru i szpitala św. Jadwigi na S tra dom iu 162. z muru w zniesiony; ma on nadto oficyn y drewniane i cm entarz d o grzebania zm arłych” lo7. W średniowiecznym K rakow ie szpital odgryw ał dużą rolę; za prepozytury S. Nakielskiego cała posiadłość została poszerzona w roku 1619. W m iędzyczasie przybyły liczne fun dacje zwłaszcza dla u bogiej szlachty w podeszłym wieku. Była to fundacja kapituły krakow skiej z roku 1691. W czasie oblężenia Krakow a przez Szwedów w roku 1656 — szpi tal i pięknie odbudow ane probostw o zgorzały. W krótce jednak odbudow ano znow u i k ościół i inne budynki dzięki staraniom prepozyta ks. W olsk iego; kon sekracja kościoła nastąpiła w roku 1674. W zakonie b ożogrobców istniała tra dycja odbywania corocznie uroczystych procesji na pamiątkę zwycięstwa grun w aldzk iego; procesja ta rozpoczynała się na Stradom iu w dniu 15 lipca i uda wała się z dziękczynieniem na W awel 158. Z końcem wieku X V I II władze K ościół św. W ita był ju ż w zm iankowany wr dokum entach z lat 1325— 1327; zachowany kościół zbudow any przez b ożogrobców w roku 1470, był restauro wany w roku 1883 i 1912 163. G otycka chrzcielnica nosi god ło b ożogrobców , obok którego znajduje się — częściow o tylko czytelna — data „1 5 ..” . W r o c i e r y ż w K ieleck im posiadał kościół pod wezwaniem N. P. Maryi i wspom niany jest wr roku 1326; w okresie lat 1394— 1875 stanowił włas ność zakonu b ożogrobców 164. Zachow any k ościół wzniesiony został przez m ie ch ow itów wr roku 1801 według p rojek tu Franciszka Żabiszew skiego z J ędrzejo wa, a fasadę wykonał kamieniarz Adam R ojew ski z M iechow a. Z zabytków związanych z bożogrobcam i znajdują się w kościele: obraz w otyw ny z roku 1548, austriackie zajęły wszystkie budynki i dokonały ich przerobienia pod kątem potrzeb urzędów . W' zamian bożogrobcy otrzym ali pojezu icki kościół i klasztor na którym przedstawiona jest klęcząca przed krucyfiksem H elena z Łączkiew ic z synem Stanisławem, m iechow itą, profesorem U niwersytetu Jagiellońskiego. św. Barbary na Małym Rynku, odebrane po kasacie. K ościół, szpital i szkoła przestały istn ieć; pozostała p o nich jedyn ie w skarbcu katedry na W awelu Krzyż b ożogrobców znajduje się jeszcze na rokok ow ej m onstrancji z roku 1760 i relikw iarzu z roku 1806; relikw iarz ten jest kopią G robu Chrystusowego szklanica św. Jadwigi, wykonana z kryształu na podstawie ze srebra złoconego. C h e ł m n a d R a b ą posiadał bardzo stare osadnictwo na w yniosłym w Jerozolim ie 165. 155 St. T o m k o w i c z, dz. cyt., s. 59— 80. 156 M. T o b i a s z, H istoryczny rozw ój sieci w odn ej K rakow a, s. 26, 36, 60. 157 St. T o m k o w i e z, dz .cyt., s. 59— 80; St. K u r a ś , O rdynacje i usta w y w iejskie, K raków 1960 s. 69 p od a je przykład uposażenia klasztoru św. Jad wigi wsią Niskową w nowosądeckiem . 158 T o m k o w i c z, dz. cyt. 159 Ks. B u 1 i ń s k i, dz. cyt., s. 483. 160 J. E. D u t k i e w i c z, Poiviat bocheński. Katal. Zabytk. Szt. w P olsce, t. I z. 2 s. 5. 161 Kod. D ypl. Malop. II s. 40. 162 Ks. B u 1 i ń s k i, dz. cyt., s. 483. 163 Z. B o c z k o w s k a, dz. cy t., s. 40. 164 Kat. Zabytk. Szt. w P olsce, t. III z. 3 s. 42. 105 tamże. Żarnowiec w Olkuskim, miasto z czasów panowania Kazim ierza W iel k iego; oprócz kościoła parafialnego Ż arnow iec posiadał kościół pod wezwaniem św. K rzyża ze szpitalem, ufundowany przez biskupa kijow skiego Michała Trestkę w roku 1403 166. R ek torow i kościoła i szpitala, oddanego m iechow itom , nie w olno było w ch odzić w obow iązki kościoła parafialnego, ch oć miał prawo odprawiania nabożeństw, kazań i dzwonienia. W ładysław Jagiełło nadał szpita low i 4 łany pola na przedm ieściu z obow iązkiem odprawiania przez proboszcza-bożogrob cę dw óch mszy św. tygodn iow o za duszę królow ej Jadwigi (p on ie działki) i na intencję jeg o oraz królow ej Anny (sob oty). W roku 1422 b o żo grobcy w Żarnow cu otrzym ali na rzecz swego szpitala sołectw o wsi Gebartowa W ola z dwom a w olnym i łanami !67. Jagiełło zw olnił sołectw o od wszelkich p o datków i dodał jeszcze dochody z karczm y pod Grzemiątną, leżącej przy trakcie P iotrków -K raków . M iechow itom w Żarnow cu nadał jeszcze król praw o posta wienia dw óch m łynów , żądając w zamian mszy św. za duszę zm arłych żon i rodziny królew skiej. Przy tymże kościele pow stało w roku 1426 Bractw o Li terackie, założone przez biskupa Trestkę oraz m iechow itów Jana, prepozyta kościoła szpitalnego w Żarnow cu, Stanisława z M iechowa oraz szereg osób duchow nych i św ieckich — w tym kilku mieszczan żarnow ieckich 168. Celem bractwa były w spólne uczynki m iłosierne, nabożeństwa, narady ,.by m odlitwam i, mszami św., jałm użnam i, nabożeństwam i żałobnym i — zasłużyć sobie na zba wienie duszy” m . Bractw o zostało zatw ierdzone przez biskupa O leśnickiego wraz z udzieleniem 40-dniow ego odpustu za w spółudział w m odlitw ie w k o ściele b ożogrobców czy też w akcie m iłośierdzia. W szpitalu b yło m iejsce dla sześciu ubogich, a uposażenie dla nich było następujące: „...p ró cz tego co ofiarow ał Jagiełło i 2 młynów w Baryczy i Jagielni, był dom , ogród, z którego czynsz czerpali ludzie pobożni w spierani przez kuratorów w ybieranych z m iejskich rajców ” 17°. Zw yczajem średniow iecznym do szpitala-przytułku w ch odziło się przez kaplicę św. Krzyża, której kolatorem był klasztor m iechow ski, a obow iązki pełn ili przysłani stamtąd zakonnicy. P ro w adzili oni często procesy ze starostami żarnow ieckim i, którzy — będąc prze ważnie innow iercam i — zagarniali dwa łany uposażeniowe. K ościółek św. K rzyża miał tylko prezbiterium murowane (pozostałość dawnej k a p licy ), a nawę dobudow ano drewnianą (nawa posiadała p olich rom ię). W roku 1817 kościół spalił się wraz ze szpitalem dla ubogich. W związku z kasatą m iechow itów nie został odbudow any. Należy zaznaczyć, że niektórzy z b ożogrobców byli p ro boszczam i k ościoła parafialnego w Żarnowcu. Chodów w M iechowskim , posiadał parafię założoną przez biskupa Zawiszę z K urozw ęk na prośbę dziedzica wsi, Krzeslawa, kasztelana sądeckiego, w roku 1381 171. Biskup uposażył k ościół w C hodow ie dziesięciną ze wsi Tczyce. 166 Ks. J. W i ś n i e w s k i , Miasto Ż arnow iec, Marjówka O poczyńska 1933 • s. 11. 167 168 169 170 171 tam ie. tam ie. tam ie. tam ie. K od. D ypl. Malop. III s. 337. Chodów należący przed tym do parafii uniejow skiej — łącznie z W olą Chodowską przynależną do parafii w Szreniawie — otrzym ał samodzielną parafię i k ościół, którego proboszczam i byli b ożogrobcy m iechow scy aż do roku 1823. Michał Radom szczanin, proboszcz m iechow ski, w ykupił C hodów od Dobiesława z G rybow a m iędzy rokiem 1395 a 1424. W ieś lokowana na prawie niem ieckim za czasów Kazim ierza W ielkiego (r. 1363) posiadała 10 łanów k m ie c y c h 172. K ościół pod wezwaniem św. Jana Chrzciciela wybudow any był z drzewa ok oło roku 1381 i proboszczam i je g o byli m iechow ici ja k o w łaściciele wsi — przy puszczalnie od roku 1404. Od roku 1417 parafia podlegała konw entow i b o żo grobców u św. Jadwigi na Stradom iu w K rakow ie. B ożogrobcy posiadali w Chodow ie folw ark klasztorny i karczm ę, przynoszącą — według Długosza — pół grzywny dochodu. Po kasacie m iechow itów wieś przeszła na rzecz skarbu K ró lestwa Polskiego w' roku 1818. W rocim owice w M iechow skim , wieś wzm iankowana w roku 1327, miała w połow ie wieku X V k ościół drewniany pod wezwaniem św. Andrzeja 173. P roboszcz pobierał dziesięcinę w w ysokości 10 grzyw ien; uposa żony był rolą, łąkami, karczm ą — dającym i dochodu 21/* grzywny. W późn iej szym czasie wieś stała się własnością b ożogrobców m iechow skich. W roku 1748 wystawili tam kościół murowany. R ob oty przy kościele prow adził mistrz kra kowski, M aciej Cepigowski. Na drewnianej dzw onnicy zawieszony jest dzwon z datą 1530. ŚLĄSKIE KOŚCIOŁY I SZPITALE W PROWINCJI MAŁOPOLSKIEJ W roku 1190, Bolesław W ysoki założył w N y s i e szpital i oddał go b o żo grobcom z M iechowa. M iechow ici byli w ięc pierwszym zakonem w Nysie posia dającym kościół i szpital (dzisiejsza ulica P rudnicka), spalone przez husytów w roku 1434. Później otrzym ali — na tzw. Rynku Solnym — plac pod budow ę nowego kościoła i szpitala, oddali zaś wszystko jezuitom w roku 1622. W latach 1719— 1730 wybudow ali jed en z najwspanialszych k ościołów na Śląsku pod wezwaniem św. św. Piotra i Paw ła; um ieszczony w głów nym ołtarzu obraz M. B. C zęstochow skiej dow odził ich ów czesnej polskości. Szpital bożogrobców , z drewnianą wieżą krytą gontem , dochow ał się po dzień dzisiejszy. Od prepozytury w Nysie zależne były śląskie parafie i k ościoły szpitalne w R aciborzu, Ząbkow icach śląskich i D zie rż o n io w ie 175. W roku 1226, w ójt biskupi W alter i mieszczanin P iotr B ogocz w Nysie poczyn ili darow izny na rzecz szpitala bożogrobców . Przed rokiem 1249 powstała placów ka m iechow itów w G łogow ie, R aciborzu (r. 1295), w D zierżoniow ie (1292— 1301). Wszystkie śląskie filie zakonu b ożogrobców podlegały zwierzchnictw u prepozyta m iechow - Rzym ie w r. 1Ó84 opatow i m ie c h o w s k i^ Relikwiarz z wieku X V I-f(« ofiarow any w 1R iym .e T 'j . w J -H ' tras ks. kardynałow i A ndrzejow i Batorem u, wyobrażający kaplicę Umie 1-2 Slown. G eogr. K ról. P ol. t. I s. 612. 173 Ks. N a w a r r a, dz. cy t., s. 302; Z. B o c z k o w k a , dz. c y t.9 s. 43. 174 T. S i 1 n i c k i, D zieje i u strój K ościoła na Śląsku do końca tv. X IV . [w :] Historia Śląska t. II. K raków 1939 s. 115; Dr Ł a z i n k a, Nysa dawniej * dziś, O pole 1949 s. 33. 175 S i l n i c k i , dz. cyt., s. 115. skiego i wyłam ały się spod niego d op iero w roku 1334 17ł. Od tego czasu b o żo grobcy z diecezji w rocław skiej pod porządkow ali się prepozyturze zakonu w Pradze. Od roku 1500 Nysa była siedzibą m iechow itów gałęzi czesko-m oraw- kowski Muskata z nadania W acława II (r. 1301); tam też wydal H enryk w kwietniu lub maju 1312 r. przyw ilej dla Henryka z Kieżm arku na Spiszu, na założenie na prawie niem ieckim wsi w pobliżu rzeki D onicz (D unajec) skiej z przełożonym generalnym w osobie prepozyta zakonu. O derwanie się w m iejscu Priuicz. P o stłumieniu buntu — m iechow itów m iechow skiego ju ż nie p ow rócił, umarł na wygnaniu, może w kouw ecie w Pra dze w r. 1314” 18°. śląskich od macierzystego klasztoru w M iechowie łączyło się z odpadnięciem od Polski Śląska Piastow skiego na rzecz Czech, wskutek zre proboszcz H enryk do klasztoru zygnowania z niego przez Kazim ierza W ielkiego, nie m ogącego sprostać k oa licji I aktem jest, że diecezja krakowska w wieku X I obejm ow ała tereny Spiszą krzyżacko-czcskiej. O bce wpływy oddziałały na M iechów tylko w roku 1311, tj. w okresie buntu w ójta A lberta; kiedy w roku 1345 chciano obsadzić prepo- i że w pływ y polskie sięgały daleko na południe tj. na dzisiejsze pogranicze zyturę w M iechow ie Czechem z pochodzenia — H enrykiem z M iędzyrzecza; nie polsko-słow ackie. W pierw szej połow ie wieku X II kasztelania spiska przeszła w ręce węgierskie jako posag Judyty, córk i Bolesława K rzyw oustego; d op iero dopuścił do tego K azim ierz W ie lk i177. P o ba n icji b ożogrobców w latach 1311— 1314 M iechów cierpiał na brak zakonników i wtedy — za zgodą pa w ięc przypadkiem , że po obu stronach K arpat działali b ożogrob cy, których w roku 1412 część Spiszą pow róciła do Polski w drodze zastawu lsl. Nie było nych. W tych w arunkach utrata przez M iechów zw ierzchnictw a nad placów kam i pierwsze enklawy m iechow skie należy przyjąć na wiek X III. Przypuszczenie takie nasuwa architektura sakralna k ościołów we Frydm anie, K rem pachach, w diecezji w rocław skiej była z politycznych w zględów uzasadniona prądami z obronnym i wieżam i typow ym i dla budow nictw a m iechow itów , które odbijają odśrodkow ym i ciążącym i w stronę Pragi. B y t o m i C h o r z ó w pozostały w ramach ob edien cji prepozytów m ie od architektury innych k ościołów nad Dunajcem . P on adto, założenie parafii w K rem pachach w roku 1278, a we Frydm anie z końcem wieku X III, m ożnaby chow skich, wiązać z bożogrobcam i. Architektura ta jest identyczną z budow lam i m iecho triarchy jerozolim sk iego P iotra — sprow adzono do Polski szesnastu braci zakon leżały bow iem na terenie diecezji krakowskiej i biskupi nie dopuścili do oderwania się ich od M iechow a. W skutek tego obie filie m iecho w itów w itów , tj. w C horzow ie i w B ytom iu, b yły w ramach organizacji wewnętrznej zakonu — p o podziale na 4 prow in cje — zaliczane do p row in cji m ałopolskiej Siedzibą m iechow itów na Spiszu był L e n d a k z gotyckim kościołem z wieku X V . B yć m oże, że pierw szy k ościół w Lendaku był rom ański, gdyż b o żo b ożogrobców . Bytom i Chorzów , sąsiadujące ze sobą m iejscow ości, otrzym ały od K azi z czasów hitlerow skich nie zaprzecza w pływ ów polskich m iechow itów na Spiszu m ierza, syna Władysława O polskiego, ok oło roku 1300 klasztor i szpital dla i zależności od klasztoru w M iechow ie, przytaczając w iadom ość z roku 1313 ubogich ch orych , z bogatym uposażeniem l78. K ościół parafialny w Chorzowie pod wezwaniem św. Marii Magdaleny posia dał p roboszczy-b ożogrob ców z tytułu patronatu klasztoru m iechow skiego. Na Canonicus H ierosolym itanus et Praepositus Mechoviensis de P olon ia ” 183. Poza przedm ieściu Bytom ia, w pobliżu bramy obron n ej, z której prow adził trakt do K rakow a, znajdowała się kaplica szpitala m iechow itów . B ożogrob cy byli w łaścicielam i wsi Chorzowa (H arzów ) i Dąbia, graniczących z Bozbarkiem , (k ościół św. K rzyża). grobcy przybyli tam z M iechowa w roku 1244 >82. Nawet literatura niemiecka 0 wygnanym przełożonym , który nazwany jest w dokum encie „F r. Henricus Lendakiem bożogrobcy m ieli parafie w Łapszach Niżnych i H anuszow cach; w Łapszach Niżnych parafia istniała ju ż od roku 1174 i nie jest w ykluczone, że do tego czasu należy odnieść usadowienie się m iechow itów na Spiszu 184. W roku 1962 przeprow adzone zostały w lesie w Falsztynie pierwsze w ykopy archeologiczno-architektoniczne przez Zakład H istorii A rchitektury Starożytnej 1 Średniow iecznej Politechniki K rakow skiej przy w spółudziale K atedry A rch e o przedm ieściem Bytomia. KOŚCIOŁY NA SPISZU W literaturze h istorycznej utarło się przekonanie, że filie klasztoru m ie chow skiego pow stały na Spiszu d op iero po buncie w ójta Alberta, kiedy w ypędze ni przez Łokietka b ożogrobcy szukali w górach sch ron ien ia 179. Pogląd oparty jest na fak cie, że w ygnany wówczas proboszcz m iechowski H enryk „przebyw ał w r. 1312 na Spiszu, m oże w zamku P alocsy, który posiadał tam biskup kra i?® tam że. 177 Z. K a c z m a r c z y k , Monarchia K azim ierza W ielk iego, Poznań 1947 s. 209. , , 178 Ks. M. W o j t a s , A kia w izytacji dekanatów b ytom skiego i pszczyń sk iego w r. 1598, K atow ice 1938 s. 80— 82. - n r 179 E. D ł u g o p o l s k i , W ładysław Ł o k ietek na tle swoich czasów, Wrocław 1951 s. 146. na dzisiejszej Słow aczyźnie, np. w K ieżm arku logii P olskiej Uniwersytetu Jagiellońskiego. Badania potw ierdziły podanie krą żące wśród tam tejszej ludności o istniejącej tam średniow iecznej osadzie, znisz czonej w czasie najazdu Tatarów w roku 1241 Badania ujaw niły fundam enty m urow anej budow li — kościoła czy klasztoru — które ze w zględu na fragm en taryczne odsłon ięcie nie mogą b yć jeszcze szczegółow o opisane. M iejsce badań 180 tamże. u■S,OM’ ac/ a 1 Słowacy, p od redakcją W ładysława S e m k o w i c z a , K ra ków 1937 t. I s. 102. s c h u r e r - W i e s e, D eu tsch e K unst in der Zips, 1938 s. 149. 183 tamże. IM T. S z y d ł o w s k i , Pow iat n ow otarski, Katal. Zabytlc. Szt. w P olsce, l - I z. U s. 6, 11, 14. ■ Badania prowadził kierow nik Zakładu M. Tobiasz przy w spółpracy rc teologów : Marii Cabalskiej, Olgi H irsch -^ a rch a łow sk iej i Oszywa. (P racocy naukowi K atedry A rch eol. P ol. U J.). znajduje się przy drodze Falsztyn-Niedzica, w pobliżu Frydmana i Łapsz Niż- Paw łow o, Przysieka, Cielisow o, U jazd, Jelem no, Stasin, z jeziorem C ubuchow o, nych. Poniew aż literatura historyczna, pam iętnikarska oraz w izytacje biskupie Łoziny, K on ikow o z jezioram i, młynam i, ogrodam i, sadami i w olnością p o lo nie w spom inają nigdzie o osadzie, kościele w zględnie klasztorze w Falsztynie wania na bobry 189. W roku 1280 darow izna ta została potw ierdzona przez Prze po przeprow adzeniu pierw szych badań źródłow ych nasuwa się przypuszczenie, że jest to m iejsce pierwszego osadnictwa b ożogrob ców — być m oże w „lesie, mysława II i uwolniona od wszelkich ciężarów . W wieku X V I I I m iechow ici w pobliżu D unajca” , którego nie m ógł odszukać Fr. Piekosiński 18,i. O czywiście W roku 1478 — z pow odu nieprzestrzegania obow iązków fundacyjnych w zakresie pracy szpitalnej — doszło do sporu; prepozyt-m iechow ita odm ów i! m iechow ici m ogli na tym terenie działać w oparciu o polską i słow acką ludność i dlatego ich uplasowanie się na tak ważnym szlaku w pływ ów diecezji krakow posiadali ju ż ty lk o: Ujazd, Jezierzany, Z dziech ow o i K onikow o. prawa ju rysdykcji biskupow i Jakubow i ze Sienna i kapitule gnieźnieńskiej. Po m owę polityczną. Przynależność do M iechowa niektórych spiskich parafii z p od pożarze Gniezna w roku 1512 nie odbudow ano szpitala i w roku 1548 kapituła gnieźnieńska nakazała w ypełnianie w oli fundatorów , gdyż k ościół św. Jana nie w ójnym uległ pożarow i. W ob ec oporu prohoszcza-h ożogrobcy i długiego procesu zakoń skiej, sięgających do Lew oczy, Spiskiej K apituły i K ieżm arku, m iało swą w y krzyżem bożogrobców , utrzymana jeszcze tacjach placów ek zakonu, dow odziła w wieku X\ III wysuniętego daleko w w izy na połu dn ie osad czonego karami kościelnym i — za zezw oleniem Zygmunta Augusta odebrano nictwa polskiego, które w czasach późniejszych znalazło się pod panowaniem b ożogrobcom kilka wsi uposażeniow ych. D och ody z nich przeznaczono na zało węgierskim 187. żenie seminarium archidiecezjalnego. W wyniku protestów i interw encji klasz toru m iechow skiego król w roku 1570 cofnął zezw olenie na odebranie fundacji. / B. P row incja W ielkopolska Sprawa przybrała ponow nie ostry ob rót w roku 1600; arcybiskup Karnkowski P row incja ta posiadała szereg placów ek m iechow itów istniejących niemal od uzyskał od króla Zygmunta III W azy zezw olenie na ponow ne przeznaczenie fundacji na cele seminarium. W tedy to opat m iechow ski Fogelw eder zw rócił zarania ich przybycia do Polski. D ow odziło to ich w ielkiej popularności w w ie się o pom oc do kardynała Cinthiusa i ten sprzeciw ił się. W’ roku 1610, proboszcz ku X II wśród w pływ ow ych osób, które bogatym i fundacjam i mogły ich ściągnąć kościoła św. Jana. Tyburcjusz Jaskłowski, założył z własnych — a nie zakonu — do kluczow ych punktów tej dzielnicy. G n i e z n o . Już w latach 1163— 1198 Gniezno posiadało dw ie placów ki. funduszów szpital św. M arty; nie oddał go jednak swym braciom klasztornym lecz pow ierzył opiekę nad nim penitencjarzom gnieźnieńskim. Z końcem wieku K ościół drewniany pod wezwaniem św. Krzyża zbudował poza miastem w roku 1179 biskup lubuski, a ów czesny kanonik gnieźnieński Przecław 188. K ościół się budową drewnianego szpitala w roku 1730, szpital pow stał przy kościele W II kapituła wszczęła pon ow nie spór o fundację szpitalną; spór ten zakończył w jeziorze B ieleckim . Kult dla tego krucyfiksu był zakorzeniony wśród m iejsco św. Jana; trzym ano w nim ubogich poddanych z wsi klasztornych. Sprawa ciąg nęła się dalej przez różne instancje aż w reszcie sejm warszawski w roku 1767 uchwalił zajęcie dóbr b ożogrobców przez arcybiskupa dla w ypełnienia założeń w ej ludności. W chwili zapadnięcia decyzji o likw idacji kościoła bożogrobców , w którym krzyż ten byl um ieszczony, w ybuchły wśród tam tejszej ludności swych delegatów do Rzymu i Św. Rota stanęła po stronie arcybiskupa i k api ten oddany został m iechow itom , którzy w uposażeniu otrzym ali dw ie wsie. W kościele znajdował się krucyfiks owiany legendą wyłow ienia go przez rybaków fundacyjnych. W ob ec dalszego sprzeciwu m iechow itów obie strony wysłały w roku 1829 rozruchy. W wieku X III przenieśli się b ożogrobcy na przedm ieście G rzybow o, gdzie tuły. w ybudow ali klasztor i kościół pod wezwaniem św. Jana Chrzciciela, utrzymując z u bogiej m łodzieży szlacheckiej. Gdy w roku 1804 rząd pruski odebrał m iecho jednak nadal w swym ręku także i kościół św. Krzyża. K ażdorazow y prepozyt- witom ostatnią wioskę — układ ten przestał istnieć. K lasztor gieźnieńskich b o żogrobców zw inięto w roku 1821, w ramach kasaty całego zakonu. Przed kasatą m iechow ita kościoła św. Jana Chrzciciela był kanonikiem kapituły gnieźnień skiej i miał m iejsce w stallach katedry. B ożogrobcy gnieźnieńscy utrzymywali stalą łączność z klasztorem w M iechow ie, prowadząc — zgodnie z regułą — szkolę i szpital, uposażone przez Przemysława i Bolesława, książąt w ielk opol skich w roku 1245. Z wsi uposażeniow ej Kantryn, pow stało w wieku XV miasteczko Grzy b ow o, nazwane tak od proboszcza W acława G rzybow skiego. K ościół św. Jana Chrzciciela otrzym ał w roku 1243 liczne w sie: Z dziech ow o, Lachow o, Im ielno, l8* St. Z a k r z e w s k i, Najdaw niejsze d zieje klasztoru cystersów w S z c z y rzycu. B ozpr. W ydz. Hist. Fil. Ak. Um. t. X L I. s. 55. 187 Zachow ane w skarbcu kościoła m iechow skiego w izytacje b ożogrobców z wieku X V III-g o nazywają kościoły spiskie prow incją węgierską. 188 St. K a r w o w s k i , G niezno, Poznań 1892 s. 173. roku 1780 przyszło do u gody; b ożogrobcy gnieźnieńscy zobow iązali się żywić w szpitalu czterech ubogich i utrzym ywać w szkole rawskiej sześciu w klasztorze gnieźnieńskim mieszkało — oprócz prepozyta — jeszcze najm niej sześciu zakonników , z których dw óch b yło kaznodziejam i. G r o d z i s k W i e l k o p o l s k i — w edług Długosza — był wsią daro waną m iechow itom przez Eudoksję, żonę Mieszka Starego; założono tam klasz tor dla dwunastu bożogrobców , nigdy jednak stan osobow y tej liczby nie osiągnął 19°. Parafia miała istnieć ju ż przed rokiem 1160; z końcem wieku X II kościół miał wezwanie św. M ikołaja. Następny k ościół był drewniany z roku 1566; zachował się z niego obraz Matki B ożej z D zieciątkiem , w barokow ej srebrnej sukience, ufundow anej w roku 1729 przez ks. Floriana B u jdeckiego, 189 tam że. 190 Ks. B u 1 i ń s k i, dz. cyt., s. 489. późniejszego generała w M iechow ie. Zachowana do dziś świątynia została zbu Szwedów i w roku 1889. Na m iejscu drewnianego kościoła b ożogrob ców stanął dowana w latach 1802— 1806 przez m iechow itę ks. Narcyza P a trzyń sk iego1M. Pyzdry. — Przyw ilejem Przemysława ielkopolskiego z roku 1290 w roku 1663 murowany, konsekrow any w roku 1724 199. W kościele zachował otrzym ały fun dację szpitala kierow anego przez m iechow itów 192. K ościółek b o żo kiego; na stopie tego kielicha znajduje się przedstaw ienie jerozolim sk iego Grobu g rob ców uległ ruinie i dlatego w roku 1745 wybudow ali na jeg o m iejscu nowy, Chrystusa. Zachow ały X V I— X V III. zarządzając nim d o kasaty w roku 1819. Jako uposażenie szpitala otrzym ali od Przemyslaw-a praw o now ej lokacji wsi K antrino (K ętrzyn?) z uwolnieniem od wszelkich pow inności na rzecz księcia. S ę p o l n o należało do m iechow itów przed rokiem 1360, w którym wnieśli się k ielich b ożogrobców z roku 1763, ufundowany przez W aw rzyńca K o ry c Wyszogród i się rów nież Rębów resztki bib lioteki m iechow itów z wieku posiadały k ościoły parafialne, nad którym i prawo patronatu oddał m iechow itom , w roku 1320, W acław książę na P łock u , skargę na dziedzica o zalanie ląk dziekana wskutek wybudow ania śluzy przez a potw ierdził pow yższe biskup p łock i F lo r ia n :M. W W yszogrodzie była prepozytura, k tórej podlegała parafia w R ębow ie, wsi królew skiej. Hektora z P a k o ś c i183. Jako zadośćuczynienie za szkodę otrzym ali patronat nad k ościołem św. B artłom ieja z 8 w łókam i ziem i i 4 ogrodam i; wzamian m iecho W ładysława i Bolesława, książąt dobrzyńskich, k ościół i szpital w roku 1321, Rypin, położon y n iedaleko Torunia w ziem i dobrzyńskiej, otrzym ał od 1764 spłonęła do których klasztor w M iechow ie wysyłał swych zakonników 201. Biskup płock i Itaplica m iechow itów nosząca wezwanie św. Krzyża. S i e r a d z posiadał kościół pod wezwaniem św. Ducha wraz z przytułkiem zatw ierdził bogate uposażenie szpitala-przytułku okolicznym i wsiami. M iech o w ici posiadali drewniany k ościółek pod wezwaniem św. Ducha za murami grodu •dla starców i u bogich, którym kasztelan sieradzki, Marcin z K alinow ej, dał ■w roku 1417 wieś G arbów i oddał szpital pod zarząd m iech ow itów ,M. K ról X IV w znieśli kościół pod wezwaniem N. P. Maryi oraz śś. P iotra i Pawła — W ładysław Jagiełło dodał jeszcze dwa lany na przedm ieściu, ale odebrał wieś dwaj m iechow ici Volkm arus i Petrus. M odłów w kaliskim , należącą do bożogrobców . W ą g ł c z e w w Sieradzkiem posiadał k ościół parafialny, ob ok którego ■— D ucha; w roku 1325 oddano je m iechow itom z obediencją i jurysdykcją klasz w latach 1626— 1629 — toru w M iechow ie w ici zobow iązali się do wystawienia młyna i śluzy. W roku rodzina Łubieńskich (Stanisław i M aciej — biskupi, (przyłączon y p o kasacie zakonu d o kościoła parafialnego). Z początkiem wieku Kolo i Sumin otrzym ały k ościoły ze szpitalami p od wezwaniem św. (K oło w d iecezji gnieźnieńskiej, Sumin w diecezji p ło c wany, osadzając w nim m iechow itów (M aciej Łubieński był proboszczem m ie kiej) 202. Fundatorem tej wsi. chow skim ) oraz klasztor dla proboszcza i czterech zakonników a dla uposa żenia tej placów ki nabyła wieś za 12 tysięcy 1,s. W roku 1786 proboszcz tam 1325 tejszy, A ndrzej Skow roński, sprawił piękne organy i chór dla muzyki 196. G o s t y ń od roku 1301 posiadał szpital z fun dacji potom ków M ikołaja, wsiami w ziem i dobrzyńskiej 20s. K ościół b ożogrobców uległ zniszczeniu w czasie w ojen szw edzkich; odbudow ał go proboszcz W ojciech Stecki z Pińczow a. K o syna P rzedpełka, w ojew ody p ozn a ń sk iego197. Drugi kościół gotycki, w ybudo ściół nosił wezwanie św. K rzyża, był przy nim szpitał-przytułek dla czterech wany w wieku X V , posiada wieżę obronną w ysokości 40 m etrów ; być m oże, że osób. Posiadał filialny k ościółek w Szczutow ie. W roku 1822 parafię przyłączono do d iecezji chełm ińskiej. W ojciech — kanonik gnieźnieński, M arcin — jezuita) wystawiła k ościół m uro wybudowana ona została przez b ożogrobców , którzy osiedli tu przed ro Suminie był Franczko Sch cnko, w łaściciel biskup Mazowiecka p ło ck i Florian, uposażając ich pięciom a miała parafię obsadzoną ok o ło roku 1320 przez konwent m iechow ski, uposażoną dziesięcinam i i jedną wsią 2W. P rócz probosz cza mieszkało tam także sześciu zakonników z M iechow a, zajm ujących się pracą C. Prow incja m azowiecka — niegdyś ważne miasto handlow e na Mazowszu — w w pow iecie b rodn ickim , b yło wsią biskupią, w k tórej w roku osadził m iechow itów Rawa kiem 1505. Skaryszów Górzno parafii po pedagogiczną. K ościół ich, pod wezwaniem św. Ducha, p o kasacie zakonu zam ie M iechow ie był najstarszym punktem osadnictwa b ożogrob ców ; w latach 1170— 1187 com es Radosław oddał im miasto p ołożon e nad rzeczką K obylanką niony na ybór w roku 1945. i byli w je g o posiadaniu do roku 1785 98. Po zniszczeniu przez Tatarów , Ska Ł ę g o w i c e otrzym ały w roku 1412 k ościół wystawiony przez arcybiskupa M ikołaja Trąbę, który oddal go m iechow itom 205. ryszów otrzym ał lok a cję na prawie średzkim , w roku 1433 nadanie prawa ewangelicki, uległ zniszczeniu w czasie działań w ojennych m agdeburskiego. Miasto zniszczyły pożary w roku 1655 tj. w czasie najazdu 1.1 K atalog Zabytk. Szt. w P olsce, t. V , pow . pleszewski. 1.2 N a k i e l s k i , ilz. cyt., s. 216. 193 Slotvn. G eogr. K ról. Pol. t. X s. 470. 194 N a k i e l s k i , dz. cyt., s. 401. 195 Slourn. G eogr. K ról. Pol. t. X I I I s. 161. 194 tamże. 197 Slown. G eogr. K ról. P ol. t. II s. 747. 198 N a k i e l s k i , dz. cyt., s. 107. 199 200 201 202 203 204 ■or‘ s. 434. Katal. Zabytk. Szt. w P o lsce, t. III z. 10 s. 38— 40. N a k i e l s k i , dz. cyt., s. 244. tam że, s. 248— 9. tamże. tam że, s. 251. Ks. P ę k a l s k i , dz. cy t., s. 87. J. K o r y t k o w s k i, A d n o ta cje do L iber B en eficioru m Łaskiego, t. I D. Prow incja przemyska Przeworsk otrzym ał pierwszą placów kę m iechow itów w roku 1394, ufundowaną przez biskupa przem yskiego Mateusza 206. Jan z Tarnowa otrzym ał w roku 1394 pozw olenie od Władysława Jagiełły na przekształcenie wsi P rze w orsk na m iasto; w tedy to nastąpiło przekazanie bożogrobcom parafii pod w ez waniem św. K atarzyny. W latach 1430— 1743 w ybudow ali b ożogrobcy gotycki kościół otoczony murami obronnym i z wieżą obronną w ysokości 40 m etrów , charakterystyczną dla budow nictw a tego zakonu 207. D o kościoła należały filia l ne: św. Katarzyny, który był pierwszym k ościołem parafialnym i od roku 1387 stał na Małym Rynku oraz Matki Boskiej Śnieżnej, usytuowany poza murami miasta. W klasztorze, uposażonym bogatym i wsiami, przebyw ało najm niej sześciu zakonników z M iechowa aż do roku 1845, tj. do śm ierci ostatniego proboszcza-bożogrobcy. Leżajsk p o nadaniu mu praw m iejskich w roku 1397, otrzym ał kościół parafialny p od wezwaniem św. T rójcy ufundow any przez W ładysława Jagiełłę 208. W roku 1439 przejęli parafię m iechow ici, utrzym ując ją do roku 1839. Przy kościele parafialnym została wybudowana wysoka w ieża, sześciokondygnacjow a, zwieńczona kopułą z latarnią — typowa dla k ościołów i klasztorów b ożog rob ców w Polsce, Giedlarowa w łańcuckiem , wieś biorąca swą nazwę od sołtysa leżaj skiego Giedlara, fundatora kościoła św. M ikołaja. Po dostatnim uposażeniu pa rafia została oddana — przy w spółudziale W ładysława W arneńczyka — zakon nikom w M iechow ie w roku 1439 209. Rudołowice — Tuligłowy posiadały k ościoły wybudowane przez dziedzica M ikołaja M zurow skiego, którego spadkobiercy niezadow oleni z proboszczy św ieckich oddali ją w zarząd m iechow itom w roku 1446 21°. W T uligłow ach był kościół filialny. W roku 1624 Tatarzy zniszczyli kościół w R u dołow icach , odbudow ał go proboszcz-m iechow ita w roku 1646. Urzejowice, wieś pod Przew orskiem , w łasność Jana Ilk o i M ikołaja Szenko (w spólnie z matką Stachną), posiadała k ościół parafialny, nad którym praw o patronatu oddano b ożogrobcom w roku 1412 211. G n i e w c z y n , wieś w pow iecie łańcuckim , z kościołem drewnianym spa lonym przez Tatarów w roku 1624; kościół odbudow any został przez proboszczym iechow itów do roku 1 7 2 4 212. 206 B i e 1 o w s k i, dz. cyt., t. II s. 886; ks. W ł. S a r n a , Episkopat p rze m yski, Przem yśl 1902 s. 34. 207 F. M ł y n e k i J. B e n b e n e k , P rzew orsk, Warszawa 1960 s. 44. 208 J. D e p t o w s k i, Leżajsk, Warszawa 1959 s. 52. 209 N a k i e l s k i , dz. cyt., s. 463. 210 ta m ie, s. 480. 211 tam że, s. 386. 212 Słown. G eogr. K ról. P ol. t. II s. 626. Biskup Tomasz Nowiński ostatni generał zakonu bożogrobców m iechow skich zmarły w r. 1830. (Z b iory b. opactwa w M iechowie) 14. K O LLEG IA I SZK O ŁY M IE CH O W ITÓW O R A Z ICH PRO FE SO RO W IE Działalność m iechow itów obejm ow ała rów nież szkolnictw o przy parafiach znajdujących się pod icłi zarządem. Prócz szkolnictwa parafialnego bożogrobcy m iechow scy prow adzili kollegia i licea, do których p rofesorów dostarczał klasztor w M iechow ie, wysyła jący wybitniejsze jednostki na studia zagraniczne. B ożogrobcy studiowali przede wszystkim na W szechnicy Jagiellońskiej i nie mal każdy zakonnik wysyłany przez M iechów do objęcia stano wiska proboszcza posiadał tytuł doktora teologii; to samo doty czyło kaznodziejów zakonu. K anonicy m iechowscy wysuwali się więc na czoło zakonów polskich, mając w swym gronie jednostki w ysoko w ykształcone w filo z o fii i teologii. Z tego tytułu die cezje — mimo w yjęcia m iechow itów spod jurysdykcji biskupiej — ubiegały się o obsadzanie probostw zakonnikam i z M iechow a, co można zaobserw ow ać zwłaszcza w wiekach X IV i X V . Tom asz z Olkusza (prepozyt m iechow ski w latach 1508— 1542) zorganizował kollegiutn m iechow skie, w którym kształciła się m ło dzież do stanu duchow nego 2IS. Ustanowił także fundację — zabez pieczoną na dobrach m iechowskich — dla dw óch uczniów kształ cących się na U niwersytecie w K rakow ie i m ieszkających w bursie Jeruzalem, co w roku 1533 potw ierdził biskup krakowski. W roku 1620 kapituła m iechowska postanow iła zapewnić d och od y z W rocim ow ic na kształcenie m iechow itów w K rakow ie, d och od y zaś z Gniezna przeznaczyła na stale wysyłanie zakonników na studia teologiczne do Rzym u; dzięki temu Nakielski doktoryzow ał się w R z y m ie 214. W ielu zakonników nosiło tytuły bakalarzów, mi strzów, d oktorów teologii, filo zo fii i prawa. Łubieńscy, tworząc fundację w W ągłczew ie, ustanowili w roku 1629 warunek p ro wadzenia szkoły pod kierunkiem m iechow ity z ukończonym uni wersytetem w K rakow ie. W Rawie M azow ieckiej — p o kasacie zakonu jezu itów — przekazano m iechow itom prow adzenie kollegium łącznie z jeg o uposażeniem . Przy klasztorze św. Jadwigi na Stradomiu w K rakow ie erygow ano w roku 1744 studium teolo giczne i filozoficzn e. Jeszcze w średniowieczu istniały now icjaty i szkoły klasztorne w Gnieźnie, Przeworsku, na Stradomiu w K rakow ie, p rzygoto wujące — niezależnie od klasztoru w M iechow ie — przyszłych kanoników Stróżów Grobu Chrystusowego. Spośród grona wielu wykształconych i uczonych m iechow itów należy w ym ienić kilku, którzy zasłużyli się dla dobra zakonu; byli nim i: Stanisław S t o i° n, d ok tor dekretów kościelnych, prawa cyw ilnego i kanoniczne8°ł prepozyt m iechow ski w latach 1383— 1395, mianowany ' l3 Ks. G a c k i , 14 tamże. dz. cyt., s. 231. przez patriarchę Ferdynanda wikariuszem i generalnym p rzeło żonym zakonu w prow incjach słow ia ń sk ich '10. Pozostaw ił ręk o pis w ykładający ewangelię na cały rok kościelny. Stanisław B e d a z Łow icza, d ok tór teologii, w ykładający pism o św. w M iechow ie. Na synodzie piotrkow skim (r. 1557) w y głosił do dygnitarzy mowę w obronie K ościoła. Był autorem kilku drukow anych prac z zakresu teologii 216. Samuel N a k i e l s k i z Lelowa (1584— 1652), odbył studia w K rakow ie i w R zym ie; do zakonu m iechow itów wstąpił w roku 1611, był kaznodzieją w Przew orsku, kustoszem i prow incjałem w M iechow ie; od roku 1616 piastował godność rektora klasztoru św. Jadwigi na S trad om iu 217. W ydał dzieła podstawowe dla historii zakonu: Miechovia 1634, D e Antiąuitate Ordinis Canonicorum Custodum SS. Sepulchri Domini Hierosolymitani, 1625. Jakub R a d l i ń s k i , generalny przełożony w M iechow ie, d ok tór teologii, p rofesor w klasztorze m iechow skim ; opublikow ał szereg prac: Norma vitae apostolicae, 1732; Grób Wielkopiątkoivy, Lublin 1730; Corona Urbis et Orbis, K raków 1748; Fundamenta scientiarum, K raków 1755, Speculum S. Augustini ritae apostolicae, K raków 1759 i wiele innych z zakresu teologii i filo z o fii !1. Franciszek P iotr P ę k a l s k i , d ok tór teologii, lektor języków wschodnich na U niwersytecie Jagiellońskim , od roku 1830 p ro fesor nadzwyczajny tej uczelni, rządca kollegium św. Barbary w K rakow ie, Kanonik Grobu Chrystusowego; opublik ow ał: Nabo żeństwo na cześć patronów polskich. K raków 1852; Ż yw ot św. Wojciecha, K raków 1858; O początku, rozkrzewieniu i upadku zakonu X X Kanoników Stróżów Grobu Chrystusowego, K raków 1867 oraz szereg innych prac 219. 13. K A S A T A ZAK O N U M IECH OW ITÓW W RO K U 1819 W roku 1817, biskupi K rólestwa Polskiego p rzedłożyli rządow i memoriał postulujący o kasatę szeregu klasztorów, opactw i świec kich prebend, w związku z pom niejszonym i przez rozbiory i w ojny d ochodam i kurii, kapituł i seminariów 22°. W w yniku petycji, p rzedłożonej przez Aleksandra I ówczesnem u papieżow i, Piu sow i V II, została wydana w roku 1818 bulla upow ażniająca arcy biskupa warszawskiego, Fr. M alczewskiego — jako delegata apo stolskiego — do podjęcia ważnych d ecyzji w sprawie utrzymania, 815 Ks. P ę k a l s k i , dz. cyt., s. 142— 158. 216 tamże. 217 tam że; ks. M. N o w o d w o r s k i , E ncykl. K ościeln a , Warszawa 1883 t. X V s. 451. 218 tamże. 219 tamże. 220 tam że, s. 124— 141. zniesienia czy erek cji instytucji kościelnych. Zadecydow ano kasatę zakonu m iechow itów i w roku 1819 wydelegow ano do M iechowa komisarza, który obliczył d och od y klasztoru na 24.000 zł. poi. rocznie. Ponieważ w tym czasie b yło 12 zakonników i 18 klery ków — rząd wyznaczył 16.000 zł. p oi. na koszty utrzymania 30 osób na rok 1819/20, a resztę p olecił w płacić do kasy rządo wej. Następnie — w drodze licytacji — w ypuszczono na okres dwunastoletni 9 wsi klasztornych, pozostałych z dóbr zakonu. Papież Pius V II dowiedziawszy się, że bulla jego została użyta do generalnej kasaty wszystkich klasztorów i opactw', wysłał breve do ks. H ołow czyca, nominata arcybiskupa, ażeby w-ykonano supresję zakonów zgodnie z zakresem b u lli221. Początkow o postan ow io no pozostaw ić b ożog rob ców w M iechow ie, cystersów w Jędrzejo wie, benedyktynów na Łysej Górze, kartuzów w Gidlach i n orber tanki w Im bram owicach. Dla M iechowa w yznaczono roczny doch ód w kw ocie 17.400 zł. poi. dla przełożon ego, sześciu księży i 8 kle ryków . Poniew aż dobra były skonfiskow ane, brakło finansowych m ożliw ości do cofn ięcia kasaty zakonu. Nastąpiła też likwidacja biblioteki. Biskupi Królestwa P olskiego nic nic otrzym ali, gdyż wszystkie sumy wpływ ające z kasaty zakonów przeszły do kasy skarbu ogólnego. Rząd pruski wykorzystał kasatę do zlikw idow a nia b ożog rob ców na terenie sw ojego zaboru zabierając im dobra i majątki. W zaborze austriackim początkow o zakony pozostały nienaruszone, a to: w Przeworsku, Leżajsku i kollegium św. Bar bary w K rakow ie (p ojezu ick ie). Senat W olnego miasta Krakow a — p o kom isyjnym zbadaniu sprawy kollegium przy św. Barbarze — przystąpił do likw idacji m iechow itów i zajął budynek wr roku 1839, oddając go U niwersytetowi w roku 1842. W Przeworsku i w Leżajsku — p o wym arciu proboszczów -m iechowritów i pom im o żyjących jeszcze zakonników — obsadzono te parafie duchownym i diecezjalnym i, likw idując ostatecznie pozostałe klasztory b o żo grobców . W M iechow ie do roku 1830 pozostał na prepozyturze ostatni generał m iechow itów , biskup Tom asz N owiński; p o jego śmierci był jeszcze — do roku 1852 — proboszczem Baltazar Chwalbiński, kanonik-tniechowita. Od jeg o śm ierci proboszczam i m iechow skim i byli księża diecezjalni. ZAK O N CZE N IE Dziwnym zbiegiem ok oliczn ości do dzisiaj pozostało wiele miejsc zabytkow ych w Z iem i Świętej bez op iek i i tylko znaki p o dw ójnego krzyża bożog rob ców wskazują, że niegdyś działali tam przedstawiciele Stróżów G robu Chrystusowego. Charakterystyczny tamże. przykład stanowi inaly, opuszczony k ościółek na nadbrzeżnej skale jeziora Genezaret, u stóp góry Ośmiu Błogosław ieństw. Skała wewnątrz kościółka miała b yć m iejscem nauk Chrystusowych. Dzisiejsze rozdarcie Jerozolim y na część arabską i izraelską, roz dzielone pasem ziem i zniszczonej przez działania w ojenne, nasuwa przypuszczenie, że reaktywow anie bożog rob ców na terenie B liskie go W schodu dla idei, dla której zostali pow ołani — m iałoby pełne uzasadnienie historyczne i służyłoby m iłośnikom zabytków wszyst kich wyznań, z uwzględnieniem opieki samarytańskiej nad piel grzymami ze świata chrześcijańskiego. Dawne opactw o w M iechow ie, owiane bogatą przeszłością i ideą służby p odw ójn ego czerw onego krzyża dla bliźnich, dzięki opiece władz duchow nych dźwiga się z ruiny i jest w idom ym zna kiem powiązania w idei m iłosierdzia oraz m iłości bliźniego Polski z Bliskim W schodem . EUGENIUSZ BAU TRO POGLĄD MISTRZA WINCENTEGO K A D Ł L B K A NA STOSUNEK W Ł A D Z Y DUCHOWNEJ I ŚWIECKIEJ Zagadnienie stosunku władzy duchow nej ( sacerdotium) do świeckiej ( imperium , regnum) w ypełniało w znacznej części p oli tyczną myśl i akcję średniowiecza, w yczerpując się w polem ikach często jałow ych i m onotonnych, a w każdym razie nazbyt długo trwałych i drobiazgow ych. Cechą ogólną tych k on cep cji było to, że m yśliciele tej epoki p odzielili się na dwa ob ozy : kościelny (pa pieski, kurialiści, kanoniści) i państwowy (św iecki, laicki, cesar ski, im perialiści, regaliści, legiści), które zrazu zwalczały się spo kojnie i łagodnie, a późn iej — w miarę, jak na w idow ni politycznohistorycznej zatarg obu potęg ów czesnych: duchow nej i świeckiej stopniował się i przybierał postać walki zaciętej — we form ie ostrej i bezw zględnej. Każde silniejsze wystąpienie przedstawicieli obu władz: papieża i cesarza (późn iej króla fran cuskiego) odbijało się echem w literaturze, w yw ołując p o to k pism p olitycznopolem icznych, z których każde starało się stanąć w o b ro nie tez sw ojego obozu i je uzasadnić, a zbić twierdzenia i argu menty strony przeciw nej. Cała ta literatura uzyskała cechę okazyjności, poniew aż powstawała i mnożyła się z okazji pociągnięć historycznych papieży w zględnie cesarzy lub królów . Zatem m o ment aktualności, jak zresztą we wszystkich pismach p olem icz nych, nadał wszystkiemu piętno nazbyt w ybitne, a przez to i szkodliwe. Walka obu obozów toczyła się o to, która z obu p otęg ma m ieć zw ierzchnie władztwo, a w ięc, czy papież czy cesarz (k ró ł). Obie strony w ych odziły z principium unitatis et totalitatis, rzą dzącego światem, a w ięc i społecznością ludzi, ale inaczej inter pretowały tę zasadę. Każda z nich operowała argumentami, za czerpniętym i z Pisma św. i dzieł O jców K ościoła oraz posługiwała SIę oficjaln ym i enuncjacjam i papieży w zględnie cesarzy, tylko
https://openalex.org/W2115302092	https://zenodo.org/record/2261975/files/article.pdf	English	null	THE MIDLAND RAILWAY COMPANY'S HARBOUR AT HEYSHAM, LANCASHIRE (INCLUDING APPENDIX AND PLATE).	Minutes of proceedings	1,906	public-domain	7,624	229 229 Papers.] ABERNETHY ON HEYSHAN HARBOUR. ABERNETHY ON HEYSHAN HARBOUR. Papers.] ( P a p No. 3635.) ( P a p No. 3635.) Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. By GEORGE NEILL ABERNETHY, M. Inst. C.E. When the embankments were completed and consolidated, the space left under the timber viaduct was closed by successive horizontal layers of rock and clay until high-water of neap-tides was reachetl, after which the space was tipped up to the full height of the embankment as rapidly as possible and the water was drawn off from the inclosed area through the sluice-pipe in the temporary dam. These precautions proved effectual, and by April 1899 the sea, had been entirely excluded from the inclosed area. For drainage purposes during the progress of the works, the contractors excavated a. sump in the sandstone rock at the eastern end of the harbour, and erected two rocker-pumps. The water was conducted from these pumps by shoots and discharged over embankment No. 2. Little The first work undertaken was the construction of two embank- ments, or breakwaters, Nos. 1 and 2, to inclose and reclaim the site of the harbour ; the material for these embankments, composed of fairly soft sandstone rock, with some clay and shaley marl, was obtained from Heysham banks, the high ground immediately ad- joining the site of the works. The progress of these embankments was at first slow, owing to the difficulty of obtaining the material in sufficient quantity and of getting a good face on the rock, and also owing to the fact that at high-water the face of the cliff was washed by the tide. I n constructing the embankments, a trench was first cut in the sand and filled with sandstone rock to form a toe, and to prevent erosion by the sea ; two rock-tips were then started on each embankment, the space between the tips being filled with clay and mar1 ; but later on, and after getting farther seaward, it was found better and more expeditious to tip a single rock-bank from two lines of rails, and to side-tip the clay and marl on the inner face. Pro- vision had also to be made for protecting the heads of these tips during stormy weather by keeping in reserve a set of wagons filled with stone ; with the aid of these wagons damage to the rock-tips during the construction of the works was almost entirely prevented. Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. By GEORGE NEILL ABERNETHY, M. Inst. C.E. By GEORGE NEILL ABERNETHY, M. Inst. C.E. TOWARDS the end of the year 1891, the late Mr. James Abernethy, Past-President Inst. C.E., was consulted by the Midland Railn-ay Company as to the construction of a dock at Heysham, in Morecambe Bay, in connection with their railway system, and in his preliminary and subsequent reports was assisted by the Author, then in partner- ship with him. The Midland Railway Compzny had for years experienced difficulty, and were incurring considerable expense, in maintaining their existing pier- and. harbour-accommodation at Morecambe, where it was only possible for steamers and vessels to enter and leave the harbour near the period of high-water ; more- over, the channel leading to Morecambe was difficult of navigation. Mr. Abernethy was impressed with the natural advantages which the proposed site afforded, as being in close proximity to Heysham Lake, a deep-water channel having a depth of 40 feet at low-water of ordinary spring-tides. The scheme first proposed was a dock of some 30 acres in area, with a lock and entrance, situated in much the same position as the present breakwater-heads ; but in 1895, after further consideration of the project with the Company’s Engineer, the late Mr. J. Allen McDonald, M. Inst. C.E., it was decided, in view of the particular nature of the traffic to be dealt with, to alter the design to that of a tidal harbour, the site being inclosed by embankments practically in the position indicated on the general plan, Fig. 1, Plate 4. g I n 1896 the Midland Railway Company obtained an Act for the construction of the harbour. I n March of that year Mr. Janlei; Abernethy died, and the Author was appointed by the Midland Railway Company Joint Engineer with the late Mr. J. Allen McDonald, the Company’s Chief Engineer. The preparation of the det,ailed plans was at once commenced, and in July 1897 the contract Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. 230 ABERNETHY ON IIEYSHAM HARBOUR. [Selected was put out to tender and was let to Messrs. Price and Wills, of Westminster. was put out to tender and was let to Messrs. Price and Wills, of Westminster. The first work undertaken was the construction of two embank- ments, or breakwaters, Nos. By GEORGE NEILL ABERNETHY, M. Inst. C.E. 1 and 2, to inclose and reclaim the site of the harbour ; the material for these embankments, composed of fairly soft sandstone rock, with some clay and shaley marl, was obtained from Heysham banks, the high ground immediately ad- joining the site of the works. The progress of these embankments was at first slow, owing to the difficulty of obtaining the material in sufficient quantity and of getting a good face on the rock, and also owing to the fact that at high-water the face of the cliff was washed by the tide. I n constructing the embankments, a trench was first cut in the sand and filled with sandstone rock to form a toe, and to prevent erosion by the sea ; two rock-tips were then started on each embankment, the space between the tips being filled with clay and mar1 ; but later on, and after getting farther seaward, it was found better and more expeditious to tip a single rock-bank from two lines of rails, and to side-tip the clay and marl on the inner face. Pro- vision had also to be made for protecting the heads of these tips during stormy weather by keeping in reserve a set of wagons filled with stone ; with the aid of these wagons damage to the rock-tips during the construction of the works was almost entirely prevented. The area inclosed by the embankments is 150 acres, and being all on tidal land, with a range of tide of about 20 feet, provision had to be made for the ebb and flow of the tide as the tips approached each other. This was effected by the construction of a timber viaduct having a clear opening of 300 feet, at a distance of 2,300 feet from the Near Naze, this point being selected as being the most shelteretl. A temporary dam of clay, with a heavy rough rock facing, was tipped across the site of the entrance, a 6-foot pipe with sluice-valves being laid through the dam for draining the area when finally closed. By GEORGE NEILL ABERNETHY, M. Inst. C.E. The area inclosed by the embankments is 150 acres, and being all on tidal land, with a range of tide of about 20 feet, provision had to be made for the ebb and flow of the tide as the tips approached each other. This was effected by the construction of a timber viaduct having a clear opening of 300 feet, at a distance of 2,300 feet from the Near Naze, this point being selected as being the most shelteretl. A temporary dam of clay, with a heavy rough rock facing, was tipped across the site of the entrance, a 6-foot pipe with sluice-valves being laid through the dam for draining the area when finally closed. When the embankments were completed and consolidated, the space left under the timber viaduct was closed by successive horizontal layers of rock and clay until high-water of neap-tides was reachetl, after which the space was tipped up to the full height of the embankment as rapidly as possible and the water was drawn off from the inclosed area through the sluice-pipe in the temporary dam. These precautions proved effectual, and by April 1899 the sea, had been entirely excluded from the inclosed area. For drainage purposes during the progress of the works, the contractors excavated a. sump in the sandstone rock at the eastern end of the harbour, and erected two rocker-pumps. The water was conducted from these pumps by shoots and discharged over embankment No. 2. Little 231 Papers.] Papers.] ABERNETHY ON HETSHAhI HARBOUR. difficulty was experienced in dealing with the water, and the site remained remarkably dry throughout. difficulty was experienced in dealing with the water, and the site remained remarkably dry throughout. y y g Excavation in the harbour ,was then commenced, the material taken out being used for completing the embankments to their finished width. In the first instance they were 30 feet in width at the top, and the material was side-tipped until a width of 600 feet at the root and 170 feet at the ends was obtained. It was then found advisable to heighten the outer embankment from 10 feet to 14 feet above high-water of ordinary spring-tides as a further protection against storms and extraordinarily high tides. The original proposal was for a depth of w-ater within the harbour of 7 feet below low-water of ordinary spring-tides, but subsequently this wa,s increased to 14 feet. By GEORGE NEILL ABERNETHY, M. Inst. C.E. The harbour-works consist of a South quay 1,600 feet in length, a North quay 900 feet in length, and a fish-stage 300 feet in length. The east end of the harbour is in the solid rock, finished with a 3-to-1 slope, and that portion between the end of the North quay and the seaward end of the embankment is a tipped rock-slope with :I hand-packed rough rubble face. The original contract-plans comprised the construction of a wharf, 600 feet in length, on the inner face of embankment No. 3, and a central pier, 900 feet by 150 feet, to accommodate the passenger-service, but after much consideration by the traffic department this central pier was abandoned and provision for the passenger-traffic was made by increasing the length of the quay at embankment No. 3, and by the construction of the North quay, It was originally intended that the foundations of the walls should be put in by means of timbered trenches, but shortly after the harbour excavation was commenced it \vas found that the material stood with such a steep face that it was possible to include the foundations of the South and Nort,ll quays in the harbour excavation, and to dispense with timbered trenches. The walls are constructed of 7-to-1 Portland-cement concrete with a 12-inch face of 5-to-1 concrete, the material used being fine ballast from Walney Island ancl selected stone from Heysham banks. In the larger sections of the walls, sandstone plums were placed, precautions being taken to ensure each plum being sufficiently surrounded with concrete. All the walls have a coping of granite. p g g South Qttny, on EmbanLtnetlt No. I.-As this quay >%-as intended to be used for the passenger- and cattle-traftic from Ireland, and for the Isle of Man steamers, provision had to be made for landing con- veniently at any state of the tide, the rise and fall of which is 27 feet, and as a t this time there had been a change in the Managership Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. 232 ABERSETHY ON IIEPSIIAJI IIACBOUR. By GEORGE NEILL ABERNETHY, M. Inst. C.E. [Selected [Selected of the Company, which involved some alterations in the traffic requirements, openings were left in the wall a t intervals to provide for the subsequent construction of the passenger- and cattle-subways. A heavy timber staging was constructed along the whole face of the quay, with continuous landings to give access from the steamers to the subways, and also to take the impact of steamers coming along- side and to minimize the risk of damage either to them or to the wall itself. The staging was constructed of pitch-pine piles, 12 inches by 12 inches, with front fenders of Karri timber, the whole being tied by diagonal and cross bracing, and secured to wrought-iron brackets previously built into the wall. The foot of each upright rests in cast-iron socket built into the concrete toe of the wall; the whole staging is well and thoroughly tied back into the wharf by long bolts securing the wrought-iron brackets on which the cross-timbers rest. In view of future extension as the traffic develops, it was decided to construct a lock-entrance, 80 feet in width, with a caisson- sill and sluices, immediately beyond the end of the South quay, with a quay-wall, now called the west wall, extending up to the end of roundhead No. 1. This entrance has been closed by a temporary dam. Beyond the South quay the sandstone rock dipped mpidly, and the whole of the west wall foundations had to be constructed in timbered trenches. North Quay, on Embanknwnt No. 2.-On the opposite side of the harbour the North quay was constructed. This quay being almost exclusively used for goods from Cork and from Scotland, it was not thought necessary to provide extensive accommodation for passengers, and accordingly the front staging was dispensed with, and Karri fender-piles were bolted to the face of the quay for steamers to lie against. A single subway for passengers was provided, to afford convenient access at about half-tide level. Romdheads.-Simultaneously with the formation of these wharved, the construction of two roundheads was commenced. From the result of the horings which had been taken, it was supposed that the clay strata met with at a depth of 10 feet below the harbour-bottom was sufficiently good to form a secure foundation ; accordingly the whole area of these heads and wing-walls was inclosed by close timber piling, and excavation was started inside. Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. By GEORGE NEILL ABERNETHY, M. Inst. C.E. It was found, after going to a depth of 20 feet, that it was impossible to carry out this work as originally intended, the running sand and silt rising up in the bottom of the trench as fast as it could be taken out; it was therefore decidwi to sink monoliths, and one of these was accordingly put down a t roundhead No. 1 ; this monolith was 17 feet in width by 24 feet in length, and was built of concrete On a steel shoe, The ABERNETHY ON IIEYSIIABI IIARBOUB. 233 Papers. J Papers. J steel shoe was constructed by Messrs. Handyside and Company, of Derby, and weighed 8 tons. The sinking of this monolith was effected without much difficulty by excavating with a Priestman grab and by hand-labour inside ; when the solid rock was reached the bottom was concretecl up for a thickness of 12 feet, and the remaining portion was filled with sand to within 8 feet of the top, when concrete W;LS again employecl. It had been intended to sink a number of these monoliths, side by side, over the whole area of the roundhead, but owing to the satisfactory manner in which the first had gone down it was decided to form each roundhead of a single circular monolith. These were the largest monoliths which had ever been used, and were constructed in the following manner :- A steel shoe was first built up of plates, angle-bars and tee-bars, stiffened at a height of 3 feet 3 inches above the cutting-edge by gusset-plates and angle-bars ; the shoe was 55 feet in diameter over the skin-plates, the circumferential sections being 8 feet in depth and 8 feet in width at the top. Owing to the great weight wllicll this shoe had to carry, special gusset-plates and angle-bars were used for stiffening the cutting-edge, with intermediate stiffeners of lighter scantling between the main bays, which were spaced 4 feet apart. The outer edge of the shoe was plated for a depth of 3 feet 3 inches ; the remainder, after being shuttered externally, was filled with 3-to-1 concrete preparatory to sinking. Brickwork in 2-to-l cement- mortar was then built on the shoe, this being found more convenient and more expeditious than concrete, whilst it also admitted of great weight being brought to bear on any particular part of the monolith where found necessary. Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. By GEORGE NEILL ABERNETHY, M. Inst. C.E. As the brickwork proceeded, the material (generally sand) was excavated from the interior partly by grabs bu chiefly by manual labour, as it was found, especially at the greater depth, that the sand was too hard for the grab to grip properly. The monoliths are divided into four divisions or compartments by internal cross-walls, carried on girders attached to the upper portion of the steel shoe a t such a height that men could pass from one compartment to the other. Pumps were kept working in each compartment. Rock was reached at a depth of 42 feet G inches below the level of the harbour-bottom, the tohl depth sunk through the sand being 75 feet. The lower portion of the monolith WRS then sealed with G-to-l concrete to a height of about 15 feet, after which sand was employed to fill the interior LIP to the level of the harbour bottom. A topping of 13 feet of concrete was then added, and 011 this the upper portion of the roundhead was constructed, of G-to-1 concrete within shuttering. These circular monoliths are 55 feet in diameter and 75 feet in depth, the weight of the steelwork being 73 234 ADERFETIIY ON HEYSHAN IIARBOUE. [Selected tons, and the total weight of the monolith, with the brickwork completed, 6,000 tons. It was found that when this large monolith had been sunk to the rock it was only 5 inches out of the perpen- dicular, and that sunk on the site of roundhead No. 2 was almost equally satisfactory. Temporary Lighti?zg.-On the commencement of the works, two temporary timber dolphins were erected at the low-water line of the entrance-channel for the purpose of lighting, as required by the Trinity Board. On the completion of the works these dolphins were removed and a Pintsch gas-buoy, to burn for 3 months, was placed on the side of the south dolphin. Lighthouses.-A lighthouse has been erected on the southern roundhead, not only to indicate the entrance to the harbour, but also to guide vessels up Heysham Lake, and for this purpose a leading lighthouse has been placed on the Near Naze. The lighthouse on the roundhead is constructed of cast-iron plates f inch in thickness ; the focal plane of the optical apparatus is 16 feet 4 i inches above the surface of the roundhead, and 30 feet 4 inches above high-water of ordinary spring-tides. Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. By GEORGE NEILL ABERNETHY, M. Inst. C.E. The tower is 6 feet 10 inches in diameter at the top, and 8 feet 11 inches at the base. An outer gallery is fixed on cast-iron brackets and provided with a balcony. The lantern-floor consists of chequered iron plates supported on rolled steel joists. The tower is secured by sixteen holding-down bolts, 13 inch in diameter by 6 feet in length, with cast-iron anchor-plates 12 inches square. The lantern is 6 feet 12 inch in internal diameter and consists of a cast-iron pedestal with gun-metal ventilators. The glazing is framed in gun-metal helically-inclined frames. The dome and ball-ventilator are of copper. The illuminating apparatus consists: of a fixed dioptric lens showing over an arc of 360°, with a vertical aperture of SOo. The illuminant is gas, with tt two-wick capillary oil-burner as a stand-by. The leading light on the Near Naze is carried on an openwork structure giving a height of 46 feet from the level of the concrete base, and a height of 66 feet 6 inches from high-water to the focal plane of the optical apparatus. The concrete base for the structure is built on the Naze Rocks, and the tower is built into this base to a depth of 10 feet. The structure consists of four solid iron columns, 5 inches in diameter, with cast- steel connecting-sockets. The horizonta,l rolled-joist bracings are connected to the columns and steel sockets by wrought-iron straps. The diagonal bracing is of round iron with screw-couplings. The lantern floor, intermediate landings, and ladders are of wrought iron. The lantern is 5 feet in diameter, and consists of a mild steel pedestal, copper roof and ventilator. The illuminating apparatus ABERNETHY ON HEYSHAhf HARBOUR. 235 Papers.] consists of a holophote, with a refracting lens of 80" vertical and horizontal angle, with two reflecting prisms on the outside. The illuminant is gas, and the storage gas-holders are on the concrete foundation. The angle of illumination given by the lens is S r , which acts as a leading light in connection with the light on the roundhead. The lighthouses were supplied and erected by Messrs. Chance Brothers, of Birmingham. Passenger-Station and Gooda-Sheda.-The passenger-station has an island-platform, 600 feet in length and 47 feet in width, with a line of rails on each side, within the building ; the platform is covered for a length of 450 feet, the roof being 75 feet in width. Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. By GEORGE NEILL ABERNETHY, M. Inst. C.E. A suite of waiting- and refreshment-rooms has been constructed along the centre of the platform. For the convenience of passengers, luggage- hoists hare been provided from each of the landing-stages, at various levels to suit the state of the tide. As the sheds for the arrange- ment of goods for transit are situated close to the quay, with the passenger-station beyond, overhead gangways have been Constructed across the goods-sheds into the passenger-station, and the stairways from each landing-stage, as well as the hoists, communicate directly with this overhead gangway. Passengers will thus be completely protected from the weather. p The goods-station is nearest to the quay, and has a length of BOO feet, there being ground mailable for a further extension of 100 feet. A second goods-shed has been constructed, having a length of 175 feet. The distance between the buildings and the front edge of the quay is 27 feet 6 inches, which is sufficient for the working of large cranes. The crane-legs on one side travel on rails laid on the quay, and those on the other side on rails supported on the stanchions of the goods-station. The rolling-stock of the Midland Railway can ettsily pass along the quays under the cranes, the width between the centres of the crane-rails being 31 feet 2 inches. I n connection with the design of the buildings, serious considera- tion had to be given to the question of wind-pressure, as the harbour is exposed to south-westerly gales. An anemometer-tower was erected on the Near Naze, with complete apparatus for taking records, including a Beckley anemometer. This instrument, on the 26th February, 1903, when a train was blown off the Leven Viaduct on the opposite shore of Morecambe Bay, recorded a velocity of 100 miles an hour. It was decided to construct the buildings of steel framing with wooden boarding and with an exceptionally flat roof, the rise being G feet 6 inches, equal to about 1 in 2. It was also deemed important to limit the load on the foundations. So far as roofs and walls are concerned there is little difference between the ABERNETIIY ON IIEYSHAM IIARllOUII. 236 [Selected goods- and passenger-stations. The span of the goods-station is 59 feet, and of the passenger-station 75 feet. The whole of the framework of the goods-station is carried on steel stanchions. By GEORGE NEILL ABERNETHY, M. Inst. C.E. Each foundation consists of a block of concrete, 7 1 feet by 4 feet, with base-plates, and four 12-inch lewis-bolts for holding down the foundation-girder of the stanchions, which is 7 feet in length and is built of plates 12 inches in depth, with l2-inch flanges. To this base-girder are riveted the vertical members forming the stanchions, so arranged that the width at the base is 6 feet, tapering to 18 inches just below quay-level. Above this the stanchion continues 18 inches in width to the top. On the t,op of each stanchion is a bracket, to which is secured the continuous longitudinal girder supporting the rails on which the wheels on one side of the gantry-crane run, the wheels on the other side travelling on rails on the quay. Between the stanchions there is steel bracing carrying timbers forming the side of the building. The stanchions carry the transverse girders supporting the roof-principals. These girders are 59 feet in length over all and are of the ordinary lattice- type, 4 feet in depth. The wind-bracing is specially strong, there being 1;-inch diagonal tie-rods with screw-couplings through each girder. The girders are spaced at intervals of 25 feet, which fixed the span of the roof-principals. These are transverse to the line of the main girders and therefore parallel to the longitudinal line of the shed. There are six principals to the width of the building to each span. They are of simple construction, being built of tee-bars and tie-rods. The covering over the good-sheds is entirely of zinc, but the central part of each span in the passenger-station roof is glazed, and the side partst are of zinc. The end of the glazing over- laps the zinc at a height of l foot to provide ventilation, a suitable lip- covering being arranged to prevent the rain driving into the interio Entrance-Channel.-After the water was admitted into the harbour, the temporary dam at the entrance had to be removed ; the upper portion was taken away by means of grabs and steam-cranes, ancl when the water in the harbour had reached sea-level the work was continued by dredging. The entrance-channel was then dredged to R depth of 17 feet at low-water of ordinary spring-tides, extending from the harbour to the low-water line in Heysham Lake. By GEORGE NEILL ABERNETHY, M. Inst. C.E. These stanchions are founded on concrete carried to a depth of 21 feet below quay-level. Each foundation consists of a block of concrete, 7 1 feet by 4 feet, with base-plates, and four 12-inch lewis-bolts for holding down the foundation-girder of the stanchions, which is 7 feet in length and is built of plates 12 inches in depth, with l2-inch flanges. To this base-girder are riveted the vertical members forming the stanchions, so arranged that the width at the base is 6 feet, tapering to 18 inches just below quay-level. Above this the stanchion continues 18 inches in width to the top. On the t,op of each stanchion is a bracket, to which is secured the continuous longitudinal girder supporting the rails on which the wheels on one side of the gantry-crane run, the wheels on the other side travelling on rails on the quay. Between the stanchions there is steel bracing carrying timbers forming the side of the building. The stanchions carry the transverse girders supporting the roof-principals. These girders are 59 feet in length over all and are of the ordinary lattice- type, 4 feet in depth. The wind-bracing is specially strong, there being 1;-inch diagonal tie-rods with screw-couplings through each girder. The girders are spaced at intervals of 25 feet, which fixed the span of the roof-principals. These are transverse to the line of the main girders and therefore parallel to the longitudinal line of the shed. There are six principals to the width of the building to each span. They are of simple construction, being built of tee-bars and tie-rods. The covering over the good-sheds is entirely of zinc, but the central part of each span in the passenger-station roof is glazed, and the side partst are of zinc. The end of the glazing over- laps the zinc at a height of l foot to provide ventilation, a suitable lip- covering being arranged to prevent the rain driving into the interio E t Ch l Aft th t d itt d i t th h b goods- and passenger-stations. The span of the goods-station is 59 feet, and of the passenger-station 75 feet. The whole of the framework of the goods-station is carried on steel stanchions. These stanchions are founded on concrete carried to a depth of 21 feet below quay-level. By GEORGE NEILL ABERNETHY, M. Inst. C.E. In forming this entrance-channel 1,115,148 cubic yards of material were removed, the plant used being tm-o bucket-ladder dredgers; two sand-pumps, the ca.pacity of each hopper being 500 cubic yards ; two steam-hoppers, each of 500 cubic yards capacity ; and two dnmb- hoppers, each of 250 cubic yards capacity. Papers.] Papers.] Papers.] Contractors’ Accommodation.for the Workmen.-At the time the works were commenced Heysham was a small village, inhabited only by a few agriculturists, so that the contractors had to make arrange- ments for the accommodation of their workmen. They accordinglg constructed a village of lmts, known by the name of “ Klondyke,” consisting of three large huts, each accommodating seventy-five men ; forty-five small huts, each taking a hut-keeper and his family, and twelve men ; n cmteen and recreation-room, 90 feet in length by GO feet in width ; butcher’s, baker’s, clothier’s, and barber’s shops ; :I police office, etc. ; as well as a, special bakery capable of supplying six hundred loaves per day, and a large cafe where, besides the m u d groceries, hot meals were served to the men. All the buildings, with the exception of the hts, were lighted by acetylene ens. When in full swing there were about seven hundred men living at this settlement. In addition to the foregoing, an enterprising resident in the neighbourhood erected a collection of huts further inland, which was distinguished by the name of “ Dawson City.” Contractors’ Workshops and Plant.-A list of the workshops erected hy the contractors on the site, and of the machinery employed in carrying out the works, is given in the Appendix. Blond-Gas Electsic Pozoer Installation.-Electricity is employed for practically all the machinery. Not only is it claimed that electricity effects considerable economies in working as compared with llydraulic and steam-power for the actuation of the cranes, capstans, lifts, pumps, etc., which constitute the bulk of the power-using machinery in harbour-works, but it imparts an elasticity to the power-distribution scheme otherwise unobtainable. Where exten- sions are probable this consideration naturally has much weight, and no doubt it did much to influence the Midland Railway Company in favour of the motor drive. Mond-gas is used for the engines and is generated in a producer-plant constructed by the Power Gas Corporation, Limited. The air-blast for producing the gas is delivered by a Roots blower, or in cases of emergency by a Kiirting blower, and after passing along the air-main enters at the bottom of the air-tower. In this tower the air comes into intimate contact with the water, which has been heated by the gas cooling- tower, and is thereby saturated with water-vapour at the temperatur at which it leaves the latter. Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. 237 AbEkNBTHY ON B$Y SHAM fIhRj3OUiL Papers.] The air-tower is packed with circular earthenware tiles down which the water trickles, meeting the air as it rises to the top. It leaves the air-tower at the outlet and after passing along the air-drain, where it receives a further supply of steam from the exhaust of the engine driving the auxiliary ABER%PI'HY 09 HEYSHAhf HARBOUR. 238 [Selected [Selected machinery, it enters the external pipe of the Superheater. The mperheater consists of two legs, each having an internal and an external pipe ; the hot blast passes through the annular space between the two pipes, and is therein superheated by means of the hot gas which is passing in the opposite direction from the producer through the internal pipe of the superheater. The superheater blast then enters the producer. The producer is cylindrical in shape, and consists of an external and an internal wrought-iron shell. The internal shell is lined with fire-brick as far down as the fire-box and has the shape of an inverted cone at the bottom ; the external shell, however, is cylindrical down to the water-seal, where it is luted with water. In the upper part of the producer, immediately beneath the fuel-hopper, is an internal bell, which, being kept full of fuel, automatically maintains the proper height of fuel in the body of the producer, and thus assists in maintaining uniformity in the composition of the gas. The bell further obviates the serious fluctuation in pressure throughout the plant which would be brought about if the fuel were dropped straight into the producer, and by providing for partial distillation of the " green " fuel before it enters the main body of the producer, reduces, to some extent, the amount of tar formed. Each producer is further provided with four poke-holes for poking into the interior of the bell. A circular platform is provided for working the coal feeding-apparatus and the poke-holes. The blast entering the producer from the superheater passes all round the annular space between the internal and externa shells and enters the interior at the bottom. The resultant gas, under ordinary circumstances, when working on a good load and consuming suitable Nottinghamshire or Lancashire slack, will have approximately the following composition :- Carbonic acid (CO,) . . . . . . . 12 per cent. Carbonic oxide (CO) . . . . . . . 17 .... Hydrogen (H) . . . Papers.] . . . . . . 21 . . . . Marsh gas (CH,) . . . . . . . . 2 . . . . Nitrogen (N) . . . . . . . . . 48 . . . . -- 100 The gas, having deposited a great part of its suspended dust in the superheaters, enters the collecting-main, where more dust is separated, being removed daily from the dust-boxes provided at the bottom. From the collecting-main the gas passes into the washer, rectangular in shape, in which revolves a dasher fitted with blades which throw fine spray into the gas as it passes through. The remainder of the dust in the gas is thus thrown out and deposited in the water. The Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. ABERNETHY 09 IIEPSHBM HARBOUR. 239 Papers.] dust which sinks to the bottom of the mater may be removed, while the plant is working, by means of rakes and scoops provided for the purpose. From the washer the gas enters the bottom of the gas- cooling tower. The gas passing up the tower comes into intimate contact, on the way, with water trickling down, and gives up its heat to the cooling-water, which is then pumped to the heating-tower, in which it is cooled by the incoming air. From the cooling-tower the gas passes to the gas-holder. After leaving the holder the gas enters a fan in which it is dashed with great force against the sides, and the remaining tar is thus thrown out. The fan forces the gas through a sawdust scrubber which is packed with coarse shavings and sawdust. In this apparatus the remaining particles of tar are removed, and the gas is left in a pure condition ready for consump- tion in the engines. The coal used in the producers is bituminous slack. The slack is dumped from railway-wagons directly into the elevator-boot, and is raised by a bucket-conveyor which deposits it in a horizontal spiral conveyor running across the tops of the bunkers above the producers. Each bunker holds 10 tons. The conveyors are driven by a 73-HP. Westinghouse motor running at 1,850 revoln- tions per minute. From the scrubber the gas passes into the gas- main, which runs outside the engine-house, alongside the three 250-HP. gas-engines. The engines are of the Westinghouse vertical single-acting pattern, and run at the comparatively high speed of 200 revolutions per minute. Each gas-engine is direct-coupled to a 150-kilowatt continuous-current generator of standard Westinghouse pattern, giving a current of 325 amperes at 430 to 460 volts when working at normal speed. The storage-battery consists of two sets, each of 115 cells. Each cell has a minimum capacity of 180 amperes for G hours, with a final voltage of 1 -85 volt. Lighting.-The landing-stages, passenger-station, goods-sheds, cart- roads, sidings, engine-house, etc., are lighted by one hundred and six 4-ampere Jandus arc-lamps, each fitted with raising- and lowering- gear, and the equivalent of one thousand 16-candle-power incan- descent lamps. The lighting of the quay-side is effected by means of two-light fittings run on alternate circuits and carried in lanterns on 10-foot cast-iron pillars, spaced about 60 feet apart. Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. Water-Supply.-Water is obtained from an Artesian well by means of a pump made by Messrs. Timmins. The pump-head well is 8 feet in internal diameter, is sunk to a depth of 16 feet G inches below the surface, and is lined with cast-iron segmental tubing. A cast-iron stand-pipe of 26 inches bore, with a large body-flange, is fitted at the base of the well and carried down 22 feet 6 inches below the surface ; this and the well-bottom are made tight with concrete to prevent any Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. 240 ADERNETfiY ON HEYSllAN HARBOUR. [Selected [Selected surface-pollution percolating into the bore-hole. Below the stand- pipe, to a depth of 101 feet, the bore-hole is 24 inches in diameter, and is lined with 19-inch and 21-inch cast-iron flush-jointed lining- tubes. The annulus outside the tubes is grouted with cement, sealing the bore-hole to the base of the tubes in order to obtain the pure and deep-seated Artesian-well water. From this point downwards an 18-inch open bore is continued to a depth of about 350 feet below the surface. The bore-hole pump is 11 inches in diameter, of the combined ram and bucket type, the base of the suction being fixed at 125 feet below the surface. The pump is capable of delivering 288,000 gallons of water per day through a 6-inch main 1,550 yards in length into a reservoir of 392,850 gallons capacity, 38 feet above the rail-level of the harbour. Cmne8, Copstans, etc.-The cranes and capstans have been supplied by Messrs. Stothert and Pitt, of Bath, and electrically-equipped by the British Westinghouse Company. There are six &ton wharf- cranes, one 2-ton wharf-crane, six 30-cwt. platform-cranes in the goods-sheds, ten l-ton capstans, and two %ton capstans. Two sets of Merryweather fire-pumps, having a united capacity of 1,400 gallons per minute, are instnlled, ancl two Waywood lifts are provided to take passengers and goods to and from the upper and lower landing-stages. The harbour is designed to provide increased accommodation for goods- and passenger-traffic to Ireland, and for excursion-traffic to the Isle of Man. The Midland Railway Company have built a new fleet of steamers, with every modern convenience, which commenced running in September 1904. Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. In order more effectually to maintain the connections in Ireland, the Midland Railway Company have absorbed the Northern Counties Railway of Ireland and the Donegal line. The intention is to foster the passenger-traffic with the provinces of Ulster and Donegal and to develop the agricultural resources of the country. The harbour is also conveniently situated for railway-connection with the English Midland towns, and a new line has been constructed from the Midland main-line, so that trains run direct vid Lancaster to the harbour, alongside the steamers. Arrangements have also been made at the harbour for cattle, fish, poultry, and other traffic. The cost of the harbour-works, without buildings or equipment, has amounted to the sum of 2796,619. The Engineers for the works were the late Mr. J. Allen McDonald, M. Inst. C.E., Chief Xngineer to the Midland Railway Company, and the Author. The Engineers for the works were the late Mr. J. Allen McDonald, M. Inst. C.E., Chief Xngineer to the Midland Railway Company, and the Author. The Eesident Engineer a t the commencement of the works was The Eesident Engineer a t the commencement of the works was Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. CoxmacroKa’ WORKMHOPB AIYD 1’L.mrr. Carpenters’ a d wagon-repairing shop, 140 feet by 25 feet, in which one foreman and thirty men were employed repairing Tvsgons, hand-carts, trucks, barrows, etc. In connection with this shop ten to fifteen men were employed outside putting up shutters for concreting, sheds and general outside repairs ; these were independent of carpenters on permanent work, such as staging, piling, etc. The cement-shed was 200 feet in length by 40 feet in width, the cement being stored to a depth of about 3 feet 6 inches. This was dividecl into ten bins, each of which took about 120 tons, to facilitate cooling, testing, etc., the cetnent being shot as it came from the steamer alongside the temporary pier. On each bin was noted the date of delivery, the date of testing, and the date when ready for use. A store-shed, 100 feet by 20 feet, \vas erect,ed, and was furnished with the usual assortment of stores. An oil-shed was placed at a considerable distance away to avoid danger in case of fire. A large stone building was utilized for the storage of materials for permanent works. A large stone building was utilized for the storage of materials for permanent works. An enginepainting shed, to take one locomotive. Blacksmiths’ and fitting shops. Machine and fitting shops. Boilewmiths’ shops. Brass-moulding department. 1 Lubecker exwsvator, eulplo~ing twelve ulm. 1 12-ton M’ilson excavator. 1 lo-ton Whitaker bucket-exmvatw. 19 locomotives of various types. 21 travelling and derrick crmes. 3 Taylor concrete-mixors. y 8 stone-crushers driven by 10-HI’. a d 12-111’. ellginca. 24 pumps of different types. p p yp 15 boilers, vertical ancl locomotive. 6 grabs 15 boilers, vertical ancl locomotive. 2 steam rock-drills. 2 steam rock-drills. Portihle engines, three lO-Hl’., two l%-HP., one ~O-IIP., two 25-IIP., am1 two 30-HI’. p?,,?,Lpy ( ~ t Sump-One pair 16-inch Coruisll rocker-beau1 1)umps sLcam- driven by apur-gearing ; approximate capacity, 800 gallons per millute at 14 revo- lutions per minute. One 12-inch Wade and Cherry centrifugal pump driven by a PB-HP. engine ; approximate capacity, 2,000 gallons per Ininute. This punq) was used for high tides and wet weather. The temporary water-pipes were 53 miles in length. The contractors’ railway was about l O + miles in length. A tetnporary jetty 011 No. 2 bank \vas constructed, at which frequently two or three steamers were discharging a t one time. ABERNETHY ON HEYSHAM HARBOUR. ABERNETHY ON HEYSHAM HARBOUR. [Selected CoxmacroKa’ WORKMHOPB AIYD 1’L.mrr. It was equipped with three 3-ton crihlles, and in the busy time these were working day and night. Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. 241 ABERNETHY OS HEYSIIARI HARBOUR. Papers.] Mr. Gerald Fitz-Gibbon, M. Inst. C.E., who had had considerable previous experience under the Author in harbour- and dock-works. He left in September 1899 to take up the appointment of Chief Engineer to the Aire and Calcler Navigation, and his place was filled by Mr. Baldwin H. Bent., M.A., M. Inst. C.E., who remained until their completion. The Paper is accompanied by five drawings and two tracings, from :L selection of which Plate 4 has been prepared; and by sixteen photographs, which may be seen in the Library of the Institution. [THE ISST. C.E. VOL. CLXVI.] [THE ISST. C.E. VOL. CLXVI.] Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. 242 S E C T H O N F. F. ht © ICE Publishing all rights reserved Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ Purdue Univ Lib TSS] on [19/09/16] Copyright © ICE Publishing a Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ Purdue Univ Lib TSS] on [19/09/16]. Copyright © ICE Publishing, all rig
https://openalex.org/W2626814575	https://research-information.bris.ac.uk/ws/files/117897959/Author_version_IEEE.pdf	English	null	A Flexible 2.45-GHz Power Harvesting Wristband With Net System Output From −24.3 dBm of RF Power	IEEE transactions on microwave theory and techniques	2,018	cc-by	14,114	Adami, S. E., Proynov, P., Hilton, G. S., Yang, G., Zhang, C., Zhu, D., Li, Y., Beeby, S. P., Craddock, I. J., & Stark, B. H. (2018). A Flexible 2.45-GHz Power Harvesting Wristband with Net System Output from - 24.3 dBm of RF Power. IEEE Transactions on Microwave Theory and Techniques, 66(1), 380-395. Article 7955016. https://doi.org/10.1109/TMTT.2017.2700299 Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.1109/TMTT.2017.2700299 This is the final published version of the article (version of record). It first appeared online via IEEE at http://ieeexplore.ieee.org/document/7955016/. Please refer to any applicable terms of use of the publisher. Adami, S. E., Proynov, P., Hilton, G. S., Yang, G., Zhang, C., Zhu, D., Li, Y., Beeby, S. P., Craddock, I. J., & Stark, B. H. (2018). A Flexible 2.45-GHz Power Harvesting Wristband with Net System Output from - 24.3 dBm of RF Power. IEEE Transactions on Microwave Theory and Techniques, 66(1), 380-395. Article 7955016. https://doi.org/10.1109/TMTT.2017.2700299 Adami, S. E., Proynov, P., Hilton, G. S., Yang, G., Zhang, C., Zhu, D., Li, Y., Beeby, S. P., Craddock, I. J., & Stark, B. H. (2018). A Flexible 2.45-GHz Power Harvesting Wristband with Net System Output from - 24.3 dBm of RF Power. IEEE Transactions on Microwave Theory and Techniques, 66(1), 380-395. Article 7955016. https://doi.org/10.1109/TMTT.2017.2700299 A Flexible 2.45-GHz Power Harvesting Wristband With Net System Output From −24.3 dBm of RF Power Salah-Eddine Adami, Plamen Proynov, Geoffrey S. Hilton, Guang Yang, Chunhong Zhang, Dibin Zhu, Yi Li, Steve P. Beeby, Ian J. Craddock, Fellow, IEEE, and Bernard H. Stark Fig. 1. Block diagram of the ﬂexible RF power harvesting system (single channel). Abstract—This paper presents a ﬂexible 2.45-GHz wireless power harvesting wristband that generates a net dc output from a −24.3-dBm RF input. This is the lowest reported system sensitivity for systems comprising a rectenna and impedance- matching power management. A complete system has been implemented comprising: a fabric antenna, a rectiﬁer on rigid substrate, a contactless electrical connection between rigid and ﬂexible subsystems, and power electronics impedance matching. Various fabric and ﬂexible materials are electrically characterized at 2.45 GHz using the two-line and the T-resonator methods. Selected materials are used to design an all-textile antenna, which demonstrates a radiation efﬁciency above 62% on a phantom irrespective of location, and a stable radiation pattern. The rectiﬁer, designed on a rigid substrate, shows a best-in- class efﬁciency of 33.6% at −20 dBm. A reliable, efﬁcient, and wideband contactless connection between the fabric antenna and the rectiﬁer is created using broadside-coupled microstrip lines, with an insertion loss below 1 dB from 1.8 to over 10 GHz. A self-powered boost converter with a quiescent current of 150 nA matches the rectenna output with a matching efﬁ- ciency above 95%. The maximum end-to-end efﬁciency is 28.7% at −7 dBm. The wristband harvester demonstrates net positive energy harvesting from −24.3 dBm, a 7.3-dB improvement on the state of the art. Fig. 1. Block diagram of the ﬂexible RF power harvesting system (single channel). garments for ﬁreﬁghters [4], life jackets for Cospas-Sarsat international satellite rescue system [5], and spacesuits [6]. Flexible RF and microwave circuits have been reported using ﬂexible thin polymers [7], fabric materials [8], [9], and inkjet- printed structures using paper [10]. Fabrics are particularly relevant for wearables due to their broad acceptance, ﬂex- ibility, lightweight, and low cost. In practice, where high functionality is required, a hybrid structure is used, where ﬂexible circuits are combined with rigid sub-PCBs with the required RF and sensor-node chipsets [11]. RF wireless power transfer provides a convenient means of remotely powering these hybrid structures [12], removing the need to directly recharge batteries. This arrangement furthermore avoids the need for rigid, dense, and potentially toxic batteries. Index Terms—Boost converter, fabric antenna, fabric-to-PCB connector, Internet of Things, power management, rectenna array, rectenna impedance matching, rectennas, RF energy har- vesting, RF power transfer, smart fabric, T-resonator method, textile antenna, two-line method. University of Bristol – Bristol Research Portal General rights This document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/red/research-policy/pure/user-guides/brp-terms/ IEEE TRANSACTIONS ON MICROWAVE THEORY AND TECHNIQUES A Flexible 2.45-GHz Power Harvesting Wristband With Net System Output From −24.3 dBm of RF Power Salah-Eddine Adami, Plamen Proynov, Geoffrey S. Hilton, Guang Yang, Chunhong Zhang, Dibin Zhu, Yi Li, Steve P. Beeby, Ian J. Craddock, Fellow, IEEE, and Bernard H. Stark The combination of RF wireless power transfer and wear- able electronics is challenging for several reasons. First, the reliability of interconnections between ﬂexible and rigid subsystems needs addressing, for example by using snap-on fasteners, as reported in [13]. Second, in order to maximize the output power, and obtain a range of several meters, rectiﬁ- cation and power management circuits need to be efﬁcient down to extremely low power levels [14]. Third, with the antenna being on a moving person, the stability of efﬁciency and radiation pattern is important. Creative Commons Attribution 3.0 License. For more information, see http://creativecommons.org/licenses/by/3.0/ work is licensed under a Creative Commons Attribution 3.0 License. For more information, see http://creativecommons.o Manuscript received November 28, 2016; revised March 6, 2017 and April 13, 2017; accepted April 16, 2017. This work was performed under the SPHERE Interdisciplinary Research Collaboration (IRC) funded by the U.K. Engineering and Physical Sciences Research Council (EPSRC), Grant EP/K031910/1. (Corresponding author: Salah-Eddine Adami.) S.-E. Adami, P. Proynov, G. S. Hilton, G. Yang, I. J. Craddock, and B. H. Stark are with the Department of Electrical and Elec- tronic Engineering, University of Bristol, Bristol BS8 1UB, U.K. (e-mail: bernard.stark@bristol.ac.uk). C. Zhang was with the Department of Electrical and Electronic Engineering, University of Bristol, Bristol BS8 1UB, U.K. D. Zhu is with the College of Engineering, Mathematics and Phys- ical Sciences, University of Exeter, Exeter EX4 4QF, U.K. (e-mail: d.zhu@exeter.ac.uk). Y. Li and S. P. Beeby are with the Department of Electronics and Computer Science, University of Southampton, Southampton SO17 1BJ, U.K. (e-mail: yi.li@soton.ac.uk; spb@ecs.soton.ac.uk). Color versions of one or more of the ﬁgures in this paper are available online at http://ieeexplore.ieee.org. Digital Object Identiﬁer 10.1109/TMTT.2017.2700299 Color versions of one or more of the ﬁgures in this paper are available online at http://ieeexplore.ieee.org. IEEE TRANSACTIONS ON MICROWAVE THEORY AND TECHNIQUES IEEE TRANSACTIONS ON MICROWAVE THEORY AND TECHNIQUES 2 2 TABLE I TABLE I CHARACTERIZATION RESULTS OF VARIOUS SUBSTRATE MATERIALS AT THE 2.45-GHz BAND CHARACTERIZATION RESULTS OF VARIOUS SUBSTRATE MATERIALS AT THE 2.45-GHz BAND Fig. 2. Photograph of the proposed RF harvesting wristband system comprising two rectennas of that shown in Fig. 1. Fig. 3. Cross-sectional SEM image view of a standard polycotton fabric with screen-printed interface layer. The roughness of the fabric material is reduced to few micrometers. Fig. 2. Photograph of the proposed RF harvesting wristband system comprising two rectennas of that shown in Fig. 1. end-to-end RF/dc efﬁciency). This system is implemented as a wristband, as shown in Fig. 2. This paper presents advances in various areas: antenna, rectiﬁer, interconnection design, and power management. It validates the approach through both simulation and measurement of individual components and through experimental results from a complete system implementation. Fig. 3. Cross-sectional SEM image view of a standard polycotton fabric with screen-printed interface layer. The roughness of the fabric material is reduced to few micrometers. with FR4 and Duroid-5880 used as baselines. The selected materials cover a wide range of application areas with a focus on purely fabric materials. Foam materials were not considered here, although they were used in many other research papers to design fabric antennas [8]. Polyester and Cotton fabrics are at the top of our selection as they are widely used in garment industry. The list also includes an UV-curable polyurethane acrylate-based standard interface material (Fabink-UV-IF1) and a waterproof interface material (Fabink-UV-IF010), both from Smart Fabric Inks Ltd. These are used in [12], to pla- narise the surface of fabrics prior to the silver screen printing of antennas. Fig. 3 shows a cross-sectional scanning electron microscope (SEM) image of a polycotton fabric with screen- printed interface material. It can be seen that the rough surface of fabric is reduced to only few micrometers. Kapton polyimide laminate from DuPont is also characterized. The paper is organized as follows. Section II presents the characterization at 2.45 GHz of various materials. Section III presents the design of the fabric antenna. Section IV shows the design of the fabric-to-rigid PCB coupling interface. Section V presents the design of the rectiﬁer. Section VI presents the power management circuit. Finally, in Section VII, the end-to-end system is presented along with experimental results. I. INTRODUCTION F LEXIBLE RF electronics has many applications in on- body and off-body communications for: healthcare [1], RFID body-centric sensing [2], wearables [3], protective F Manuscript received November 28, 2016; revised March 6, 2017 and April 13, 2017; accepted April 16, 2017. This work was performed under the SPHERE Interdisciplinary Research Collaboration (IRC) funded by the U.K. Engineering and Physical Sciences Research Council (EPSRC), Grant EP/K031910/1. (Corresponding author: Salah-Eddine Adami.) S.-E. Adami, P. Proynov, G. S. Hilton, G. Yang, I. J. Craddock, and B. H. Stark are with the Department of Electrical and Elec- tronic Engineering, University of Bristol, Bristol BS8 1UB, U.K. (e-mail: bernard.stark@bristol.ac.uk). S.-E. Adami, P. Proynov, G. S. Hilton, G. Yang, I. J. Craddock, and B. H. Stark are with the Department of Electrical and Elec- tronic Engineering, University of Bristol, Bristol BS8 1UB, U.K. (e-mail: bernard.stark@bristol.ac.uk). This paper reports, for the ﬁrst time, a ﬂexible and wearable end-to-end RF-harvesting system, that is designed to operate over a range exceeding 2 m at Wi-Fi transmit power levels (100-mW EIRP). A single channel of this system is illustrated in Fig. 1. The ﬂexible fabric antenna is coupled to the rigid rec- tiﬁer and boost converter, using broadside-coupled microstrip lines. This system is optimized to operate at a power of −20 dBm in the center of the 2.4-GHz ISM band. This power level gives a good balance between transmission range (about 2 m from a 100-mW EIRP typical Wi-Fi source [15]) and available dc power for the load (about 1 μW, hence a 10% C. Zhang was with the Department of Electrical and Electronic Engineering, University of Bristol, Bristol BS8 1UB, U.K. C. Zhang was with the Department of Electrical and Electronic Engineering, University of Bristol, Bristol BS8 1UB, U.K. D. Zhu is with the College of Engineering, Mathematics and Phys- ical Sciences, University of Exeter, Exeter EX4 4QF, U.K. (e-mail: d.zhu@exeter.ac.uk). Y. Li and S. P. Beeby are with the Department of Electronics and Computer Science, University of Southampton, Southampton SO17 1BJ, U.K. (e-mail: yi.li@soton.ac.uk; spb@ecs.soton.ac.uk). Color versions of one or more of the ﬁgures in this paper are available online at http://ieeexplore.ieee.org. igital Object Identiﬁer 10.1109/TMTT.2017.2700299 B. Discussion and Analysis of the Results Fig. 4. Examples of samples characterized using the two-line and the T-resonator methods. In this section, the line losses αTyp (dB/m) considering a typical microstrip line of width w = 3 mm over a h = 1.6 mm height substrate, are compared for all materials. Fig. 5. Characterization results of various materials at 2.45 GHz: loss tangent versus relative permittivity. Contours of constant microstrip line dielectric loss are also plotted considering a typical microstrip line of width w = 3 mm over a h = 1.6 mm height substrate. 1) Interface Material: The interface material was applied in a previous work to polycotton to smooth its surface and make it ready for silver screen printing of conductive lay- ers [12]. This strengthens the adherence of the conductive layer (especially when the antenna is bent) and reduces surface roughness. The dielectric loss associated with this structure is, however, about 22.4 dB/m, which is high compared to the reference FR4 (7.6 dB/m) and Duroid-5880 (0.3 dB/m). Regarding pure interface material samples, the results show a relative permittivity of 3.26 and a loss tangent of 0.063. These values are slightly higher than those of the polycotton-interface material sandwich (εr = 3.23 and tanδ = 0.06). Considering that 2/3 of the thickness of the sandwich is interface material, it is apparent that the interface layer is the main contributor to the high dielectric loss of the structure. Interface materials should therefore be used sparingly, and further researched for lower-loss variants. Fig. 5. Characterization results of various materials at 2.45 GHz: loss tangent versus relative permittivity. Contours of constant microstrip line dielectric loss are also plotted considering a typical microstrip line of width w = 3 mm over a h = 1.6 mm height substrate. 2) Woven Fabrics: Cotton and woven polyester fabrics are widely used in garment industry. Both exhibit similar loss tangent values of 0.028. However, the 8.5 dB/m of loss for cotton is higher than the 7.6 dB/m for polyester, as a result of cotton’s higher relative permittivity [21]. Both materials appear to be comparable with FR4 (7.6 dB/m), which make them good candidates for a wide range of low-cost RF con- sumer electronics applications. The surface of woven fabrics is smooth and therefore suitable for screen printing. A. Characterization Method The two-line method [16], [17] is used here to characterize various textile and other ﬂexible materials, as shown in Table I, 3 ADAMI et al.: FLEXIBLE 2.45-GHz POWER HARVESTING WRISTBAND Fig. 4. Examples of samples characterized using the two-line and the T-resonator methods. Loss tangent was not extracted from the T-junction samples as a speciﬁc calibration kit need to be designed for each sample, which is quite impracticable considering the number of samples tested here. The extracted loss tangent of FR4 using the two-line method was 0.017 which is very similar to what was found in [19]. The other materials in our table have similar or even higher loss tangent than FR4. Therefore, loss tangent for those materials is estimated to have similar or a better accuracy than the one obtained with FR4. B. Discussion and Analysis of the Results The weave pattern of fabrics can have an effect on the density of ﬁbers in the fabric which could affect the high-frequency electrical characteristics of the fabric. A systematic characterization of the fabric material of interest is essential in order to achieve an accurate design. For each material, two microstrip lines of different lengths are produced (see Fig. 4). The relative permittivity εr and loss tangent tanδ are calculated from the measured S-parameters. Table I gives the results of the experimental characterization. The results of the characterization are also plotted in Fig. 5, with contours of constant dielectric attenuation. The two-line method gives an estimation of the material characteristics for a relatively wide frequency range. However, the resulting tanδ can appear to be a nonmonotonic function of frequency [12], [17], as a consequence of multiple reﬂexions and standing waves in the relatively small samples under test. Nonphysical behavior can also appear when the two-line method is used on substrates with low dielectric loss (such as Duroid-5880). Averaging [17] or polynomial ﬁtting [12] is normally required to smooth the ﬁnal results. A second veriﬁcation using a narrowband technique (e.g., antenna efﬁ- ciency [18], T-resonator [19], ring resonator [20], resonant cavity, and cavity perturbation [21]) is recommended when an accurate characterization is required for a given frequency. As a validation in this paper, the T-resonator method is used to estimate the permittivity of the samples measured with the two-line method. T-resonator samples are designed to resonate around 2.45 GHz using the results from the two-line method (see Fig. 4). The results of the T-resonator characterization are summarized in Table I. One can see the excellent match with the results found using the two methods. 3) Polymers: Kapton has a loss below 5 dB/m. Although the tested sample had a thickness of 1 mm, thin 125-μm ﬂexible kapton is available. Thus, RF circuits and antennas can be designed on thin Kapton and integrated with clothing or any application requiring ﬂexibility, with better performance than FR4 (e.g., STMicroelectronics M24LR04E NFC/RFID ﬂexible tag). 4) Felts: Polyester felt is considered here. It should be noted that felt fabric has a rough and irregular surface which is nonoptimal for screen printing. The relative permittivity of felt is very close to 1 (air) due to its porosity (∼40% air in volume). B. Discussion and Analysis of the Results The materials selected for the design of the present harvester are synthetic, which makes them more immune to variations in environmental conditions. 5) Effect of Environmental Conditions: The humidity and temperature do have an effect on the high-frequency parame- ters of fabric materials, as a proportion of water (depending on the relative humidity level and temperature) is absorbed by the fabric ﬁbers. Detailed studies were performed in [22] and [23]. The increase in relative humidity translates to an increase in the amount of water in the ﬁbers, which increases by consequence the dielectric constant and the loss tangent of the fabric. The variations of temperature have the same effect [23]. Fig. 7. Layout of the all-fabric antenna. All conductive parts are made from ﬂexible copper fabric. One of the conclusions from [22] was that natural ﬁbers are much more sensitive to humidity than synthetic ﬁbers. The materials selected for the design of the present harvester are synthetic, which makes them more immune to variations in environmental conditions. The experiments in this paper were all realized in an air-conditioned laboratory environment. However, no precise record was made of temperature and humidity levels. The accurate quantiﬁcation of the effect of a given relative humid- ity and temperature level requires a dedicated study and is outside the scope of this paper. III. ANTENNA DESIGN Patch antenna with ground plane is very suitable for the realization of fabric wearable antennas. Also, a full ground plane has been shown to reduce the speciﬁc absorption rate value by a factor of 3 when compared to an antenna without ground plane [24]. Furthermore, a ground plane results in a thinner overall system when compared to other shielding options, such as air or fabric spacing gaps [25], electromag- netic band gap [26], and artiﬁcial magnetic conductor [27]. In this paper, a planar multilayer-fabric patch antenna is chosen with proximity-coupled feed [28]. Fig. 7. Layout of the all-fabric antenna. All conductive parts are made from ﬂexible copper fabric. fabric layers. As only a small quantity of adhesive is required, no degradation of the ﬂexibility was noticed. The antenna in Fig. 7 was optimized using ADS Momen- tum. The length of the patch resonant at 2.45 GHz is L p = 50 mm and its width Wp was optimized to achieve a good 50- match [see Fig. 6(a)]. The width of the 50- microstrip feed line is W f = 7 mm, its length L f impacts both the antenna’s resistance and reactance [see Fig. 6(b)]. The ﬁnal design and the optimized parameters are given, as shown in Fig. 7 and a photograph of a fabricated prototype is shown in Fig. 8. The ground plane extends 10 mm beyond the patch edges in order to improve the isolation from the immediate environment. The study in Section II suggested the use of polyester felt and woven polyester for their reduced loss. Early design simulation (using ADS Momentum with pure copper as the conductive material) showed that two layers of polyester felt and one layer of woven polyester achieve a radiation efﬁciency above 70%. This 3.7-mm multilayer structure also improves the bandwidth, which makes the antenna more robust when bent or worn. The thin 0.33-mm woven polyester is interlaced between the polyester felt fabrics and will be used later as a dielectric for the broadside coupling with the rectiﬁer. A ﬂexible conductive fabric (80-μm-thick woven copper- plated polyester ﬁber fabric, from LessEMF Inc. with 0.18 /sq at 2.45 GHz [8]) is used for the patch, the feed line, and the ground plane. An adhesive spray (3M Display Mount) was used for mechanical bonding between the various B. Discussion and Analysis of the Results Polyester felt has a dielectric loss of 5.8 dB/m, IEEE TRANSACTIONS ON MICROWAVE THEORY AND TECHNIQUES 4 Fig. 6. Simulated antenna impedance, ZAnt = RAnt + j×XAnt, and input response S11 as function of the patch width Wp, and the length of the microstrip feed line L f . (a) Variation of Wp with constant L f = 21 mm. (b) Variation of L f with constant Wp = 44 mm. The impedance of the antenna is calculated 10 mm from the edge of the patch in the x-axis direction, as shown in Fig. 7. which is better than FR4 (6.7 dB/m). Polyester felt shows lower dissipation than woven polyester primarily due to its higher proportion of air. We conclude from this paper that polyester felt is the fabric with the lowest loss. Its relatively high nominal thickness (1.65 mm) is an additional advantage to improve antenna’s performance such as efﬁciency and bandwidth [18]. However, due to its porosity, felt is susceptible to compression that could detune the RF circuit or antenna. As Polyester felt is not suitable for silver screen printing, the use of conductive fabric is preferred for the conductive parts of the antenna. Fig. 6. Simulated antenna impedance, ZAnt = RAnt + j×XAnt, and input response S11 as function of the patch width Wp, and the length of the microstrip feed line L f . (a) Variation of Wp with constant L f = 21 mm. (b) Variation of L f with constant Wp = 44 mm. The impedance of the antenna is calculated 10 mm from the edge of the patch in the x-axis direction, as shown in Fig. 7. 5) Effect of Environmental Conditions: The humidity and temperature do have an effect on the high-frequency parame- ters of fabric materials, as a proportion of water (depending on the relative humidity level and temperature) is absorbed by the fabric ﬁbers. Detailed studies were performed in [22] and [23]. The increase in relative humidity translates to an increase in the amount of water in the ﬁbers, which increases by consequence the dielectric constant and the loss tangent of the fabric. The variations of temperature have the same effect [23]. One of the conclusions from [22] was that natural ﬁbers are much more sensitive to humidity than synthetic ﬁbers. IV. FABRIC-TO-RIGID PCB INTERFACE DESIGN Establishing an RF connection between fabric and a rigid PCB is one of the main sources of failure in smart electronic ADAMI et al.: FLEXIBLE 2.45-GHz POWER HARVESTING WRISTBAND 5 Fig. 9. Simulation of the broadside coupling performance. (a) 3-D view of the broadside-coupled microstrip lines. (b) S21 with variable h (L = 7 mm). (c) S21 with variable L (h = 0.33 mm). Fig. 8. Fabricated prototype of the all-fabric antenna. Fig. 8. Fabricated prototype of the all-fabric antenna. textile applications [29]. Speciﬁcally, at RF where insertion loss and stability of the interconnection are of most important. In order to avoid the use of fragile coaxial cable connection, metallic snap-on buttons have been proposed with a work- ing frequency range up to 5 GHz and insertion loss better than 0.8 dB [13]. Similarly, conductive hook and loop fastener was proposed in [30] with insertion loss about 1 dB at 4 GHz. Both techniques can be used to allow the user to unmount the electronic module from the fabric if needed. A nongalvanic interconnection was proposed in [31] using magnetic coupling between two coils, one on fabric and the other on a ﬂexible PCB. The drawback of that proposed system is related to the inherent complex impedance of the transformer at both terminals, i.e., fabric and PCB, which reduces the operational bandwidth of the transition according to the Bode–Fano Criterion [21]. An additional drawback is the noncompatibility with a 50- standard connection, making experimental tests more challenging. Fig. 9. Simulation of the broadside coupling performance. (a) 3-D view of the broadside-coupled microstrip lines. (b) S21 with variable h (L = 7 mm). (c) S21 with variable L (h = 0.33 mm). a baseline for the broadside coupling. S21 is calculated from 10 MHz up to 10 GHz for various values of L and h in order to give a wide overview of the operation bandwidth of the coupling. As shown in Fig. 9, S21 is improved with increased coupling area (by increasing L) and with reduced thickness due to the increase in the equivalent capacitance. We present herein two techniques that are expected to per- form better in term of bandwidth and 50 compatibility: slot- coupled microstrip lines and broadside-coupled (or stacked) microstrip lines [21]. Both techniques are convenient and easy to implement in this type of application and are well-known to the microwave community. IV. FABRIC-TO-RIGID PCB INTERFACE DESIGN The novelty therefore is the combination of fabric and rigid material. We deﬁne the bandwidth for an insertion loss of 1 dB. For a thickness h (0.33 mm), the bandwidth extends from 1.8 GHz up to over 10 GHz. More improvement regarding the bandwidth can be mainly achieved by reducing the thickness h. A larger bandwidth from 850 MHz to over 10 GHz is achieved with h/2 (175 μm), and from 400 MHz to over 10 GHz with h/4 (≈85 μm), as seen in Fig. 9(b). Slot-coupled microstrip line is a better option if the PCB is to be placed back-to-back with the fabric antenna. In order to preserve the ﬂexibility of the antenna, the microstrip line feeding the antenna must be slot-coupled to the second microstrip line on PCB at the extremity of the antenna. This implementation is different from an aperture-fed antenna, in which case the PCB is to be placed at the back-center of the antenna, which reduces its ﬂexibility. Using this technique, the operational bandwidth can be made very large using a multiresonant slotline resonator [32]. The nominal thickness h (0.33 mm) gives an insertion loss of 0.7 dB at 2.45 GHz. This performance is largely sufﬁcient for our application. The parameters shown in Fig. 9(a) are, thus, employed for experimental implementation. V. RECTIFIER DESIGN The most critical part of a wireless power transfer receiver is the rectiﬁer because its efﬁciency dominates the system efﬁciency at low power levels. The rectiﬁer efﬁciency is function of all these: frequency, level of the input power, diode type and rectiﬁer circuit topology, substrate, input matching with the antenna, and output dc load. Broadside-coupled microstrip line is a better option if the PCB and the antenna face the same direction, which is the case in our application since the rectiﬁer uses microstrip lines. Here a series capacitive coupling is created between the two microstrip lines. Its effect on the impedance of the line can be minimized by improving the coupling, i.e., increasing the equivalent capacitance. This way, the impedance at both input and output terminals stays mainly real, leading to an improvement of the matching bandwidth. The rectiﬁer presented here was optimized to operate at 2.450 GHz. A Duroid-5880 substrate (εr = 2.2, tanδ = 0.0009) was used in order to reduce dielectric loss in the input and output microstrip matching circuit. A Schottky diode (SMS7630 with a very low forward voltage V f = 60 to 120 mV at 0.1 mA) was selected in order to optimize the efﬁciency around −20 dBm. However, this diode suffers from a considerable reverse leakage (Ileak = 5 μA) Full-wave electromagnetic simulation using ADS Momen- tum is used to optimize the structure proposed in Fig. 9(a). The input is the 50- microstrip line from the antenna. Woven polyester with a nominal thickness h of 0.33 mm is used as IEEE TRANSACTIONS ON MICROWAVE THEORY AND TECHNIQUES 6 Fig. 10. Harmonic Balance optimization using ADS at −20 dBm input power. For each set of the three variables: Rs, Xs, and RL, a harmonic balance simulation is run to evaluate the RF/dc efﬁciency. RL = 5 k, RS = 20 , and XS = 260 are found to maximize the RF/dc efﬁciency at −20 dBm. Fig. 11. Layout of the rectiﬁer on Duroid-5880 (35 × 23 mm) including the fabric-to-PCB coupling interface (top view is to scale). All vias are constructed from 0.3-mm radius metallic wire. Fig. 10. Harmonic Balance optimization using ADS at −20 dBm input power. For each set of the three variables: Rs, Xs, and RL, a harmonic balance simulation is run to evaluate the RF/dc efﬁciency. V. RECTIFIER DESIGN RL = 5 k, RS = 20 , and XS = 260 are found to maximize the RF/dc efﬁciency at −20 dBm. and a very low breakdown voltage (VB = 2 V), which implies that the rectiﬁer efﬁciency will be reduced at higher power levels. A different diode with lower reverse current and higher VB (such as HSMS8250 with Ileak = 3 μA and VB = 3.8 V) would perform better when the power is above about −15 dBm, as seen in [33]. The input and output matching circuit was designed using ADS and Momentum following this procedure. Fig. 11. Layout of the rectiﬁer on Duroid-5880 (35 × 23 mm) including the fabric-to-PCB coupling interface (top view is to scale). All vias are constructed from 0.3-mm radius metallic wire. 1) Two parallel-connected open-circuit quarter-wave stubs were designed using Momentum at the ﬁrst (2.45 GHz) and second (4.9 GHz) harmonics. The stubs act as a short circuit at their design frequency, providing a low loss return path for the harmonics generated by the diode and thereby improving the rectiﬁer efﬁciency. The stubs also prevent the harmonics from propagating to the output dc terminal. Simulation showed that the presence of the second-harmonic stub improves the rectiﬁer efﬁciency by about 1% compared to a single stub conﬁguration. Both stubs are bent in order to occupy the smallest area on the board. Fig. 12. Fabricated prototype of the rectiﬁer on Duroid-5880 and the fabric-to-PCB coupling interface. Fig. 12. Fabricated prototype of the rectiﬁer on Duroid-5880 and the fabric-to-PCB coupling interface. 2) As stated above, the rectiﬁer efﬁciency depends on the input matching to the antenna and on the output load. A co-simulation in ADS is realized, in which the diode is connected to the EM model of the dual-stub ﬁlter (see Fig. 10). The stub is loaded with a resistive load (RL) and the diode is fed by a −20 dBm RF source with vari- able complex impedanceZS = RS + j ∗XS. A harmonic balance simulation (large signal simulation) is run with simultaneous 3-variable sweep on RS, XS and RL. The optimization gave the following parameters: RL = 5 k, RS = 20 , and XS = 260 . 2) As stated above, the rectiﬁer efﬁciency depends on the input matching to the antenna and on the output load. V. RECTIFIER DESIGN The 50- matching also gives access to a better accuracy when measuring rectiﬁer ADAMI et al.: FLEXIBLE 2.45-GHz POWER HARVESTING WRISTBAND 7 Fig. 13. (a) Self-powered boost converter. (b) MOSFET driver comprising a 3-stage oscillator. The UB20M voltage detector [35] is an ultralow power integrated circuit with an input quiescent current of around 10 pA. consumption is only few picoamperes. The oscillator switches at FSw = 200 Hz with δ = 2%, and draws 64 nA from a 1.2-V supply. A low power linear regulator (ZSPM4141 with 20-nA quiescent current) is used to power the oscillator from the output of the boost converter VBoost; the total quiescent current, including that of the regulator, is about IQuiescent_Boost = 150 nA with VBoost = 3V. The output of the boost converter is connected to a precision source/measure unit (Keysight B2911A) conﬁgured as a voltage source. This device allows us to accurately measure the output current and evaluate the converter efﬁciency. If a storage capacitor (or supercapacitor) is to be used at the output of the boost converter, it should be precharged to about 2 V to kick-start the operation of the boost converter. Fig. 13. (a) Self-powered boost converter. (b) MOSFET driver comprising a 3-stage oscillator. The UB20M voltage detector [35] is an ultralow power integrated circuit with an input quiescent current of around 10 pA. efﬁciency as the power sources can be calibrated to about 0.1 dB uncertainty. On the other hand, if the rectiﬁer was directly matched to the antenna, radiated measurement in anechoic chamber would have to be used for measuring the efﬁciency. In that case, the power level received by the rectenna is estimated using a reference antenna connected to a power meter. A typical 0.5 dB uncertainty on the reference antenna gain would dramatically reduce the accuracy of the efﬁciency measurement. It should be noted that the actual optimal parameters of the boost converter (FSw = 200 Hz, δ = 2%, and LBoost = 8.1 mH) do not satisfy (2) for RBoost_in = 4 k as a result of the internal loss of the boost converter not being considered in the derivation of (2). The value of LBoost was experimentally ﬁne-tuned with the boost converter being connected to the rectenna in order to achieve the required optimal impedance RBoost_in = 4 k. A. Patch Antenna Measurement Simulated and measured input responses S11 for the fabric antenna are provided in Fig. 14. The antenna works well at the intended frequency band. This is supposed to be 2 MHz around 2.45 GHz, because no speciﬁc bandwidth is required for the receiving antenna harvesting a single tone continuous wave signal (black rectangle in Fig. 14). The measured S11 matches well the simulation, with a shift of about 10 MHz in the resonant frequency, which may be due to the connector not modeled and the fabrication tolerances. These can be expected to be below 0.5 mm, resulting in a maximum possible frequency shift of about 25 MHz. RBoost_in = 2LBoostFSw δ2 1−VRect VBoost (1) (1) where LBoost = 8.1 mH is the inductance of the boost converter, FSw is the switching frequency, and δ the duty ratio of the oscillator that drives the MOSFET. Here VBoost is relatively high compared to VRect (ratio of 26 at −20 dBm RF power), so only the ﬁrst term in (1) remains, i.e., RBoost_in will only depend on LBoost, FSw, and δ where LBoost = 8.1 mH is the inductance of the boost converter, FSw is the switching frequency, and δ the duty ratio of the oscillator that drives the MOSFET. Here VBoost is relatively high compared to VRect (ratio of 26 at −20 dBm RF power), so only the ﬁrst term in (1) remains, i.e., RBoost_in will only depend on LBoost, FSw, and δ The antenna was tested with tight bending radii in the E- and H-planes [see Fig. 14(a) and (b)]. Contrary to what was reported in [36], the bending in the E-plane [bending axis parallel to Wp, as illustrated in Fig. 15(a)] has negligible effect whereas the bending in the H-plane [bending axis parallel to L p, as illustrated in Fig. 15(b)] shifts the resonant frequency up. A similar result was reported in [37] where antenna layers were bonded by sewing. In the present antenna, the antenna’s substrate fabric layers were bonded using hand-applied spray adhesive. The irregularity of the adhesive spread and the compressible nature of the felt fabric creates a substrate with unpredictable behavior when is deformed; bending the sub- strate creates some zones with reduced substrate thickness and some zones with increased substrate thickness (compared to RBoost_in ≈2LBoostFSw δ2 . VII. EXPERIMENTAL RESULTS Matching the rectenna’s output load to the rectenna’s opti- mal impedance RRect_optim is essential in order to maximize rectiﬁcation efﬁciency. This is achieved using the power man- agement circuit of Fig. 13. The circuit contains an inductive boost converter that operates in discontinuous conduction mode. In this mode, no feedback control loop is required as the output voltage depends only on the output load. On the other hand, the input impedance of the boost converter in this mode of operation is given by [34] V. RECTIFIER DESIGN The vias connecting the ground plane of the antenna to the ground plane of the rectiﬁer can be mechanically supported by a rigid plate at the bottom of the multilayer structure (Fig. 11). If a via is to be used to connect the input microstrip line from the antenna to the rectiﬁer circuit, no mechanical support is possible as the microstrip line is wrapped by fabric layers at both sides, Hence the beneﬁt of the broadside coupling which provides a practical and mechanically robust RF connection. The matching efﬁciency Matching is deﬁned here as Matching = rect(RBoost_in) rect(RRect_optim) (3) (3) which is the ratio between actual and maximum achievable rectiﬁer efﬁciency, when the boost converter provides perfect impedance matching. V. RECTIFIER DESIGN A co-simulation in ADS is realized, in which the diode is connected to the EM model of the dual-stub ﬁlter (see Fig. 10). The stub is loaded with a resistive load (RL) and the diode is fed by a −20 dBm RF source with vari- able complex impedanceZS = RS + j ∗XS. A harmonic balance simulation (large signal simulation) is run with simultaneous 3-variable sweep on RS, XS and RL. The optimization gave the following parameters: RL = 5 k, RS = 20 , and XS = 260 . rigid PCB of the rectiﬁer. The electromagnetic waves in the rectiﬁer circuit are conﬁned inside the low loss Duroid-5880 substrate, resulting in an improved rectiﬁ- cation efﬁciency. An L-network microstrip matching is used to transform the antenna’s 50- impedance to the diode’s optimal complex input impedance. The width of microstrip matching line is gradually reduced in order to reach the required highly inductive impedance while minimizing the effect on the input 50- matching. The layout of the proposed rectiﬁer is depicted in Fig. 11 and a photograph of a fabricated prototype is shown in Fig. 12. 3) A multilayer structure including fabric layers and a 1.57-mm Duroid-5880 layer is constructed in Momen- tum, as depicted in Fig. 11. The parameters for the broadside coupling are those found in the previous opti- mization. Two vias (made from a 0.3-mm radius metallic wire) are used to connect the ground plane of the antenna on fabric to the ground plane of the rectiﬁer. These vias do not need to be ﬂexible as they are connected to the 3) A multilayer structure including fabric layers and a 1.57-mm Duroid-5880 layer is constructed in Momen- tum, as depicted in Fig. 11. The parameters for the broadside coupling are those found in the previous opti- mization. Two vias (made from a 0.3-mm radius metallic wire) are used to connect the ground plane of the antenna on fabric to the ground plane of the rectiﬁer. These vias do not need to be ﬂexible as they are connected to the The 50- matching of the antenna and the rectiﬁer facilitates experimental measurement using standard measurement equip- ment. Eliminating the matching network would not improve the rectiﬁer efﬁciency considerably as the matching circuit on Duroid-5880 substrate has very low loss. A. Patch Antenna Measurement (2) (2) The oscillator controlling the boost converter is a ring oscillator, comprising a cascade of three inverters. Each inverter uses the UB20M voltage detector chip developed at the University of Bristol. As shown in the datasheet [35] the open-drain output is activated when the input is close to the detection threshold of about 600 mV. The current IEEE TRANSACTIONS ON MICROWAVE THEORY AND TECHNIQUES 8 Fig. 14. Measured and simulated S11 of the antenna in free space, on body, and with various bending scenarios. (a) Bending in E-plane. (b) Bending in H-plane. (c) Wrist bending. (d) Shoulder bending. The width of the desired operation band is 20 MHz centered at 2.45 GHz with S11 below −10 dB (black rectangle). 1 of the antenna in free space, on body, and with various bending scenarios. (a) Bending in E-plane. (b) Bending in H-plane. ding. The width of the desired operation band is 20 MHz centered at 2.45 GHz with S11 below −10 dB (black rectangle). Fig. 14. Measured and simulated S11 of the antenna in free space, on body, and with various bending scenarios. (a) Bending in E-plane. (b) Bending in H-plane. (c) Wrist bending. (d) Shoulder bending. The width of the desired operation band is 20 MHz centered at 2.45 GHz with S11 below −10 dB (black rectangle). Fig. 14. Measured and simulated S11 of the antenna in free space, on body, and with various bending scenarios. (a) Bending (c) Wrist bending. (d) Shoulder bending. The width of the desired operation band is 20 MHz centered at 2.45 GHz with S Fig. 16. Fabric antenna mounted on the phantom’s body. Fig. 15. Experimental setup for E/H bending of the antenna. (a) Bending in the E-plane. (b) Bending in the H-plane. Fig. 15. Experimental setup for E/H bending of the antenna. (a) Bending in the E-plane. (b) Bending in the H-plane. Fig. 16. Fabric antenna mounted on the phantom’s body. the nominal 3.7-mm thickness). Despite these variations, the antenna is robust to bending radii as small as 21 mm. When measured on body, the antenna demonstrates excellent robustness for the various scenarios shown in Fig. 14(c) and (d): wrist, back, ﬂat, and E- and H-planes bending. It can be seen that the antenna is operating as expected with the cross-polarization component being below about −20 dB for the E- and H-plane patterns [see Fig. 17(a)]. A. Patch Antenna Measurement When the antenna is mounted on the phantom’s chest and wrist, the pattern appears to be relatively similar to the reference large ground plane’s pattern [see Fig. 17(b) and (c)]. The level of back radiation increases because of the relatively small ground plane of the antenna which extends only 10 mm beyond the patch. Full-3-D radiation pattern measurements were undertaken in the anechoic chamber of the University of Bristol, as depicted in Fig. 16. The efﬁciency is accurately measured by com- parison with a reference monopole antenna following the procedure described in [25]. The antenna was initially mounted on a large ground plane (about 35 × 35 cm) as a reference best condition scenario, then measured on the chest and the wrist of a human body phantom (see Fig. 16). A maximum directivity of 9.5 dBi and an efﬁciency (calculated from measured 3-D radiation pattern) of 73% are ADAMI et al.: FLEXIBLE 2.45-GHz POWER HARVESTING WRISTBAND 9 Fig. 18. Measured and simulated rectiﬁer’s input response at various power levels. Fig. 17. Measured normalized power radiation pattern of the fabric antenna in various conﬁgurations. (a) Large ground plane. (b) Phantom’s chest. (c) Phantom’s wrist Scale: 10 dB/div Range: 0 to −40 dB at center Fig. 18. Measured and simulated rectiﬁer’s input response at various power levels. and construction parameters. Only all-fabric antennas were considered, except Moro et al. [8] whose design uses a polymer-based expanded rubber as substrate. The antennas’ aperture efﬁciency εap is calculated considering no impedance or polarization mismatch [18]. Also we deﬁne the antenna’s thickness-normalized aperture efﬁciency ε∗ ap as the antenna’s aperture efﬁciency εap divided by the antenna’s thickness ha ε∗ ap = εap ha . (4) (4) This parameter provides a better ﬁgure of merit, as it takes into account the thickness of the antenna. It can be seen that textile antennas in general provide comparable or even better performance than their rigid coun- terparts (e.g., FR4). In this paper, as the required bandwidth for power transfer is only 2 MHz, the fabric antenna can operate with a bending radius as small as 21 mm in both E- and H-planes. Other reported antennas are designed for communication, and therefore, are required to support a wider bandwidth. For this reason, they are demonstrated with lower curvature. TABLE II MEASURED AND SIMULATED FABRIC ANTENNA’S PERFORMANCE AT 2.45 GHz TABLE II MEASURED AND SIMULATED FABRIC ANTENNA’S PERFORMANCE AT 2.45 GHz A. Patch Antenna Measurement The all-fabric antenna of this paper has the highest on-body measured efﬁciency of 62%, the highest on-body measured gain of 6.8 dBi, the highest on-body aperture efﬁciency of 1.3 (considering purely fabric antennas, i.e., excluding [8]), and the highest thickness-normalized aper- ture efﬁciency of 0.34. Fig. 17. Measured normalized power radiation pattern of the fabric antenna in various conﬁgurations. (a) Large ground plane. (b) Phantom’s chest. (c) Phantom’s wrist. Scale: 10 dB/div. Range: 0 to −40 dB at center. Fig. 17. Measured normalized power radiation pattern of the fabric antenna in various conﬁgurations. (a) Large ground plane. (b) Phantom’s chest. (c) Phantom’s wrist. Scale: 10 dB/div. Range: 0 to −40 dB at center. Fig. 17. Measured normalized power radiation pattern of the fabric antenna in various conﬁgurations. (a) Large ground plane. (b) Phantom’s chest. (c) Phantom’s wrist. Scale: 10 dB/div. Range: 0 to −40 dB at center. B. Fabric-PCB Transition and Rectiﬁer Measurement The rectiﬁer efﬁciency is measured considering the RF power level at the interface fabric-PCB (reference plan 2 in Fig. 12). An RF power meter was initially used to estimate the RF power at the input of the SMA connector (at reference plane 1 in Fig. 12). Next, the loss in the fabric microstrip line, connecting the two reference planes is deducted (about 0.13 dB estimated using simulation). The measured recti- ﬁer efﬁciency includes, thus, the loss from the interface fabric-PCB. measured when the antenna is mounted on a large ground plane, which ﬁts well with the simulation (Table II). The maximum gain is indirectly calculated as the product of the measured directivity and efﬁciency as 8.1 dBi. The gain and the efﬁciency reduce to 7.3 dBi and 66% on the chest, and to 6.8 dBi and 62% on the wrist. The proximity of the phantom has little effect on the antenna performance of input response, gain, radiation efﬁciency, and radiation pattern. Measured and simulated S11 responses are given in Fig. 18 for various power levels. Measurements are realized using a standard VNA (Rohde & Schwarz ZVL6) where its output Table III gives an overview of reported fabric antennas, showing their electrical performance as well as their design IEEE TRANSACTIONS ON MICROWAVE THEORY AND TECHNIQUES ADAMI et al.: FLEXIBLE 2.45-GHz POWER HARVESTING WRISTBAND ADAMI et al.: FLEXIBLE 2.45-GHz POWER HARVESTING WRISTBAND 11 Fig. 20. Measured and simulated rectiﬁer efﬁciency at 2.45 GHz as function of the resistive load for RF power levels from −35 to 0 dBm. Fig. 21. Rectiﬁer of this paper compared with the state of the art at 2.45 GHz: Lorenz’15 [42], Song’15 [41], Olgun’11 [33], and Suh’02 [44]. Also, integrated circuit implementations for 900 MHz are shown: Power- cast [45], Stoopman’14 [43], and Le’08 [46]. The rectiﬁer in this paper is optimized for −20 dBm, where its efﬁciency of 33.6% is about 17% higher than that of Olgun’11. Fig. 20. Measured and simulated rectiﬁer efﬁciency at 2.45 GHz as function of the resistive load for RF power levels from −35 to 0 dBm. Fig. 21. Rectiﬁer of this paper compared with the state of the art at 2.45 GHz: Lorenz’15 [42], Song’15 [41], Olgun’11 [33], and Suh’02 [44]. Also, integrated circuit implementations for 900 MHz are shown: Power- cast [45], Stoopman’14 [43], and Le’08 [46]. The rectiﬁer in this paper is optimized for −20 dBm, where its efﬁciency of 33.6% is about 17% higher than that of Olgun’11. of 56% is achieved at 2.45 GHz. At −20 dBm, the efﬁciency is 33.6% at 2.45 GHz and 34.2% at 2.4 GHz, these values are the highest reported efﬁciencies at −20 dBm. At −30 dBm, a maximum of 9.6% is obtained at 2.4 GHz. Fig. 20 shows the measured and simulated rectiﬁer efﬁ- ciency as function of the resistive load for power levels from −35 to 0 dBm at 2.45 GHz. At each power level, a maximum efﬁciency is obtained for a given value of the resistive load, this can be considered as the rectenna’s optimal load impedance. The optimal impedance RRect_optim is 2.6 k at 0 dBm, 4 k at −20 dBm, and 7.2 k at −35 dBm. The boost converter (Section VI) is designed to match the rectenna at −20 dBm, i.e., to 4 k. As the change in the rectiﬁer’s optimal impedance is relatively small, a good matching is expected for a wide range of RF power. 3) The layout of the rectiﬁer was designed carefully in Momentum with: a) optimized close meshing for better accuracy and b) accurate placement of the ports. IEEE TRANSACTIONS ON MICROWAVE THEORY AND TECHNIQUES 10 10 TABLE III COMPARISON OF THE PROPOSED FABRIC ANTENNA WITH PREVIOUS REPORTED WORK TABLE III COMPARISON OF THE PROPOSED FABRIC ANTENNA WITH PREVIOUS REPORTED WORK TABLE III Fig. 19. Measured and simulated rectiﬁer’s efﬁciency at various power levels. Fig. 19. Measured and simulated rectiﬁer’s efﬁciency at various power levels power is set to various discrete levels. Simulations were realized using the ADS LSSP simulation module (large-signal S-parameters, also called power-dependent S-parameters) which allows the user to set the power level of the source. The rectiﬁer is well-matched at −20 and −10 dBm (mea- sured S11 < −10 dB at 2.45 GHz). The rectiﬁer is slightly mismatched at −30 dBm (S11 = −8.5 dB), as this power level is outside the design range. The input response S11 changes with the RF power as a result of nonlinear variations of the complex input impedance of the diode with the RF power. In fact, when the RF power level at the input of the diode changes, the operation point of the diode on its nonlinear I–V curve changes accordingly, leading to a change in the real impedance of the diode. The imaginary part of the impedance change is due to the consequent nonlinear change in the currents and voltages on the parasitic capacitances and inductances around the ideal diode. The resonance shifts to high frequency with increased power level. The simulation ﬁts well to the measurement especially at −20 dBm. Both the antenna and the rectiﬁer are well matched to 50 at the frequency and power levels of interest, which mitigates any matching loss when they are connected. Fig. 19 shows the simulated and measured RF/dc efﬁciency of the rectiﬁer as Fig. 19. Measured and simulated rectiﬁer’s efﬁciency at various power levels. function of the frequency. The same nonlinear behavior of the diode makes the matching to shift to higher frequencies with increased power level. At −10 dBm a maximum efﬁciency ADAMI et al.: FLEXIBLE 2.45-GHz POWER HARVESTING WRISTBAND 3) The layout of the rectiﬁer was designed carefully in Momentum with: a) optimized close meshing for better accuracy and b) accurate placement of the ports. 4) In the optimization process, ﬁnding the optimal input impedance of the diode was realized with a variable load resistance. This way, a better accuracy for the optimal operation point is achieved (Section V). Lorenz’15 [42] achieves remarkable rectiﬁer efﬁciencies at extremely low power levels using a custom designed hetero- junction backward tunnel diode. Their efﬁciency is reported to be below that of this paper at −20 dBm. The integrated circuit rectiﬁers of Fig. 21 are optimized for high sensitivity rather than efﬁciency. Therefore the rectiﬁer of Stoopman’14 [43] would provide higher open-circuit voltage at −20 dBm than the rectiﬁer in this paper. The design reported by Song’15 [41] is a multiband rectiﬁer with a higher efﬁciency at frequency bands below 2.45 GHz. However, for the sake of a fair comparison, only their 2.45-GHz results have been plotted here. The measured efﬁciency appears to be slightly higher than the simulated one (Figs. 19 and 20) which could be explained by the fact that the spice model of the diode is limited espe- cially at high power levels above −10 dBm. This limitation also appears in [41]. Also, the efﬁciency is very sensitive to the source power level accuracy. In fact, a 0.1-dB shift in the source power could change the efﬁciency by 2%. Reported measured efﬁciencies against RF power are plotted in Fig. 21. Solid lines show rectiﬁers operating at 2.45 GHz, using discrete diodes (see labels in Fig. 21). Dashed lines show integrated rectiﬁers with the highest reported efﬁciencies; these operate at 0.9 GHz. Of those using commercially available diodes, the rectiﬁer presented herein has the highest efﬁciency at power levels below −16 dBm. At high power levels, about 0 dBm, the efﬁciency of the present rectiﬁer starts to reduce, as expected, as the dc output voltage approaches the breakdown voltage of the diode. The same phenomenon is present in the other reported Schottky-diode-based designs. The present rectiﬁer has also a wide power operating range, over three orders of magnitude. The maximum efﬁciency is 64.6%, achieved at 2.45 GHz, −2 dBm RF power level, and with 3-k load. The high efﬁciency achieved around −20 dBm is a result of the following factors. C. Wristband With Parallel- and Series-Connected Rectennas C. Wristband With Parallel- and Series-Connected Rectennas The RF harvesting wristband of Fig. 2 comprises two rectennas, with a 40-mm curvature radius. The goal is to combine and use them in the most efﬁcient way. The two rectennas can operate in three different ways: operate indepen- dently, connected in parallel or connected in series. A base- line performance level is ﬁrst established without the use of power management circuits, for the case when one rectenna faces the RF power source, while the other one faces in the opposite direction (see Fig. 22). This conﬁguration emulates 1) Use of Duroid-5880 substrate with extremely low dielec- tric loss. 2) Unlike other systems such as Song’15 [41] in which the rectiﬁer was designed for a wide range of RF power levels, the rectiﬁer of this paper was locally optimized at −20 dBm. 12 IEEE TRANSACTIONS ON MICROWAVE THEORY AND TECHNIQUES 12 Fig. 25. Experimental setup for the measurement of the complete system. This setup corresponds to one channel where the other channel is identical. For the overall system, the output terminals of the two boost converters are connected in parallel to the output Battery. Fig. 22. Measurement setup for the harvesting wristband. Fig. 25. Experimental setup for the measurement of the complete system. This setup corresponds to one channel where the other channel is identical. For the overall system, the output terminals of the two boost converters are connected in parallel to the output Battery. The received RF power is the power at the input of the rectiﬁer, measured using a reference calibrated rectenna. This is made from a fabric antenna (identical to the one on the wristband harvester) connected to rectiﬁer with known RF/dc transfer characteristic. A lookup table is used to estimate the RF power level at the input of the rectiﬁer from the output dc voltage of the reference rectenna. Fig. 24 also shows the relative power of each combined system, deﬁned as the total dc output of a system divided by the output of the rectenna facing the transmitter. A relative power of 1 would signify that the rectenna facing away from the transmitter has no impact on the system. Fig. 22. Measurement setup for the harvesting wristband. Fig. 23. Measurement setup for testing various rectenna interconnection scenarios for the harvesting wristband: single, parallel, and series. The value of each resistor is 4 k. C. Wristband With Parallel- and Series-Connected Rectennas It is apparent, that when the two rectennas are separate, only the rectenna facing the transmitter generates dc power while the other rectenna generates almost no power. When connected in parallel or in series, the relative power is below 1, showing that power is lost due to connecting to the rectenna facing away from the transmitter. Up to 50% of the potential output is seen to be lost, which is a result of voltage reversal in the series connection, and reverse leakage current in the parallel connection [47]. This loss appears in direct-connected systems whenever one rectenna receives less power than the other. Fig. 23. Measurement setup for testing various rectenna interconnection scenarios for the harvesting wristband: single, parallel, and series. The value of each resistor is 4 k. Fig. 24. Measured dc power and relative power in various interconnection scenarios (see Figs. 22 and 23). p The use of 2 and 8 k as optimal impedances for the series and the parallel conﬁgurations, respectively, is an approxi- mation. In fact, it has been veriﬁed experimentally that for the parallel unbalanced conﬁguration, the second rectiﬁer not receiving RF power has little impact on the optimal impedance. While for the series conﬁguration, the change in the optimal impedance is more pronounced but does not reach two times the optimal impedance of a single rectenna. This does not affect the fact that a considerable proportion of power is lost in the other rectenna not receiving any RF power. A simple calculation can show that doubling or dividing the optimal impedance of the rectenna by 2 only reduces the output dc power by about 10% (can also be seen in Fig. 20). Therefore, we estimate that the results presented in Fig. 24 are quite accurate. Fig. 24. Measured dc power and relative power in various interconnection scenarios (see Figs. 22 and 23). a scenario where the wristband is worn by a user. As shown in Fig. 22, due to the curvature of the wristband, the recten- nas consequently face opposite directions. The wristband is positioned 2 m away from the transmitter, in a realistic indoor environment. The two rectennas are ﬁrst measured separately with a 4-k load, and then connected in parallel with a 2-k load and in series with an 8-k load (see Fig. 23). The dc power output against received RF power is shown in Fig. C. Wristband With Parallel- and Series-Connected Rectennas 24, for the two individual rectennas, and for the two combined systems. D. Complete RF Wristband Using Power Management D. Complete RF Wristband Using Power Management A power management circuit per rectenna permits the separation of rectenna channels to avoid the loss shown in the previous section, and ensures that each rectenna is working at its maximum power point. For the overall system, two identical channels exist with the output terminals of the two boost converters being connected in parallel. The diagram of one channel is shown in Fig. 25. ADAMI et al.: FLEXIBLE 2.45-GHz POWER HARVESTING WRISTBAND 13 Fig. 26. Measured efﬁciencies as function of the received RF power. The results shown here correspond to the setup in Fig. 25. Fig. 27. Measured impedances, and zone where matching efﬁciency exceeds 95%. This zone is derived from the results of Fig. 20. Fig. 27. Measured impedances, and zone where matching efﬁciency exceeds 95%. This zone is derived from the results of Fig. 20. Fig. 26. Measured efﬁciencies as function of the received RF power. The results shown here correspond to the setup in Fig. 25. COMPARISON OF LOW-POWER RF HARVESTERS COMPRISING RECTIFIER AND POWER MANAGEMENT CIRCUIT COMPARISON OF LOW-POWER RF HARVESTERS COMPRISING RECTIFIER AND POWER MANAGEMENT CIRCUIT COMPARISON OF LOW-POWER RF HARVESTERS COMPRISING RECTIFIER AND POWER MANAGEMENT CIRCUIT Using the notation of Fig. 25, the rectiﬁer efﬁciency is Rect = PRect PRF = IRectVRect PRF (5) the boost converter efﬁciency is Boost = PBoost −PQuiescent_Boost PRect = IBatVBat IRectVRect (6) and the end-to-end efﬁciency is End−to−end = Rect Boost. (7) COMPARISON OF LOW-POWER RF HARVESTERS COMPRISING RECTIFIER AND POWER MANAGEMENT CIRCUIT Using the notation of Fig. 25, the rectiﬁer efﬁciency is Using the notation of Fig. 25, the rectiﬁer efﬁciency is Rect = PRect PRF = IRectVRect PRF (5) (5) the boost converter efﬁciency is the boost converter efﬁciency is Boost = PBoost −PQuiescent_Boost PRect = IBatVBat IRectVRect (6) and the end-to-end efﬁciency is End−to−end = Rect Boost. (7) Boost = PBoost −PQuiescent_Boost PRect = IBatVBat IRectVRect (6) and the end-to-end efﬁciency is (7) End−to−end = Rect Boost. Measured rectiﬁer, boost, and end-to-end efﬁciencies against received RF power are shown in Fig. 26. levels and therefore has a higher minimum power threshold and reduced range. The system reported in this paper was optimized for −20 dBm, a power at which none of the reported rigid systems can operate. The rectiﬁer efﬁciency increases monotonically with the RF power from 32% at −20 dBm to 56% at −5.6 dBm. This agrees well with the results of Fig. 21, where the rectiﬁer is investigated in isolation. Results are provided up to −5.6 dBm, as the source saturates at this point, although from Fig. 21 it is apparent that the rectiﬁer’s efﬁciency continues to increase. The efﬁciency of the boost converter is positive from −24.3 dBm and achieves a maximum of around 57% at −14.2 dBm. The end-to-end efﬁciency reaches a maximum of 28.7% at −7 dBm. REFERENCES [21] D. M. Pozar, Microwave Engineering, 4th ed. Hoboken, NJ, USA: Wiley, 2011. [1] R. R. G. Perron, G. C. Huang, and M. F. Iskander, “Textile electro- magnetic coupler for monitoring vital signs and changes in lung water content,” IEEE Antennas Wireless Propag. Lett., vol. 14, pp. 151–154, 2015. [22] C. Hertleer, A. V. Laere, H. Rogier, and L. V. Langenhove, “Inﬂuence of relative humidity on textile antenna performance,” Textile Res. J., vol. 80, no. 2, pp. 177–183, Jan. 2010. [23] J. Lilja, P. 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Fig. 27 shows the rectenna’s optimal load impedance, the boost converter’s input impedance, and the impedance range where the matching efﬁciency exceeds 95%. The boost converter impedance is seen to provide at least 95% matching over the entire working range, from −24.3up to −5.6 dBm. Table IV compares the results of this paper, with those of reported low-input-power RF harvesters that comprise a rectenna and an impedance-matching power management cir- cuit. The ﬂexible system developed in this paper has the lowest RF power threshold of −24.3 dBm, and the highest matching efﬁciency of >95%, despite being the only ﬂexible system. The quiescent consumption of 150 nA achieved in this paper is comparable with the best quiescent current achieved by the integrated design of [48]. The system of [49] achieves a higher end-to-end efﬁciency, as it is optimized for higher power This work assumes that the wearable should be thin, ﬂexible, and close to the body. The 64-mm-wide wristband provides a 10-mm ground plane overlap, which is sufﬁcient to minimize the coupling with the body, and the resulting loss. In this case, the textiles with the lowest loss have been selected. For more constrained envelopes, where, due to a reduced antenna ground plane, the coupling with the body is higher, the best overall performance is not necessarily obtained using the lowest loss materials, and thicker structures may be required [39]. 14 ADAMI et al.: FLEXIBLE 2.45-GHz POWER HARVESTING WRISTBAND degree from the University of Bristol, Bristol, U.K., in 1993, for research on the design and ﬁnite-difference time-domain modeling of printed antenna elements. [38] S. Yan and G. A. E. Vandenbosch, “Radiation pattern-reconﬁgurable wearable antenna based on metamaterial structure,” IEEE Anten- nas Wireless Propag. Lett., vol. 15, pp. 1715–1718, 2016, doi: 10.1109/LAWP.2016.2528299. d i d [39] S. J. Boyes, P. J. Soh, Y. Huang, G. A. E. Vandenbosch, and N. 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His current research interests include practical antenna and system design for a variety of communications and radar applications such as ground penetrating radar, performance evaluation of antennas in mobile radio, electrically small elements, active/tuneable elements, and vehicle-mounted conformal antennas. [40] Y. Ouyang and W. J. Chappell, “High frequency properties of electro- textiles for wearable antenna applications,” IEEE Trans. Antennas Propag., vol. 56, no. 2, pp. 381–389, Feb. 2008. [41] C. Song, Y. Huang, J. Zhou, J. Zhang, S. Yuan, and P. Carter, “A high- efﬁciency broadband rectenna for ambient wireless energy harvest- ing,” IEEE Trans. Antennas Propag., vol. 63, no. 8, pp. 3486–3495, Aug. 2015. [42] C. H. P. Lorenz et al., “Breaking the efﬁciency Barrier for ambi- ent microwave power harvesting with heterojunction backward tun- nel diodes,” IEEE Trans. Microw. Theory Techn., vol. 63, no. 12, pp. 4544–4555, Dec. 2015. Guang Yang received the Ph.D. degree from the Bio-inspired VLSI Circuits and Systems Group, Department of Bioengineering, Imperial College London, London, U.K., in 2012. During 2013–2016, he was a Research Associate with the Electrical Energy Management Group, Uni- versity of Bristol, Bristol, U.K. His current research interests include auditory (cochlea) modeling and signal processing, speech enhancement and psy- chophysical evaluation, and low-power analog IC design for biomedical and energy harvesting appli- cations. [43] M. Stoopman, S. Keyrouz, H. J. Visser, K. Philips, and W. A. ADAMI et al.: FLEXIBLE 2.45-GHz POWER HARVESTING WRISTBAND 15 [32] X. Guo, L. Zhu, J. Wang, and W. Wu, “Wideband microstrip-to- microstrip vertical transitions via multiresonant modes in a slot- line resonator,” IEEE Trans. Microw. Theory Techn., vol. 63, no. 6, pp. 1902–1909, Jun. 2015. Plamen Proynov received the B.Sc. and M.Sc. degrees in automation and control engineering from the University of Varna, Varna, Bulgaria, in 2005 and 2008, respectively, and the Ph.D. degree from the Electrical Energy Management Group, University of Bristol, Bristol, U.K., in 2014. [33] U. Olgun, C. C. Chen, and J. L. Volakis, “Investigation of rectenna array conﬁgurations for enhanced RF power harvesting,” IEEE Antennas Wireless Propag. Lett., vol. 10, pp. 262–265, 2011. Since 2014, he has been a Post-Doctoral Research Associate in energy management, harvesting, and wireless power transfer with the Department of Elec- trical Engineering, University of Bristol. He is cur- rently involved in the SPHERE project, where he is investigating sensor-powered electronics and RF power transfer in healthcare applications. His previous research interests included kinetic energy harvesting and renewable micropower sources. His current research interests include low- power power management, energy harvesting, and wireless sensors. Since 2014, he has been a Post-Doctoral Research Associate in energy management, harvesting, and wireless power transfer with the Department of Elec- trical Engineering, University of Bristol. He is cur- rently involved in the SPHERE project, where he is investigating sensor-powered electronics and RF power transfer in healthcare applications. His previous research interests included kinetic energy harvesting and renewable micropower sources. His current research interests include low- power power management, energy harvesting, and wireless sensors. [34] T. Paing, J. Shin, R. Zane, and Z. Popovic, “Resistor emulation approach to low-power RF energy harvesting,” IEEE Trans. Power Electron., vol. 23, no. 3, pp. 1494–1501, May 2008. [35] G. Yang, C. Zhang, B. Stark, P. Proynov, and S.-E. Adami, “Voltage detector,” U.K. Patent Application GB 1607304.1, Apr. 27, 2016. [36] T. Kellomäki, “Analysis of circular polarization of cylindrically bent microstrip antennas,” Int. J. Antennas Propag., vol. 2012, 2012, Art. no. 858031, 8 pp. pp [37] N. Amaro, C. Mendes, and P. Pinho, “Bending effects on a textile microstrip antenna,” in Proc. IEEE Int. Symp. Antennas Propag. (APSURSI), Jul. 2011, pp. 282–285. Geoffrey S. Hilton received the B.Sc. degree from the University of Leeds, Leeds, U.K., in 1984, and the Ph.D. REFERENCES Haagenson, S. Noghanian, P. de Leon, and Y. H. Chang, “Textile antennas for spacesuit applications: Design, simulation, man- ufacturing, and testing of textile patch antennas for spacesuit appli- cations,” IEEE Antennas Propag. Mag., vol. 57, no. 4, pp. 64–73, Aug. 2015. [28] L. Inclan-Sanchez, J. L. Vazquez-Roy, and E. Rajo-Iglesias, “Proximity coupled microstrip patch antenna with reduced harmonic radiation,” IEEE Trans. Antennas Propag., vol. 57, no. 1, pp. 27–32, Jan. 2009. [7] S. J. Cho, Y. H. Jung, and Z. 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Rogier, F. Alimenti, P. Mezzanotte, and L. Roselli, “Wearable textile antenna magnetically coupled to ﬂexible active electronic circuits,” IEEE Antennas Wireless Propag. Lett., vol. 13, pp. 209–212, 2014. ADAMI et al.: FLEXIBLE 2.45-GHz POWER HARVESTING WRISTBAND ADAMI et al.: FLEXIBLE 2.45-GHz POWER HARVESTING WRISTBAND Serdijn, “Co-design of a CMOS rectiﬁer and small loop antenna for highly sensitive RF energy harvesters,” IEEE J. Solid-State Circuits, vol. 49, no. 3, pp. 622–634, Mar. 2014. [44] Y.-H. Suh and K. Chang, “A high-efﬁciency dual-frequency rectenna for 2.45- and 5.8-GHz wireless power transmission,” IEEE Trans. Microw. Theory Techn., vol. 50, no. 7, pp. 1784–1789, Jul. 2002. [45] Powercast Corp. P1110, accessed on Jun. 28, 2016. [Online]. Available: http://www.powercastco.com/ cations. [46] T. Le, K. Mayaram, and T. Fiez, “Efﬁcient far-ﬁeld radio frequency energy harvesting for passively powered sensor networks,” IEEE J. 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His current research interests include analog IC design, mainly on power management ICs. Dibin Zhu received B.Eng. degree in information and control engineering from Shanghai Jiao Tong University, Shanghai, China, in 2004, and the M.Sc. degree in RF communication systems and Ph.D. degree in electrical and electronic engineering from the University of Southampton, Southampton, U.K., in 2005 and 2009, respectively. Dibin Zhu received B.Eng. degree in information and control engineering from Shanghai Jiao Tong University, Shanghai, China, in 2004, and the M.Sc. degree in RF communication systems and Ph.D. degree in electrical and electronic engineering from the University of Southampton, Southampton, U.K., in 2005 and 2009, respectively. He is currently a Lecturer with the Energy Harvest- ing Research Group, University of Exeter, Exeter, U.K. He has been a Post-Doctoral Researcher with the University of Bristol, Bristol, U.K., since 2013. He is part of the Sphere IRC and part of the Electrical Energy Management Research Group. His current research interests include the development of safe and reliable end-to-end solutions for far-ﬁeld wireless charging. ADAMI et al.: FLEXIBLE 2.45-GHz POWER HARVESTING WRISTBAND His current research interests include energy harvesting from various sources, e.g., vibration, sound, wind, human movement, solar, RF, wireless power transfer, etc., and their applications, as well as advanced materials for energy harvesting applications. Dibin Zhu received B.Eng. degree in information and control engineering from Shanghai Jiao Tong University, Shanghai, China, in 2004, and the M.Sc. degree in RF communication systems and Ph.D. degree in electrical and electronic engineering from the University of Southampton, Southampton, U.K., in 2005 and 2009, respectively. He is currently a Lecturer with the Energy Harvest- ing Research Group, University of Exeter, Exeter, U.K. His current research interests include energy harvesting from various sources, e.g., vibration, sound, wind, human movement, solar, RF, wireless power transfer, etc., and their applications, as well as advanced materials for energy harvesting applications. Dibin Zhu received B.Eng. degree in information and control engineering from Shanghai Jiao Tong University, Shanghai, China, in 2004, and the M.Sc. degree in RF communication systems and Ph.D. degree in electrical and electronic engineering from the University of Southampton, Southampton, U.K., in 2005 and 2009, respectively. He is currently a Lecturer with the Energy Harvest- ing Research Group, University of Exeter, Exeter, U.K. His current research interests include energy harvesting from various sources, e.g., vibration, sound, wind, human movement, solar, RF, wireless power transfer, etc., and their applications, as well as advanced materials for energy harvesting applications. Salah-Eddine Adami received the engineering Diploma degree in electrical engineering from the National Institute for Applied Sciences, Lyon, France, in 2010, and the Ph.D. degree from the Ecole Centrale de Lyon, Lyon, in 2013. He has been a Post-Doctoral Researcher with the University of Bristol, Bristol, U.K., since 2013. He is part of the Sphere IRC and part of the Electrical Energy Management Research Group. His current research interests include the development of safe and reliable end-to-end solutions for far-ﬁeld wireless charging. Salah-Eddine Adami received the engineering Diploma degree in electrical engineering from the National Institute for Applied Sciences, Lyon, France, in 2010, and the Ph.D. degree from the Ecole Centrale de Lyon, Lyon, in 2013. He has been a Post-Doctoral Researcher with the University of Bristol, Bristol, U.K., since 2013. He is part of the Sphere IRC and part of the Electrical Energy Management Research Group. His current research interests include the development of safe and reliable end-to-end solutions for far-ﬁeld wireless charging. Salah-Eddine Adami received the engineering Diploma degree in electrical engineering from the National Institute for Applied Sciences, Lyon, France, in 2010, and the Ph.D. degree from the Ecole Centrale de Lyon, Lyon, in 2013. ADAMI et al.: FLEXIBLE 2.45-GHz POWER HARVESTING WRISTBAND Salah-Eddine Adami received the engineering Diploma degree in electrical engineering from the National Institute for Applied Sciences, Lyon, France, in 2010, and the Ph.D. degree from the Ecole Centrale de Lyon, Lyon, in 2013. He has been a Post-Doctoral Researcher with the University of Bristol, Bristol, U.K., since 2013. He is part of the Sphere IRC and part of the Electrical Energy Management Research Group. His current research interests include the development of safe and reliable end-to-end solutions for far-ﬁeld wireless charging. Salah-Eddine Adami received the engineering Diploma degree in electrical engineering from the National Institute for Applied Sciences, Lyon, France, in 2010, and the Ph.D. degree from the Ecole Centrale de Lyon, Lyon, in 2013. He is currently a Lecturer with the Energy Harvest- ing Research Group, University of Exeter, Exeter, U.K. His current research interests include energy harvesting from various sources, e.g., vibration, sound, wind, human movement, solar, RF, wireless power transfer, etc., and their applications, as well as advanced materials for energy harvesting applications. IEEE TRANSACTIONS ON MICROWAVE THEORY AND TECHNIQUES IEEE TRANSACTIONS ON MICROWAVE THEORY AND TECHNIQUES 16 Yi Li received the B.Eng. degree in electronic engi- neering from the University of Warwick, Coventry, U.K., in 2008, and the M.Sc. degree in microsystem technology and Ph.D. degree in printed smart fabrics from the University of Southampton, Southampton, U.K., in 2009 and 2013, respectively. Ian J. Craddock (M’09–SM’10–F’16) is currently a Full Professor with the University of Bristol, Bristol, U.K., and the Director of the ﬂagship “SPHERE” centre (www.irc-sphere.ac.uk) comprising approxi- mately 100 researchers and clinicians involved in Internet of Things (IoT) technology for health. He has been involved in healthcare technology for 15 years and founded a company that is currently completing trials of a CE-marked breast imaging device based on his research. He is currently a Managing Director with the Telecommunications Research Lab, Toshiba, Bristol, where he is responsible for a portfolio of both internal and collaborative communications, IoT, and smart city research. He has authored over 150 papers and serves on the healthcare strategy board for the U.K.’s largest engineering funder. He possesses more than three years work expe- rience at the University of Southampton. In addi- tion, he has also focused on various novel func- tional materials formulation development for print- able smart fabrics research, including conductor, semiconductor, PV inks, bioelectrodes, metal oxides, and dielectrics. His current research interests include printed fabric-based antennas, capacitors, transistors, solar cells, speakers, and stimulation electrodes. Steve P. Beeby received the B.Eng. (Hons.) degree in mechanical engineering from the University of Portsmouth, Portsmouth, U.K., in 1992, and the Ph.D. degree in MEMS resonant sensors from the University of Southampton, Southampton, U.K., in 1998. He was a Principal or Co-Investigator on an addi- tional 18 projects and has coordinated two European Union research projects. He is currently the Head of the Electronics and Electrical Engineering Research Group. He leads the U.K.’s Energy Harvesting Net- work. He is currently leading three U.K. funded research projects. He is a Co-Founder of Perpetuum Ltd., a University spin-out based upon vibration energy harvesting formed in 2004, Smart Fabric Inks Ltd., and D4 Technology Ltd. He has co-authored/edited four books including Energy Harvesting for Autonomous Systems (Artech House, 2010). He has given 14 invited talks and has over 200 publications and 10 patents. He has an h-Index of 39 with >9500 citations. His current research interests include energy harvesting, e- textiles, MEMS, and active printed materials development. Bernard H. Stark received the M.S. degree in elec- trical engineering from the Swiss Federal Institute of Technology, Zurich, Switzerland, in 1995, and the Ph.D. degree in engineering from Cambridge University, Cambridge, U.K., in 2000. IEEE TRANSACTIONS ON MICROWAVE THEORY AND TECHNIQUES Bernard H. Stark received the M.S. degree in elec- trical engineering from the Swiss Federal Institute of Technology, Zurich, Switzerland, in 1995, and the Ph.D. degree in engineering from Cambridge University, Cambridge, U.K., in 2000. University, Cambridge, U.K., in 2000. He was a Junior Research Fellow with St. Hugh’s College, Oxford, U.K., and a member of the Control and Power Group, Imperial College, London, U.K. He is currently a Reader in electrical and electronic engineering with the University of Bristol, Bristol, U.K., and a member of the Electrical Energy Man- agement Research Group. His current research interests include renewable power sources and power electronics. Dr. Beeby was the recipient of two prestigious EPSRC Research Fellowships to investigate the combination of screen-printed active materials with micro- machined structures and textiles for energy harvesting and was also awarded a Personal Chair in 2011. He is currently the Chair of the International Steering Committee for the PowerMEMS Conference series.
https://openalex.org/W4229825797	https://scindeks-clanci.ceon.rs/data/pdf/0085-6320/2021/0085-63202102538B.pdf	Latin	null	Technofeudalism illustrated by Trump's Twitter suspension and Australia vs. Google and Facebook dispute	Sociološki pregled/Sociološki pregled	2,021	cc-by-sa	12,410	Љубиша М. Бојић1 Дамир Г. Зејнулаховић2 Милош В. Јанковић3 Универзитет у Београду, Институт за филозофију и друштвену теорију Београд (Србија) Љубиша М. Бојић1 Дамир Г. Зејнулаховић2 Милош В. Јанковић3 Универзитет у Београду, Институт за филозофију и друштвену теорију Београд (Србија) 316.774:621.39 Прегледни научни рад Примљен 04/05/2021 Измењен 25/05/2021 Прихваћен 30/05/2021 doi: 10.5937/socpreg55-32105 1 ljubisa.bojic@instifdt.bg.ac.rs 2 damir.zejnulahovic@instifdt.bg.ac.rs 3 milos.jankovic@instifdt.bg.ac.rs 4 Oвaј рaд je рeaлизoвaн уз пoдршку Mинистaрствa прoсвeтe, нaукe и тeхнoлoшкoг рaзвoja Рeпубликe Србиje прeмa Угoвoру o рeaлизaциjи и финaнсирaњу нaучнoистрaживaчкoг рaдa. 1 ljubisa.bojic@instifdt.bg.ac.rs Увод Технолошки развој, био он оличен у иновацијама у процесу производње или, као у случају који ћемо разматрати овде, у појави нових начина комуникације, нужно утиче на читаву друштвену реалност, мења устаљене облике друштвених односа и узрокује појаву нових друштвених феномена које је потребно испитати. Тако, још сре- дином прошлог века, Дебор примећује да спектакл, односно „друштвени однос међу људима посредован сликама” (Debord, 2003, str. 18) постаје основа живота у свим посеб- ним аспектима информисања. Наравно, Дебор ово везује, пре свега, за значај телеви- зије која се појављује као најбитнији преносник знакова и порука, док би паралелу у данашњици најпре пронашли у социјалним (друштвеним) медијима које, као што ћемо даље у раду видети, постају преносник без којег је немогуће замислити друштво. Почетком текуће 2021. године, у јеку пандемије коронавируса, два догађаја су уздрмала светску јавност, указујући да је по среди потенцијални трансфер моћи између држава и високотехнолошких компанија. Наиме, прво је почетком јану- ара компанија Твитер одлучила да суспендује профил одлазећег председника САД, Доналда Трампа, са те мреже због наводног говора мржње (Twitter, 2021). Друго, мул- тинационална медијска компанија Фејсбук извршила је снажан притисак на Аустра- лију да измени предлог закона који би компанију приморао да плати медијским изда- вачима за њихов садржај објављен на овој друштвеној мрежи (BBC, 2021). Ова два догађаја задобила су глобалне размере, актуализовала расправу о ути- цају глобализације на суверенитет националних држава и посведочила да свет улази у нови технократски или технофеудални светски поредак. Постоје три школе мишљења које дају различите одговоре на питање државног суверенитета у контексту глобализације. Хиперглобалисти сматрају да је улога државе прилично смањена и да је нарочито угрожен њен економски суверенитет. С друге стране, скептици на глобализацију гледају као на продукт самих држава, те тврде да је њихова улога и моћ остала непромењена. Између ова два теоријска пола налазе се трансформационалисти. Улога државе, кажу они, нити је ослабила, нити остала нетакнута, већ се значајно променила (Reddy, 2012, str. 61–64). Посебно место заузима литература која се фокусира на мултинационалне ком- паније. Док одређени аутори препознају утицај компанија на државни суверенитет (Vernon, 1971), други сматрају да је он неоправдано наглашен (Kline, 2006). Постоји мишљење и да је до осамдесетих година двадесетог века држава и даље имала при- мат, али је крај старог и почетак новог миленијума донео значајне промене, међу којима нове технологије и „појаву електронски умрежене светске економије”, чиме је суверенитет држава заиста постао упитан (Kobrin, 2009, str. 3). ТЕХНОФЕУДАЛИЗАМ НА ПРИМЕРИМА ТРАМПОВЕ СУСПЕНЗИЈЕ СА ТВИТЕРА И СПОРА АУСТРАЛИЈЕ СА ГУГЛОМ И ФЕЈСБУКОМ 4 Сажетак: Друштвени медији постају незаобилазан комуникациони алат данашњице, укључујући и политичку комуникацију. Самим тим, технолошке ком- паније које њима управљају имају велику моћ. Свака њихова интервенција у јавној сфери може да има далекосежне последице. Фокусирамо се на два случаја утицаја технолошких компанија на медије, изборни и легислативни процес, као основне чиниоце демократије. Разматрамо импликације забране Доналду Трампу да користи Твитер, као и окршај медијске политике Аустралије са Фејсбуком и Гуглом. Дола- зимо до закључка да су технолошке компаније у овим случајевима преузеле моћ која је претходно припадала судској и легислативној грани власти, у смислу одређивања шта је говор мржње, као и регулацију медија. Самим тим, угрожени су информи- сање и суверенитет држава у којима се спроводе ове интервенције. Ови догађаји указују да свет улази у период доминације технолошких компанија, који можемо назвати технократијом или технофеудализмом. Даља разматрања треба да иду у правцу дефинисања друштвених медија као јавног добра у коме утицај треба да имају друштва, а не само технолошке компаније који су власници ових комуника- ционих платформи. Кључне речи: друштвени медији, суверинитет, технолошке компаније, техно- кратија, технофеудализам 538 Социолошки преглед, vol. LV (2021), nо. 2, стр. 538–561 Увод Утицај друштвених медија на политичке процесе је у константном порасту, па тако одређена истраживања говоре да на Западу кандидати на изборима у све већем броју користе Твитер, како би се промовисали и остварили контакт са потен- цијалним гласачима (Enli, 2017; Enli & Skogerbo, 2013; Graham, Jackson, & Broesma, 2016). Поред недвосмисленог значаја које у свакодневном животу имају друштвени медији као једaн од, ако не и примарни, начин комуникације између људи, оно што посебно привлачи политичаре је чињеница да путем друштвених медија, пре- васходно Твитера, могу остварити директан контакт са бирачима, заобилазећи 539 Љубиша М. Бојић, Дамир Г. Зејнулаховић, Милош В. Јанковић, Технофеудализам на... уредничку политику традиционаних медија. На тај начин политичари имају моћ да снажније, са више контроле, представе себе и своју поруку, као и да постигну ути- сак директне комуникације и одговорности са појединцем, што представља важну чињеницу у савременом историјском моменту, где гласови који оптужују полити- чаре за отуђеност од свакодневног живота својих грађана постају све громкији (Enli, 2017; Ouyang & Waterman, 2020). Ову улогу друштвених медија примећују и Гejнус и Вагнер (Gainous & Wagner, 2014), који наводе да друштвени медији померају поље моћи у корист политичара, где „твитовање до моћи” постаје политички стандард, нешто што је обавезно у савременом политичком тренутку и што ће свакако зна- чити још више у будућности. На овај начин друштвени медији постају незаобилазни део политичких процеса. Као што смо приказали, утицај друштвених медија на политичке процесе већ сада може да се окарактерише као кључан, док је сигурно да ће тај значај све више расти у наредним годинама. Међутим, сада је потребно у нашу анализу укључити и чиње- ницу да су ови друштвени медији заправо продукти приватних компанија које задр- жавају право њихове модерације. Како нас Варис (Varis, 2020) подсећа, иако неке тео- рије, попут семиотичке демократије, као и саме компаније које путем маркетинга желе да представе своје платформе, друштвене мреже, као браниоце демократије и про- сторе у којима је гарантован слободан говор, то није њихова ни примарна функција ни намера. Заправо, модерација није периферна функција друштвених мрежа, већ цен- трална, то јест, она је начин на који друштвене мреже спроводе своје задате циљеве на тржишту, одређујући које групе и интереси су допустиви, а који нису (Gillespie, 2018). Проблем говора мржње на интернету, с једне стране можемо посматрати из угла права на слободу говора, које гарантује амерички Устав. С друге стране, бројне јури- сдикције широм света јасно дефинишу концепт говора мржње. Увод Овде постоји свакако сукоб између широког схватања слободе говора и гаранције на достојанство поједи- наца и група, који могу бити предмет говора мржње (Heldt, 2019). Треба имати у виду и забрану цензуре од стране државе, јер је она прописана од стране многих земаља, чиме се додатно гарантује слобода говора. Једна од полазних тачака овог разматрања јесте да државе и компаније деле одговорност регулације лажних вести, говора мржње и разноразних саботажа, који се могу појавити на интернету. Овде је највећи притисак тренутно на власницима водећих друштвених медија, технолошким компанијама, које се у највећој мери налазе у Америци. У овом случају било би неадекватно априори оптужити компа- није које су власници друштвених медија за цензуру, јер је недостатак регулатива утицао на садашње стање ствари. Наравно, треба имати у виду да ово не оправдава експлицитно мешање у изборни процес, тако да технолошка компанија транспа- рентно стане на једну од страна које учествују у изборима. Овакве одлуке могу да испровоцирају жестоке реакције јавности, као и да угрозе демократију. Једна од одлука коју је усвојила компанија Фејсбук односи се на лажне вести и прописује да ће смањити видљивост оваквих дезинформација, у смислу да оне неће бити широко дистрибуиране, иако би на то имале права у складу са важећим системима за препоручивање – алгоритмима који приказују садржаје корисни- цима друштвених медија. Ове објаве се означавају као лажне, што раде независни стручњаци (Facebook, 2021). Додатно, Фејсбук је успоставио и апелациони процес 540 Социолошки преглед, vol. LV (2021), nо. 2, стр. 538–561 који омогућава спорење одлука ове компаније од угрожених страна. Апелационо тело надгледа посебан одбор (Collins, 2021). Иако наведени примери сведоче да у погледу легислативе Фејсбук успоставља стистем сличан оном државном и да тиме деимично преузима овлашћења једне гране власти, ово је само иницијатива да се хитно одговори на изазов настао у вакууму нових технологија и застарелих закона. Можемо да подведемо ово питање под кон- цепт дефинисан у литератури као „проблем много руку”, што је потешкоћа да се децидно одреди ко је одговоран у комплексним срединама, где учешће у одлучивању имају многи чиниоци. Томпсон детаљно описује ово питање, наводећи више при- мера, и покушава да утврди приступ дефинисању одговорности (Thompson, 2014). Ван Друнен такође објашњава „проблем много руку”, од централизоване до подељене одговорности (van Drunen, 2020). У складу с тим, конектовани свет односи се на онлајн платформе које имају далекосежни утицај на друштва широм света. Увод Нови концепт функционисања, који намеће интернет, узрокује трансформацију тржишта, процедура и демократских процеса (Jevtović, 2020). У анализи друштава заснованих на онјалн платформама истиче се сукоб идеолошких система и друштвених чини- лаца, што укључуује владе, компаније и цивилни сектор, као и поделу испреплетане одговорности, како би се постигла приватност, тачност, безбедност, фер окружење, доступност и демoкратичност (Van Dijck et al., 2018). Ове вредности су у ствари угро- жене у друштву где значајан утицај имају глобалне и регионалне онлајн платформе, које прожимају јавне и приватне секторе, а и многе области, као на пример инфор- мисање, транспорт, здравље и образовање. Неки од ових пороцеса тичу се локал- них питања, док многи утичу и на глобалну сферу. Имајући ово у виду, треба истаћи сукобе између мултинационалних компанија, влада и мултилатералних чинилаца као релевантне у разматрању прерасподеле моћи у ери дигиталних технологија. Феномен на који се фокусирамо у овом раду тиче се пре свега утицаја техно- лошких компанија, тј. друштвених медија на демократију и суверенитет у Сједиње- ним Америчким Државама и Аустралији, кроз примере суспензије бившег председ- ника Доналда Трампа са друштвене мреже Твитер и окршај јавне медијске политике Аустралије са технолошким гигантима Фејсбуком и Гуглом. Све ово посматраћемо кроз призму суверенитета. Суверенитет и глобализација У класичним текстовима (Bodin, 2002; Hobbes, 1961) вредност постојања суверена обично лежи у његовој моћи да гарантује мир. Савремени аутори, ипак, на много ра- зноврсније начине говоре о појму суверенитета. Политички теоретичар Харолд Ласки (Harold Laski) пита се „да ли би у држави требало да постоји једна власт без ограни- чења ма које врсте” (Laski, 1934, str. 62). Закључује да „нема сталног права на власт” и да се свака „влада мора покорити мишљењу оних који трпе последице њених аката” (Laski, 1934, str. 63). Слично је и код француског филозофа Жака Маритена (Jacques Maritain). За њега је овај појам „суштински погрешан” (Maritain, 1966, str. 29). Аутори који одбацују појам суверенитета (Duguit, 1997) или о њему говоре у негативном светлу (Laski, 1934; Maritain, 1966) обично суверенитет разумевају у бодe- новском (Jean Bodin) смислу. С тим у вези заиста јесте тешко не бити критичан према 541 Љубиша М. Бојић, Дамир Г. Зејнулаховић, Милош В. Јанковић, Технофеудализам на... овом концепту који не одговара нашем времену. Ипак, чини се да је погрешно одба- цити цео концепт због једне његове концепције. Најшира дефиниција одређује суверенитет као „врховни ауторитет на одређеној територији” (Philpott, 2011, str. 3). У својој савременој концепцији, носилац суверени- тета нити је доживотно одређен, нити има апсолутне ингеренције у свим областима (Philpott, 2011, str. 3). Из ове перспективе суверенитет сам по себи не мора бити пробле- матичан. Можемо говорити о нелегитимном суверену и нелегитимним овлашћењима, али, уколико одбацимо анархизам и успемо да оправдамо постојање политичког ауто- ритета (Perry, 2012), не можемо више говорити о нелегитимном суверенитету. Појам суверенитета у глобализованом свету користи се као контраст „појави транснациoналних мрежа приватних интереса” и „изражава фрустрацију и бес због све мањег простора колективног реосмишљавања, стварања и преобликовања инди- видуалних и групних идентитета” (Koskenniemi, 2011, str. 70). Суверенитетом се данас „артикулише нада да ћемо... своје сопствене животе држати у својим рукама” (Ibid.). Како тврди професор политичких наука Ненад Димитријевић, „покидана је суштинска модерна веза између политичке власти, демократије као политичког облика власти и права као претпостављено легитимног система друштвене координације. Демократска политика се повлачи пред приватизованим глобалним механизмима управљања који егзистирају и функционишу у ванустав- ном простору” (Dimitrijević, 2019, str. 79). Житељи савремених држава осећају се беспомоћно и због тога је прича о суве- ренитету тако присутна, јер „суверенитет указује на могућност, колико год она била ограничена и идеалистична, да, шта год се догодило, човек није само пиoн у туђим играма, већ... господар сопственог живота” (Koskenniemi, 2011: 70). 5 Free media thesis (FMT) 6 Изражено у апсолутним бројевима, Доналд Трамп је добио 4.960.000.000 долара бесплатне медијске вредности, док је Хилари Клинтон добила 3.240.000.000 долара бесплатне медијске вредности. Суверенитет и глобализација Гледамо ли на суверенитет из ове перспективе, можемо уочити да се у средишту приче налази демократска самовладавина и вредност јавне аутономије, односно пози- тивне слободе. А чак и најлибералније тумачење демократије, које полази од нега- тивне слободе као врховне вредности а демократију разумева инструментално, не допушта преиспитивање постојања саме политичке заједнице (Dimitrijević, 2017, str. 229) или могућност иступања из ње. Управо чињеница да политичку заједницу не можемо бирати, ове је либералне теоретичаре навела да инсистирају на сету основних права и слобода које носилац суверенитета мора безусловно поштовати. Само у том случају, рећи ће они, постоји обавеза поштовања обавезујућих демократских одлука. Другим речима, чак и ако максимално умањимо значај јавне аутономије, која се испољава у процесу демократске самовладавине, и значајно ограничимо опсег леги- тимних одлука које суверен може донети, једно ипак остаје неупитно: из политичке заједнице се не може иступити уколико се са одлукама већине не слажемо. Ово је минимални гарант стабилности (Ibid.). Поставимо ли сада читаву причу у одређени идеолошки контекст, видећемо да је експанзија неолибералног капитализма пресудно утицала на осећај беспомоћности житеља савремених држава. Створен је глобални економски простор слободе, али не и глобална политичка заједница. Тако је омогућено приватним економским актерима да „стално присутном претњом бекства капитала спрече да масовна народна очеки- вања постану стварност” (Slobodian, 2018, str. 155–156). Колико год се неолиберали 542 Социолошки преглед, vol. LV (2021), nо. 2, стр. 538–561 позивали на примат негативних слобода, неолиберални пројекат заправо је прекр- шио најмањи услов стабилности демократске политичке заједнице и приватним акте- рима омогућио искорак из ње. Идеолошким контекстом, који смо управо представили, заокружен је теоријски део и постављен оквир за анализу скорашњих случајева: Твитер против Доналда Трампа и Аустралија против Фејсбука. У складу са представљеним концептима, ста- вићемо моћ друштвених медија у контекст сукоба који су прожети „проблемом више руку”, а дотичу се суверенитета и идеолошких апарата. У том погледу, кроз примере ће бити разматране индикације трансфера моћи, који се назире између учесника у политичком процесу, држава и технолошких компанија. 5 Free media thesis (FMT) Љубиша М. Бојић, Дамир Г. Зејнулаховић, Милош В. Јанковић, Технофеудализам на... Љубиша М. Бојић, Дамир Г. Зејнулаховић, Милош В. Јанковић, Технофеудализам на.. Љубиша М. Бојић, Дамир Г. Зејнулаховић, Милош В. Јанковић, Технофеудализам на... Два дана након насилних протеста7 на Капитолу8, 8. јануара 2021. године, Доналд Трамп, тада још увек председник Сједињених Америчких Држава, трајно је суспендован са друштвене мреже због које је, како смо видели, добрим делом изабран на то место. У званичном обраћању Твитера, Трамп је суспендован са друштвене мреже због два твита који „глорификовањем насиља могу да инспиришу друге ... да понове криминална дела која су почињена код Капитола САД-а, 6. јануара 2021. године” (Twitter, 2021). Да подсетимо, Трамп је изгубио на изборима два месеца раније, али је све до насилних протеста на Капитолу тврдио да је заправо победио, све време храбрећи своје присталице да се не предају. Трампово одбијање признања резултата избора препознато је као један од узрока дешавања тог дана, када су његове присталице насилно ушле у зграду Конгреса. Оно што је проблематичније од уласка у Конгрес је чињеница да је петоро људи изгубило своје животе, а да је јавност могла да, како преко друштвених мрежа тако и традиционалних медија, присуствује фаталном пуцњу који је усмртио једну жену. Како год окарактерисали дешавања на Капитолу, оно што је неоспорно је да је политичко насиље, са смртним исходима, учињено како би се довео у питање резултат избора за председника Сједињених Америч- ких Држава, односно светског хегемона. Трајна суспензија Трампа са Твитера, кога је овај друштвени медиј препознао као узрочника насилних протеста, одузела му је платформу директне комуникације са његовим пратиоцима, како би се, наводно, ограничила могућност како понављања сличних догађаја, тако и могуће ескалације политичког насиља. Суспензија је произвела низ критичких реакција. С једне стране, конзервативци су суспензију препознали као напад на слободу говора и цензуру (Haroun, 2021). Поред тога, критика суспензије дошла је и из либералног табора. Кенан Малик, у свом чланку у Гардијану тврдио је да Трампово искључење може довести до праксе широких рестрикција, што већ сада злоупотребљавају аутори- тарни режими (Malik, 2021). Професор политичке теорије Џефри Хауард тврдио је да је Твитер оправдано суспендовао Трампа, али да је, са моралне тачке гледишта, неоправдано што је суспензија трајна (Howard, 2021). Наравно, било је и оних који су реакцију Твитера у потпуности подржали (Romano, 2021; Bensinger, 2021). Како би потпуније разумели разлоге за Трампову суспензију, као и функцију и суштину друштвених медија, морамо истражити Алтисерово разликовање државних репресивних апарата и државних идеолошких апарата. 8 Capitol Hill riots, где назив Капитол који ми користимо представља устаљени назив за Конгрес Сједињених Америчих Држава и где су се 6. јануара 2020. године одиграле суштинске сцене овог насилног протеста. 7 Сама природа дешавања тог дана и даље није у потпуности расветљена, тако да се могу наћи примери који дешавања називају пучем или покушајем државног удара. 7 Сама природа дешавања тог дана и даље није у потпуности расветљена, тако да се могу наћи примери који дешавања називају пучем или покушајем државног удара. 8 Capitol Hill riots, где назив Капитол који ми користимо представља устаљени назив за Конгрес Сједињених Америчих Држава и где су се 6. јануара 2020. године одиграле суштинске сцене овог насилног протеста. Сузпензија Доналда Трампа са Твитера Моћ друштвених медија нигде није била очигледнија него у случају председ- ничких избора 2016. године у Сједињеним Америчким Државама, поготово када говоримо о кампањи Доналда Трампа, победника тих избора. Иако је у финалну фазу избора ушао са, по мишљењу експерата (Ceaser, Busch, & Pitney, 2017), малим или никаквим шансама за победу против кандидата Демократске странке Хилари Клинтон, Трамп је на крају победио и постао 45. председник Сједињених Америч- ких Држава. Ова неочекивана победа дала је подстицај друштвеним научницима и политичким експертима да разраде теорије у вези са узроцима Трамповог успеха. Једна од теорија која покушава да објасни победу Трампа је теза бесплатних медија5. Ова теза постулира да је кључни допринос Трамповој неочекиваној победи дошао преко његовог коришћења друштвених медија, путем којих је успео да избегне трошкове за рекламе на традиционалним медијима, као и да оствари директну кому- никацију са потенцијалним гласачима и јавности (Khan, 2016; Le Miere, 2016; Yu, 2016). По мишљењу Франција, „Твитер је често био у центру Трампове способности да генерише бесплатну медијску пажњу” (Francia, 2017, str. 2). Поменути аутор је пока- зао да је Трамп имао за читаву трећину6 више бесплатне медијске пажње од Хилари Клинтон у завршној изборној години (Francia, 2017, str. 8). Поред овог податка, Фран- циjа додаје да је Трамп такође имао и већи број пратилаца на Твитеру, као и податак да је већи број људи потврдио да је чешће виђао Трампа на друштвеним мрежама (Francia, 2017). Међутим, ови налази не потврђују каузалну везу између веће способ- ности генерисања бесплатне медијске пажње и победе Доналда Трампа на изборима за председника. Оно што ови налази показују, додуше, је чињеница да су друштвене мреже, поготово Твитер, постале неизоставан чинилац у политичким процесима, да кроз њихову ефикасну употребу постоји простор за приступ потенцијалним гласа- чима ван традиционалних медија, као и да ће њихов значај у будућности неминовно расти, односно да ћемо засигурно видети политичаре који ће Трамповом стратегијом коришћења друштвених мрежа покушати да понове његов успех. 543 9 Иако се може чинити нелогичним концепт да приватне институције врше улогу државних идеолошких апарата, Алтисер предупређује ове критике. По његовим речима „Разлика између јавног и приватног је разлика унутар буржоаског права и валидна је у (подређеним) областима у којима буржоаско право извршава своју ’моћ’. Подручје државе му измиче јер је она ’изнад права’ ... она је предуслов за сваку разлику између јавног и приватног” (Althusser, 2009, str. 37). Када је утврдио такво разумевање односа јавног и приватног, Алтисер објашњава како се ово односи на државне идеолошке апарате. „Исто се може рећи и за државне идеолошке апарате. Није битно да ли су институције у којима се реализују ’јавне’ или ’приватне’. Приватне институције могу савршено ’функционисати’ као државни идеолошки апарати” (Althusser, 2009, 37). На овај начин конципирана разлика између јавног и приватног не постулира подређеност идеолошких апарата држави као таквој, већ репродукцији произиводног система. Држава јесте место скупљања моћи, али је и подређена тој сврси, уколико је буржоаска. Љубиша М. Бојић, Дамир Г. Зејнулаховић, Милош В. Јанковић, Технофеудализам на... Јанковић, Технофеудализам на... класе, оличене у Доналду Трампу и Џоу Бајдену, у који су, по природи изборних про- цеса, уплетене и остале друштвене класе, мада без значајног утицаја. Твитер, као при- ватна друштвена мрежа, одиграо је кључну улогу у овом процесу, па је тако, приме- ном цензуре, учврстио своју доминантну позицију унутар поља идеолошких апарата, коју је преузео од традиционалних медија. Љубиша М. Бојић, Дамир Г. Зејнулаховић, Милош В. Јанковић, Технофеудализам на... „Оно што разликује државне идеолошке апарате од државних (репресивних) апарата је следећа основна разлика: државни репресивни апарати функционишу путем насиља, док државни идеолошки апарати функционишу путем идеологије” (Althusser, 2009, str. 29–30). Тако, за Алтисера, државни репресивни апарати представљају „владу, админи- страцију, војску, полицију, судове, затворе” (Althusser, 2009, str. 27), односно једин- ство државних институција које, пре свега, путем репресије могу да санкционишу противнике. 544 Социолошки преглед, vol. LV (2021), nо. 2, стр. 538–561 С друге стране, државни идеолошки апарати, на пример, религијски, образовни, породични, правни, и за нас најбитнији, информациони, су бројни и различити, као и релативно аутономни, али уједињени у својој функцији очувања односа експлоа- тације путем репродукције и очувања хегемонијског положаја идеологије владајуће класе (Althusser, 2009). Међутим, овако идеално-типски постављена „подела рада” није сасвим тачна. Алтисер напомиње да не постоје чисто репресивни и идеолошки апарати, већ да та подела указује на примарни начин функционисања, док оба апарата садрже и секундарну функцију, односно државни идеолошки апарати секундарно делују путем репресије, а репресивни секундарно путем идеологије (Althusser, 2009). Релативна аутономија државних идеолошких апарата, такође, отвара простор, место за класну борбу. Ова посебност државних идеолошких апарата је условљена „хармонијом (која понекад шкрипи)” (Althusser, 2009, str. 37) између репресивних и идеолошких апа- рата. Ако знамо да државни апарат чини јединство репресивног и идеолошког апа- рата, са лакоћом можемо да увидимо потенцијалне сукобе и начине борбе за пре- власт над државним апаратом, кроз одвојене „подапарате”. Познавање суштине и функције државних идеолошких апарата нам омогућава да разумемо дешавања која су се одиграла почетком године у Сједињеним Америч- ким Државама. Тако, друштвене мреже можемо да сместимо у алтисеровски оквир, као нову форму, технолошки напредак у информационим државним идеолошким апаратима9. Иако социјалне мреже представљају технолошки помак и иновацију у комуника- ционој и информацијској технологији, оне потпадају под исту друштвену функцију, функцију државних идеолошких апарата. Део савременог информационог идеоло- шког апарата, друштвена мрежа Твитер, одлучила је да искористи своју секундарни начин функционисања, репресију, оличену у суспензији. Наводно се Твитер заложио за заштиту демократског процеса, како би осигурао релативно мирну предају моћи наредном председнику Џоу Бајдену. Важно је приметити да је, недељу дана након насилних протеста, активиран и државни репресивни апарат, војска, као гарант пре- даје власти. Тај чин озваничава власт над целокупним државним апаратом. Оно чему смо присуствовали почетком године у Сједињеним Америчким Држа- вама је борба за превласт над државним апаратом између две фракције владајуће 545 Љубиша М. Бојић, Дамир Г. Зејнулаховић, Милош В. Окршај медијске политике Аустралије са Фејсбуком и Гуглом Почетком 2021. године Аустралијско регулаторно тело за конкуренцију напи- сало је нацрт закона којим се покушава преусмерити део профита технолошких гига- ната, као што су Фејсбук и Гугл, ка издавачима (Beta, 2021). Прецизније, законом се жели прописати „да део новца од сваког плаћеног огласа... мора ићи и медијима уз чији садржај је оглас постављен” (Ilić, 2021). Гугл је као одговор на овај предлог запре- тио да ће се повући из Аустралије (Rujević, 2021). Другим речима, Гугл је на почетку реаговао у складу са Слободијановим наво- дима (Slobodian, 2018, str. 155–156). Претњу повлачењем капитала омогућила је управо непотпуна интеграција. Без економске интеграције, капитал не би могао слободно да шета глобалним тржиштем. С друге стране, потпуна интеграција која би подразумевала и политичку интеграцију, односно стварање европске или светске „државне” заједнице, омогућила би регулацију токова капитала и значајно ограни- чила уцењивачки потенцијал приватних актера. У политичко-демократском смислу, потпуна интеграција омогућила би минимум стабилности политичкој заједници, спречавајући њене чланове (и њихов капитал) да заједницу напусте у случају да се не слажу са легитимном већинском одлуком. За разлику од Гугла, Фејсбук је применио другачију стратегију. Претио је бло- кадом приступа информативном садржају путем своје мреже, а потом своје претње спровео у дело (Beta, 2021). На тај начин житељима Аустралије „није било омогућено да деле и гледају садржаје са вестима на овој платформи” (BBC, 2021). Блокирано је и „неколико налога Владе из домена здравља, као и хитних служби” (Ibid.). Перфидност Фејсбукове стратегије огледа се у томе што челници ове компаније рачунају на аустралијску демократску опредељеност. Имајући у виду да је инфор- мисаност житеља један од основних предуслова функционалне демократије, те да Фејсбук „убрзано постаје један од највећих извора вести на свету” (Zorzi, 2021), ова компаније ставила је власти у Аустралији у безизлазну позицију. Да бисмо је разумели, важно је присетити се савременог разумевања концепта суверенитета. У демократијама, било да је суверенитет додељен народу или парламенту, народни представници могу имати врховни ауторитет само у случају да им га народ додели демократским путем. Политички представници могу легитимно задржати овај ауто- ритет само уколико владају у прописаним оквирима и не нарушавају само демократ- ско начело. Оквири би подразумевали скуп неотуђивих права и слобода, а, примера ради, фер и слободни избори су један од елемената демократског начела. Перфидност Фејсбукове стратегије огледа се у томе што челници ове компаније рачунају на аустралијску демократску опредељеност. Окршај медијске политике Аустралије са Фејсбуком и Гуглом Имајући у виду да је инфор- мисаност житеља један од основних предуслова функционалне демократије, те да Фејсбук „убрзано постаје један од највећих извора вести на свету” (Zorzi, 2021), ова компаније ставила је власти у Аустралији у безизлазну позицију. Да бисмо је разумели, важно је присетити се савременог разумевања концепта суверенитета. б Имајући ово у виду, ево шта свака од опција која се нашла пред аустралиј- ским властима носи са собом. У првом случају парламент може одбити усвајање закона и приволети Фејсбук да омогући житељима Аустралије уредно информисање. Овом одлуком сачувала би се веза између демократског легитимитета и врховног 546 Социолошки преглед, vol. LV (2021), nо. 2, стр. 538–561 ауторитета јер се не би нарушио један од основних елемената демократије. Ипак, уко- лико претпоставимо да су житељи своје представнике бирали како би ови, између осталог, успешно решили проблем финансирања медија, онда би парламент у првом случају деловао противно вољи народа. У другом случају, уколико би аустралијски парламент усвојио закон не обази- рући се на Фејсбукове блокаде, он би довео у питање један од кључних елемената демократије (информисано грађанство) и ризиковао да поништи извор сопственог ауторитета. Посланици би и даље задржали власт, али Аустралија више не би била демократија. Другим речима, Фејсбуков ултиматум на одређени начин кида везу између суве- ренитета и демократије. Одлуче ли власти у Аустралији да делују суверено и поступе према својим уверењима, ризиковаће да озбиљно науде демократији. С друге стране, одлуче ли се да обезбеде својим житељима могућност информисања, чувајући тако саму срж демократије, власти морају играти по Фејсбуковим правилима, односно суспендовати сопствени суверенитет. Да ствар буде гора, спасавајући демократију власт би доносила одлуке противне жељама већине, а у складу са економским инте- ресом једног приватног актера. Ево како се завршила ова непријатна законодавна епизода. Фејсбук је након пар дана укинуо блокаду. Један од потпредседника компаније изјавио је да је постигнут споразум тако да се Фејсбуку и Гуглу омогући да сами одаберу којим медијима ће пружити финансијску подршку. Одмах након договора „закон је усвојен у оба дома аустралијског парламента, са новим амандманима који омогућавају да Фејсбук и Гугл не буду обухваћени новим законом” (BBC, 2021). Завршна разматрања Гашење Твитер профила Доналда Трампа показало је да идеолошки апарати могу да играју огромну улогу у осигуравању власти над државним апаратом, а да се као најзначајнији представници информационих државних идеолошких апа- рата појављују друштвени медији. Како су друштвени медији већ сад један од кључ- них чинилаца у изборним процесима, можемо закључити да ће њихова моћ над друштвеним животом у будућности само расти, ако узмемо у обзир значај улоге коју су одиграли у осигуравању власти над државним апаратом. Значајно је истаћи и да је Доналд Трамп, у тренутку укидања његовог профила са друштвеног медија Тви- тер, још увек био председник. Самим тим, одлука Твитера може да се посматра као напад на суверенитет САД. С друге стране, Гугл и Фејсбук приморали су власти једне државе која при- пада капиталистичком центру да доноси законе по економској вољи ових компа- нија. Мада се одлука аустралијског парламента може тумачити као жеља да се сачува демократија у тој држави, ово тумачење промашило би суштину проблема. А про- блем не лежи у самој одлуци, већ у понуђеним опцијама. Демократија подразумева не само избор представника који ће доносити обавезујуће одлуке, већ и ефективну моћ тих представника да доносе одлуке које утичу на животе свих чланова зајед- нице. Противници суверенитета прибојавали су се да се суверен неће „покорити мишљењу оних који трпе последице” (Laski, 1934, str. 63) његових одлука. Ми данас 547 Љубиша М. Бојић, Дамир Г. Зејнулаховић, Милош В. Јанковић, Технофеудализам на.. имамо потпуно супротан проблем. Суверен жели да поступа у складу са преферен- цијама „оних који трпе последице” и у складу са јавним интересом, али је цена так- вог поступања превисока. Држава је препустила приватном сектору контролу над ресурсима неопход- ним за њено функционисање. Док с једне стране Гуглова претња повлачењем капи- тала представља последицу делимичне интеграције, Фејсбукова реакција на нацрт закона логичан је след медијске дерегулације (Ilić, 2021). И делимична интеграција и дерегулација спровођене су са циљем сузбијања моћи државе. С обзиром да држава има монопол над легитимном применом силе, бројни либерали и неолиберали бри- нули су се само како да ова сила буде што мања. Процес разбијања државне моћи није водио њеном распарчавању, већ померању на неухватљиве економске актере који делују ван права и без легитимитета. На крају процеса, добили смо кобну ком- бинацију. Према речима Димитријевића, „демократске државе су... изгубиле много од њихове способности политичке владавине”, а истовремено су „задржале способ- ност репресије” (Dimitrijević, 2019, str. 78). Завршна разматрања Истовремено, појавили су се моћни при- ватни актери над којима више ни државе ни грађани немају никакву контролу. ј ју у у Технолошке компаније узимају од медија финансије неопходне за њихов опста- нак, иако су само преносиоци садржаја који је други креирао, чиме је угрожена функ- ција информисања у друштву. Такође, укидање профила Доналда Трампа на Тви- теру је заправо преузимање судске власти од стране технолошке компаније која, у овом случају, себи даје за право да одређује шта је говор мржње, иако он није дефи- нисан америчким законима. Истовремено се овом одлуком угрожава суверенитет САД, јер је Доналд Трамп још увек био председник у тренутку укидања његовог профила. На основу наведеног, јасно је да се додатно редефинише концепт сувере- нитета и назире трансфер моћи између држава и технолошких компанија, што ће имати велике реперкусије за демократију. Изгледа да ови догађаји указују на гло- бални улазак у нови поредак доминације технолошки компанија, који можемо на- звати технократијом или технофеудализмом. Оба разматрана догађаја указују да су друштвени медији, између осталог, и јавно добро од којег зависи демократија. Препо- ручује се да даља истраживања буду усмерена на дефинисање друштвених медија као јавног добра, што би подразумевало разграничење надлежности друштва у односу на њихове формалне власнике, као што се ради у случају етарских медија са јавном фреквенцијом. 548 Sociološki pregled / Sociological Review, vol. LV (2021), nо. 2, pp. 538–561 Ljubiša M. Bojić1 Damir G. Zejnulahović2 Miloš V. Janković3 University of Belgrade, Institute for Philosophy and Social Theory Belgrade (Serbia) 1 ljubisa.bojic@instifdt.bg.ac.rs 1 ljubisa.bojic@instifdt.bg.ac.rs 2 damir.zejnulahovic@instifdt.bg.ac.rs 3 milos.jankovic@instifdt.bg.ac.rs 4 This paper was realized with the support of the Ministry of Education, Science and Technological Development of the Republic of Serbia according to the Agreement on realization and financing of scientific research work. 2 damir.zejnulahovic@instifdt.bg.ac.rs 3 milos.jankovic@instifdt.bg.ac.rsh Introduction Technological development, either reflected in innovations in the production process or, as in the case considered here, in the emergence of new modes of communication, inev- itable affects the entire social reality, changes the established forms of social relations and causes the appearance of new social phenomena that need to be examined. Therefore, as early as mid-20th century, Debord observes that a spectacle, i.e. “a social relation among peo- ple, mediated by images” (Debord, 2003, p. 18), is becoming the essence of life in all special aspects of information. Of course, Debord connects this primarily with the importance of television that emerged as the most important transmitter of symbols and messages, while today’s parallel may be found in social media which, as it will be seen further in the paper, are becoming a transmitter without which the society would be impossible to imagine. At the beginning of 2021, at the peak of the coronavirus pandemic, two events under- mined the world public, indicating that there was a potential transfer of power between states and high technological companies. First, at the beginning of January, Twitter Com- pany decided to suspend the account of the leaving US President Donald Trump on that social medium due to his alleged hate speech (Twitter, 2021). Then, the multinational media company Facebook exerted a tremendous pressure on Australia to amend the draft law that would force this company to pay media publishers for their contents posted on this social medium (BBC, 2021). These two events reached global proportions, reviving the debate on the globaliza- tion impact on the sovereignty of national states and witnessing that the world is entering a new technocratic or technofeudal order. There are three schools of thought with different answers to the matter of state sov- ereignty in the globalization context. Hyperglobalists think that the state’s role is rather reduced and its economic sovereignty is particularly threatened. On the other hand, scep- tics see globalization as a product of the states themselves, claiming that their role and power have remained unchanged. Between these two theoretical poles there are transfor- mationalists. The role of the state, according to them, has already changed significantly (Reddy, 2012, pp. 61–64). y pp A particular place is held by the literature focusing on multinational companies. TECHNOFEUDALISM ILLUSTRATED BY TRUMP’S TWITTER SUSPENSION AND AUSTRALIA VS. GOOGLE AND FACEBOOK DISPUTE4 (Translation In Extenso) Abstract: Social media are becoming an indispensable communication tool today, including political communication. Therefore, technological companies running them have a great power. Every intervention they have in the public sphere may have far-reach- ing consequences. We will focus on two cases of the influence of technological companies on media, election and legislative processes as basic factors of democracy. We will consider the implications of the prohibition of Donald Trump’s use of Twitter, as well as the conflict of Australia’s media policy with Facebook and Google. We have reached the conclusion that in these cases technological companies took over the power that used to belong to the judicial and legislative branch of power, in terms of defining what hate speech, as well as media regulation is. Accordingly, this threatens the information system and sovereignty of the countries where these interventions are made. These events indicate that the world is entering a period of domination of technological companies, which may be called tech- nocracy or technofeudalism. Further considerations should be directed towards defining social media as a public good that should be influenced by societies and not only techno- logical companies as the owners of these communication platforms. Keywords: social media, sovereignty, technological companies, technocracy, techno feudalism 549 Ljubiša M. Bojić, Damir G. Zejnulahović, Miloš V. Janković, Technofeudalism illustrated by.. Introduction As Varis (Varis, 2020) reminds us, although some theories, such as semiotic democracy, as well as those companies that use marketing to present their platforms, try to present social media as defenders of democracy and the space where free speech is guaranteed, it is neither their primary function nor intention. In fact, moderation is not a peripheral but central function of social media, i.e. the way in which social media achieve their set goals in the market actually determines whether groups and interests are permissible or not (Gillespie, 2018). The problem of hate speech on the Internet can be observed, on one hand, from the aspect of the right to the freedom of speech as guaranteed by the US Constitution. On the other hand, many jurisdictions worldwide clearly define the concept of hate speech. There is definitely a conflict between the broad understanding of the freedom of speech and the guarantee of dignity of individuals and groups that may be subject to hate speech (Heldt, 2019). We should also take into account the prohibition of censorship by the state because it has been prescribed by many countries, thus additionally guaranteeing the freedom of speech. One of the starting points of this consideration is that states and companies share responsibility for the regulation of fake news, hate speech and various sabotages that may appear on the Internet. Here the greatest pressure is exerted on the owners of the leading social media – technological companies mostly located in the USA. In this case it would be inappropriate to accuse a priori the companies which are social media owners of censor- ship, because the absence of regulations has led to the current state of affairs. Of course, it should be taken into account that this does not justify explicit interference in the election process in such a way that a technological company transparently takes one of the sides participating in the election. Such decisions can provoke fierce reactions in the public, and threaten democracy as well. One of the decisions adopted by Facebook Company refers to fake news, undertak- ing that it will reduce the visibility of such disinformation so that it will not be widely dis- tributed, although the Company would be entitled to it in line with the applicable recom- mendation systems – algorithms that show contents to social media users. Introduction While some authors recognize the impact of companies on state sovereignty (Vernon, 1971), others think that such impact has been unjustifiably emphasized (Kline, 2006). There is also an opinion that in the 1980s the state still had primacy, but the turn of the millennia brought substantial changes, including new technologies and the “emergence of the elec- tronically networked world economy”, which definitely brought state sovereignty to ques- tion (Kobrin, 2009, p. 3). The impact of social media on political processes is constantly growing, and some studies say that the election candidates in the West increasingly use Twitter to promote and realize contact with potential voters (Enli, 2017; Enli & Skogerbo, 2013; Graham, Jack- son, & Broesma, 2016). Apart from the unambiguous significance of social media in every- day life as one of, or perhaps even primary, mode of communication among people, what particularly attracts the politicians’ attention is the fact that through social media, chiefly Twitter, they can realize direct contact with voters, thus bypassing the editorial policy of traditional media. In that way, politicians have the power to present themselves and their message much more strongly and with more control, as well as to create the impression 550 Sociološki pregled / Sociological Review, vol. LV (2021), nо. 2, pp. 538–561 of direct communication and responsibility with an individual. This is an important fact at the modern historical moment, where voices that accuse politicians of alienation from everyday life of their citizens are growing louder and louder (Enli, 2017; Ouyang & Water- man, 2020). This role of social media is also observed by Gainous and Wagner (Gainous & Wagner, 2014) who state that social media are shifting the field of power to the benefit of politicians, where “Twitting to power” is becoming a political standard, something that is obligatory at the modern political moment and which will have even more importance in the future. In that manner social media become an inevitable part of political processes. p p p As we have shown, the impact of social media on political processes can already be characterized as crucial, while that significance is certain to grow in the following years. However, now our analysis should include the fact that these social media are actually the product of private companies which reserve the right of their moderation. Ljubiša M. Bojić, Damir G. Zejnulahović, Miloš V. Janković, Technofeudalism illustrated by... Ljubiša M. Bojić, Damir G. Zejnulahović, Miloš V. Janković, Technofeudalism illustrated by... Although the above-listed examples prove that, with respect to legislation, Facebook establishes a system similar to the state system and thus partially takes over the authorities of one branch of power, this is only an initiative calling for an urgent reaction to the chal- lenge emerging in the vacuum of new technologies and obsolete laws. We can classify this matter within the concept that is defined in the literature as the “problem of many hands”, i.e. the difficulty in pinpointing decisively who is responsible in complex environments, where many factors are involved in decision-making. Thompson describes this issue in detail, list- ing a number of examples in an attempt to establish the approach in defining responsibility (Thompson, 2014). Van Drunen also explains the “problem of many hands” from central- ized to shared responsibility (van Drunen, 2020). Accordingly, the connected world refers to online platforms with a far-reaching impact on societies throughout the world. The new concept of functioning imposed by the Internet causes the transformation of markets, pro- cedures and democratic processes (Jevtović, 2020). In the analysis of the societies based on online platforms, the emphasis is placed on the conflict between ideological systems and social factors, which includes governments, companies and the civil sector, as well as the allotment of interconnected responsibility, in order to achieve privacy, security, fair environ- ment, availability and democracy (Van Dijck et al., 2018). These values are actually threat- ened in the society with a significant impact of global and regional online platforms which permeate public and private sectors, but also many areas, for example information, trans- port, healthcare and education. Some of these processes refer to local matters, while many of them also affect the global sphere. Having this in mind, we should emphasize the con- flicts between multinational companies, governments and multilateral factors as relevant in the consideration of power redistribution in the digital technologies era. The phenomenon we focus on in this paper refers primarily to the impact of techno- logical companies, i.e. of social media, on democracy and sovereignty in the United States of America and Australia, as illustrated by the suspension of former president Donald Trump’s Twitter account, and the clash between Australia’s public media policy with technological giants Facebook and Google. We will perceive all this through the prism of sovereignty. Introduction These posts are labelled as fake, which is done by independent experts (Facebook, 2021). Moreover, Facebook has also established the appellate process that enables the decisions of this com- pany to be disputed by the threatened parties. The appellate body is supervised by a spe- cial board (Collins, 2021). 551 Sovereignty and globalization In classical texts (Bodin, 2002; Hobbes, 1961), the value of the existence of a sover- eign usually lies in his power to guarantee peace. In contrast, modern authors speak of the concept of sovereignty in much more diverse ways. Political theoretician Harold Laski wonders whether “the state should have ultimate power with no limits of any kind” (Laski, 1934, p. 62). He concludes that “there is no permanent right to power” and that “every gov- ernment must subordinate to the opinion of those that suffer the consequences of its acts” (Ibid., 63). Jacques Maritain has a similar opinion in that respect. To him, this concept is “essentially wrong” (Maritain, 1966, p. 29). The authors who reject the concept of sovereignty (Duguit, 1997) or speak about it in the negative light (Laski, 1934; Maritain, 1966) usually understand sovereignty in Bodin- ian terms (Jean Bodin). To that end, it is truly difficult not to be critical towards the con- cept that does not suit our time. However, it seems wrong to reject the entire concept just because of one of its ideas. The broadest definition determines sovereignty as an “ultimate authority in a certain territory” (Philpott, 2011, p. 3). In its contemporary concept, the holder of sovereignty is 552 Sociološki pregled / Sociological Review, vol. LV (2021), nо. 2, pp. 538–561 neither appointed for life nor has absolute competencies in all areas (Philpott, 2011, p. 3). From this perspective, sovereignty itself is not necessarily problematic. We can speak of an illegitimate sovereign and illegitimate authorities, but if we reject anarchism and man- age to justify the existence of political authority (Perry, 2012), we can no longer speak of illegitimate sovereignty. The concept of sovereignty in the globalized world is used in contrast to the “emer- gence of transnational networks with private interests” and “to express frustration and anger because of the decreasing area of collective redesigning, creating and reshaping individual and group identities” (Koskenniemi, 2011, p. 70). Nowadays sovereignty serves to “articu- late the hope that we will… keep our own lives in our hands” (Ibid.). According to Nenad Dimitrijević, Professor of Political Science, “the essential modern connection has been broken between political power, democracy as a political form of power and law as an assumed legitimate system of social coordination. Democratic politics is retreating from privatized global management mechanisms that also exist and function in the unconstitutional environment” (Dimitrijević, 2019, p. Sovereignty and globalization 79). The residents of modern states feel helpless and that is why the narrative of sover- eignty is present to such an extent, because “sovereignty points to the possibility, no matter how limited and idealistic it may be, that the man, whatever happens, is not only a pawn in someone else’s games, but… the master of his own life” (Koskenniemi, 2011, p. 70). If we look at sovereignty from this perspective, we may observe that in the centre of the narrative there is democratic self-governance and the value of public autonomy, i.e. positive freedom. Even the most liberal interpretation of democracy, which starts from negative freedom as an ultimate value and understands democracy instrumentally, does not allow re-examination of the existence of the political community itself (Dimitrije- vić, 2017, p. 229) or the possibility of stepping out of it. The very fact that the political community cannot be chosen has made liberal theoreticians to insist on the set of fun- damental rights and freedoms that must be unconditionally respected by the holder of sovereignty. According to them, only in this case there is an obligation to respect bind- ing democratic decisions. g In other words, even if we maximally reduce the importance of public autonomy man- ifested in the process of the democratic self-governance, and if we substantially limit the scope of legitimate decisions that may be made by the sovereign, one thing will still remain indisputable: we can leave the political community if we do not agree with the decisions of its majority. This is the minimum guarantee of stability (Ibid.). If we put the whole narrative into a certain ideological context, we will see that the expansion of neoliberal capitalism has decisively influenced the feeling of helplessness of the residents of modern states. A global economic area of freedom has been created, but not a global political community as well. That is what enabled private economic actors to “use the ever-present threat of capital flight to prevent popular mass expectations from coming true” (Slobodian, 2018, pp. 155–156). Despite the fact that neoliberals tend to cite the pri- macy o negative freedoms, the neoliberal project has actually violated at least one condition of stability of the democratic political community and enabled private actors to step out of it. Suspension of Donald Trump’s Twitter account The power of social media has never been more evident than in the case of the 2016 presidential election in the United States of America, particularly when speaking of the campaign of Donald Trump, the winner of the election. Although, according to some experts (Ceaser, Busch, & Pitney, 2017), Trump entered the final stage of the election with little or no chance to beat the Democratic Party’s candidate Hilary Clinton, he won in the end and became the 45th president of the United States of America. This unexpected vic- tory encouraged social scientists and political experts to elaborate theories in relation to the causes of Trump’s success. One of the theories attempting to explain Trump’s victory is the free media thesis. This thesis postulates that the key contribution to Trump’s unexpected victory was his use of social media, through which he managed to avoid advertising costs in traditional media, as well as to realize direct communication with potential voters and the public (Khan, 2016; Le Miere, 2016; Yu, 2016). According to Francia, Twitter was often in the centre of Trump’s ability to generate free media attention” (Francia, 2017, p. 2). This author shows that Trump had more media attention by as much as one third5 than Hilary Clinton in the final election year (Francia, 2017, p. 8). In addition to this information, Francia adds that Trump also had a larger number of followers on Twitter, as well as that a larger number of people confirmed they more often saw Trump in social media (Francia, 2017). However, these findings do not confirm the causal connection between a greater ability to generate free media attention and Donald Trump’s victory in the presidential election. As a matter of fact, these findings show that social media, particularly Twitter, have become an indispensable factor in political processes, that through their efficient use there is also space for approaching potential voters outside traditional media, and that their significance will inevitably grow in the future, i.e. that we will definitely see politicians trying to repeat Trump’s success by applying his strategy of using social media. Two days after violent protests6 in Capitol7, on 8th January 2021, Donald Trump, still the President of the United States of America at the time, was permanently suspended from the social medium thanks to which, as we have seen, he had been elected to that position for the most part. 5 Shown in absolute numbers, Donald Trump got $4,960,000,000 of free media value, while Hilary Clinton got $3,240,000,000 of free media value. 6 The very nature of the events on that day has not been completely clarified yet, so that there are cases of labelling the events as a coup or an attempted coup. 7 Capitol Hill riots, where the name of Capitol as we use it is the usual name for the US Congress, and where the central scenes of this violent protest took place on 6th January 2020. Sovereignty and globalization The ideological context we have just presented completes the theoretical segment and establishes the framework for analyzing two recent cases: Twitter vs. Donald Trump, and 553 Ljubiša M. Bojić, Damir G. Zejnulahović, Miloš V. Janković, Technofeudalism illustrated by... Australia vs. Facebook. According to the listed concepts, we will place the power of social media within the context of the conflicts permeated by the “problem of many hands” and concerning sovereignty and ideological apparatuses. In that respect, through these exam- ples we will consider the indications of the transfer of power that may be perceived between the participants in the political process, the states and technological companies. Australia vs. Facebook. According to the listed concepts, we will place the power of social media within the context of the conflicts permeated by the “problem of many hands” and concerning sovereignty and ideological apparatuses. In that respect, through these exam- ples we will consider the indications of the transfer of power that may be perceived between the participants in the political process, the states and technological companies. Suspension of Donald Trump’s Twitter account In the official Twitter address, Trump was suspended from this social medium because of two tweets that by “glorifying violence may inspire others ...to repeat criminal acts committed in the US Capitol on 6th January 2021” (Twitter, 2021). 554 Sociološki pregled / Sociological Review, vol. LV (2021), nо. 2, pp. 538–561 Namely, Trump lost the election two months earlier, but until the violent protests in Capitol, he claimed victory, constantly encouraging his followers not to give up. Trump’s refusal to accept the election results was identified as one of the causes of the events that day, when his followers stormed the Congress building. What is more problematic than the actual storming of the Congress is the fact that five people lost their lives and that the pub- lic could, equally through social and traditional media, actually witness the fatal shot kill- ing one woman. No matter how Capitol riots may be characterized, it is indisputable that political violence with fatal outcomes was committed to question the presidential election results in the United States of America, or the world hegemon. Permanent Twitter’s suspen- sion of Trump, identified by this social medium as the causative agent of the violent pro- tests, took away the platform of direct communication with his followers, allegedly in order to limit the possibility of the repetition of such events, as well as of the potential escalation of political violence. This suspension provoked a series of critical reactions. On one hand, conservatives perceived the suspension as an attack on the freedom of speech and as cen- sorship (Haroun, 2021). Moreover, the suspension was criticized from the liberal camp as well. In his Guardian article, Kenan Malik claims that Trump’s suspension might lead to the practice of broad restrictions, which is already abused by authoritarian regimes nowa- days (Malik, 2021). Jeffrey Howard, Professor of Political Theory, claims that Twitter jus- tifiably suspended Trump, but, from the moral point of view, it was not justifiable to make such suspension permanent (Howard, 2021). Of course, there were also those who fully supported Twitter’s reaction (Romano, 2021; Bensinger, 2021). In order to understand fully the reasons for Trump’s suspension, as well as the func- tion and essence of social media, we must examine Althusser’s differentiation between state repressive apparatuses and state ideological apparatuses. 8 Although the concept of private institutions playing the role of state ideological apparatuses may seem illogical, Althusser warns against such criticism. According to him, “the difference between the public and the private is the difference within bourgeois law and is valid in (subordinated) areas where bourgeois law exercises its ‘power’. The area of the state is moving away because it is ‘beyond law’ ... it is the prerequisite for every difference between the public and the private” (Althusser, 2009, p. 37). Having established such understanding of the relation between the public and the private, Althusser explains how it refers to state ideological apparatuses. “The same may also be said about state ideological apparatuses. It is unimportant whether institutions where they are realized are ’public’ or ’private’. Private institutions can perfectly ’function’ as state ideological apparatuses” (Althusser, 2009, p. 37). Such conceived difference between the public and the private does not postulate the subordination of the ideological apparatus to the state itself, but to the reproduction of the production system. The state is a place of power concentration, but it is also subordinated to that purpose if it is bourgeois. Suspension of Donald Trump’s Twitter account “What distinguishes state ideo- logical apparatuses from state (repressive) apparatuses is the following basic difference: state repressive apparatuses function through violence, while state ideological apparatuses func- tion through ideology” (Althusser, 2009, pp. 29–30). Therefore, in Althusser’s opinion, state repressive apparatuses constitute “government, administration, army, police, courts, prisons” (Althusser, 2009, p. 27), i.e. the unity of state institutions that can sanction opponents primarily through repression. In contrast, state ideological apparatuses, for example, religious, educational, family, legal and, most importantly to us, information apparatuses, are numerous and diverse, as well as relatively autonomous, but united in their function of maintaining the exploitation relationship through reproduction and preservation of the hegemony position of the rul- ing class ideology (Althusser, 2009). However, this ideal-typical “division of work” is not completely accurate. Althusser notes that there are no purely repressive and ideological apparatuses, but that the division points to the primary method of functioning, while both apparatuses also have a secondary function, i.e. state ideological apparatuses secondarily function through repression, while repressive apparatuses secondarily unction through ideology (Althusser, 2009). The rel- ative autonomy of state ideological apparatuses also opens up the space or place for class struggle. This specific feature of state ideological apparatuses is conditioned by the har- mony (that sometimes falters)” (Althusser, 2009, p. 37) between repressive and ideological apparatuses. Knowing that the state apparatus constitutes the unity of the repressive and 555 Ljubiša M. Bojić, Damir G. Zejnulahović, Miloš V. Janković, Technofeudalism illustrated by.. ideological apparatuses, we can easily understand the potential conflicts and methods of fighting for supremacy over the state apparatus through separate “sub-apparatuses”. Being familiar with the essence and function of state ideological apparatuses enables us to understand the events at in the United States of America the beginning of the year. Therefore, we can place social media within the Althusserian framework, as a new form, as technological progress in information state ideological apparatuses8. Although social networks are technological progress and innovation in communica- tion and information technologies, they belong to the same social function, the function of state ideological apparatuses. As part of the modern information ideological apparatus, the social medium Twitter decided to use its secondary mode of functioning, repression reflected in suspension. Twitter allegedly advocated for the protection of the democratic process in order to ensure a relatively peaceful handover of power to the new President Joe Biden. Suspension of Donald Trump’s Twitter account It is important to note that, one week after the violent protests, the state repressive apparatus, i.e. the army, was activated as a guarantee of power handover. That act formal- izes power over the entire state apparatus. What we witnessed in the United States of America at the beginning of the year is the struggle for supremacy over the state apparatus between two factions of the ruling class, embodied in Donald Trump and Joe Biden, in which, by the nature of election processes, other social classes are also intertwined, but with no relevant impact. Twitter as a private social medium played the key role in this process and by applying censorship it strength- ened its dominant position within the area of the ideological apparatus – the position taken over from traditional media. Australia’s media policy clash with Facebook and Google At the beginning of 2021, Australian regulatory body for competition prepared the draft law that attempts to redirect part of the profit of technological giants such as Face- book and Google, to publishers (Beta, 2021). Specifically, the law is aimed at prescribing that “part of the money from each paid advertisement… must also be given to the media with the content of which the advertisement was posted” (Ilić, 2021). Google response to this proposal was to threaten by its withdrawal from Australia (Rujević, 2021). In other words, Google initially responded in line with Slobodian’s statements (Slobo- dian, 2018, p. 155-156). The threat by capital withdrawal was made possible by incomplete 556 Sociološki pregled / Sociological Review, vol. LV (2021), nо. 2, pp. 538–561 integration itself. Without economic integration, capital would not be able to move freely across the global market. On the other hand, complete integration that would also imply political integration, i.e. creation of the European or world “state” community, would enable the regulation of capital flows and substantially limit the blackmailing potential of private actors. In terms of politics and democracy, complete integration would ensure the mini- mum stability to the political community, preventing its members (and their capital) from leaving the community in case they did not agree with the legitimate majority decision. Unlike Google, Facebook applied a different strategy. It threatened by blocking the access to information content through its medium, and then carried out its threats (Beta, 2021). Consequently, the residents of Australia “were not able to share and watch news con- tents on this platform” (BBC, 2021). Facebook also blocked “several accounts of the Gov- ernment in the field of healthcare, as well as emergency services” (Ibid.). Perfidiousness of Facebook’s strategy is reflected in the fact that the leaders of this company relied on the democratic commitment of Australia. Having in mind that people’s being informed is one of the basic prerequisites of functional democracy, and that Face- book is “increasingly becoming one of the greatest sources of news in the world” (Zorzi, 2021), this company put the Australian authorities in a hopeless position. In order to com- prehend it, we should remember the modern understanding of the concept of sovereignty. In democracies, no matter whether sovereignty is assigned to the people or the parliament, people’s representatives can have supreme authority only if people grant it to the democrat- ically. Australia’s media policy clash with Facebook and Google Political representatives can legitimately keep such authority only if they rule within the prescribed frameworks and do not violate the democratic principle itself. The frame- works would refer to a set of inalienable rights and freedoms and fair and free elections, for example, are one of the elements of the democratic principle. Considering this, this is what each option before the Australian authorities implied. In the first case, the Parliament can refuse to adopt the law and force Facebook to provide proper information to Australians. This decision would keep the connection between dem- ocratic legitimacy and supreme authority because one of the basic elements of democracy would not be violated. However, if we assume that the people elected their representatives so that the latter could successfully resolve the problem of media funding, the Parliament would act contrary to the people’s will in the former case. In the latter case, if the Australian Parliament adopted the law without paying atten- tion to Facebook’s blockades, it would question one of the key elements of democracy (informed citizens) and risk cancelling the source of its own authority. The members of the Parliament would still keep their power, but Australia would no longer be a democracy. In other words, Facebook’s ultimatum in a certain manner breaks the connection between sovereignty and democracy. If the Australian authorities decided to act in a sover- eign manner and in line with their beliefs, they would risk causing serious harm to democ- racy. On the other hand, if they decided to provide the possibility of being informed to Aus- tralians, thus preserving the very essence of democracy, the authorities would have to play by Facebook’s rules, i.e. suspend their own sovereignty. To make matters worse, by saving democracy, the authorities would make decisions contrary to the wishes of the majority, and in line with the economic interests of a single private actor. g p This is how this unpleasant legislative episode ended. After several days, Facebook lifted the blockade. One of the company’s vice presidents stated that the agreement had 557 Ljubiša M. Bojić, Damir G. Zejnulahović, Miloš V. Janković, Technofeudalism illustrated by.. been reached, enabling Facebook and Google to choose on their own what media to sup- port financially. Final considerations The suspension of Donald Trump’s account on Twitter has shown that ideological apparatuses can play an enormous role in ensuring power over the state apparatus, while social media appear to be the most important representatives of information state ideolog- ical apparatuses. Since social media are already one of the key factors in election processes, we may conclude that their power over social life in the future will keep growing, if we take into account the importance of the role they played in ensuring power over the state appa- ratus. It is also important to point out that Donald Trump was still the official president at the moment when his account on social medium Twitter was suspended. Therefore, Twit- ter’s decision may be seen as an attack on the US sovereignty. On the other hand, Google and Facebook forced the authorities of the country that belongs to the capitalist centre to enact laws in line with the economic will of these com- panies. Although the decision of the Australian Parliament may be interpreted like a desire to preserve democracy in that country, such interpretation would miss the essence of the problem. The problem does not lie in the decision itself but in the options offered. Democ- racy implies not only the appointment of representatives who will make binding decisions, but also effective power of those representatives to make decisions affecting the lives of all members of the community. The opponents of sovereignty feared that the sovereign would not “be subordinated to the opinion of those that suffer consequences” (Laski, 1934, p. 63) of the sovereign’s decisions. Today we have a totally opposite problem. The sovereign wants to act in line with preferences of “those that suffer consequences” and, in line with the pub- lic interest, but the prince of such acting is too high. The state has given the private sector control over resources necessary for its func- tioning. While, on one hand, Google’s threat by withdrawing the capital is a consequence of partial integration, Facebook’s reaction to the draft law is a logical outcome of media dereg- ulation (Ilić, 2021). Both partial integration and deregulation were implemented with the aim of suppressing the power of the state. Since the state has the monopoly over the legit- imate exercise of power, many liberals and neoliberals took care only to keep this power as small as possible. Australia’s media policy clash with Facebook and Google Immediately after this agreement, “the law was enacted in both houses of the Australian Parliament, with new amendments making clear that Facebook and Goo- gle are not covered by the new law” (BBC, 2021). Final considerations The process of breaking the state power did not lead to its fragmenta- tion but to its shift to elusive economic actors who proceed outside law and with no legit- imacy. At the end of the process we got a fatal combination. According to Dimitrijević, “democratic states have... lost much of their capability of political rule”, while at the same time they “retained the capability of repression” (Dimitrijević, 2019, p. 78). Simultaneously, powerful private actors appeared and neither states nor citizens have any control over them. Technological companies take from the media the funds necessary for their survival, although they are only the transmitters of the content created by others, which threatens the function of information in the society. 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https://openalex.org/W2799600092	https://www.jhsci.ba/ojs/index.php/jhsci/article/download/612/622	English	null	Decrease in body mass index: Personal genotyping, individual diet, and exercise plan	Journal of Health Sciences	2,017	cc-by	4,676	Mirela Džehverović, et al. Journal of Health Sciences 2017;7(2):91-98 Mirela Džehverović, et al. Journal of Health Sciences 2017;7(2):91-98 Mirela Džehverović, et al. Journal of Health Sciences 2017;7(2):91-98 Journal of Health Sciences Open access Mirela Džehverović1, Anesa Ahatović1, Naris Pojskić1, Naida Lojo-Kadrić1, Amela Pilav1, Damir Marjanović2, Jasmina Čakar1 1Institute for Genetic Engineering and Biotechnology, University of Sarajevo, Sarajevo, Bosnia and Herzegovina, 2International Burch University, Sarajevo, Bosnia and Herzegovina Corresponding author: Mirela Džehverović, Institute for Genetic Engineering and Biotechnology, University of Sarajevo, Francuske Revolucije bb, Sarajevo, Bosnia and Herzegovina. E-mail: mirela.dzehverovic@ingeb.unsa.ba Decrease in body mass index: Personal genotyping, individual diet, and exercise plan Mirela Džehverović1, Anesa Ahatović1, Naris Pojskić1, Naida Lojo-Kadrić1, Amela Pilav1, Damir Marjanović2, Jasmina Čakar1 ABSTRACT Introduction: Single nucleotide polymorphisms (SNPs) have lately been used for prediction of metabolic processes that may be related to obesity. The aim of our study was to examine the association of SNPs of several genes with obesity and physical activity in 18 healthy volunteers. Methods: We used buccal swabs to collect and extract DNA from 18 volunteers. Pyrosequencing was used for molecular analysis of 13 polymorphisms in 10 genes (APOA2, MTHFR, MCM6, peroxisome pro liferators-activated receptor gamma, FABP2, beta-2-adrenergic receptor (ADRB)2, ADRB3, A-actinin-3, angiotensin-converting enzyme, and FUT2). The volunteers’ personal data included body mass index (BMI), dietary practice and information on daily fitness and workout routine. Association between the 13 observed gene polymorphisms and individual BMI status (normal or overweight) was analyzed. Results of the DNA analysis were used for the expert evaluation by nutritionists and physiologists to obtain optimal regulation of nutrition and exercise. The volunteers had a dietary and fitness program for 12 months which they tracked by filling in a suitable study form. Results: 14 volunteers had a moderate genetic predisposition for abdominal adipose-tissue accumula tion, while 4 of them had genotypes not associated with abdominal fat tissue accumulation. A statistically significant difference was found between the value of BMI before and after the implementation of per sonalized training and nutrition plan within the group of overweight volunteers (paired sample t=3.382; p = 0.006; exact p = 0.015). The single-locus F-test showed no association between the gene polymor phisms and BMI values. In addition, no correlation was detected between the gene polymorphisms and amount of BMI reduction prior and after the implementation of the personalized training and nutrition plan within the overweighed group of volunteers. Conclusion: Optimal nutrition and training plan are crucial for the BMI reduction as observed in the over weighed volunteers after the 12-month personalized training and individualized nutrition plan. However, the analyzed polymorphisms were not significantly associated with the obesity in this study. Key words: Single-nucleotide polymorphisms; nutrition; physical activity; body mass inde Submitted: 20 February 2017/Accepted: 28 March 2017 © 2017 Mirela Džehverović, et al; licensee University of Sarajevo - Faculty of Health Studies. 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. Corresponding author: Mirela Džehverović, Institute for Genetic Engineering and Biotechnolo Revolucije bb, Sarajevo, Bosnia and Herzegovina. E-mail: mirela.dzehverovic@ingeb.unsa.ba INTRODUCTION and heat. Some genetic variants are related to physi cal activity in reducing the amount of adipose tissue after the endurance training. Significant technological advances of single-nucleo tide polymorphisms (SNPs) have further strength ened research methodologies for genetic analyses used to predict metabolic process that might be asso ciated with diet and physical activity (1). Obesity is a major epidemic problem of the public health world wide, characterized as an excess of adipose tissue. The most commonly used measurement to deter mine weight status is body mass index (BMI) (2). The World Health Organization recommends the following BMI cut points to classify weight status in adults aged 20 or older: <18.5 kg/m2 (under weight), 18.5-24.9 kg/m2 (normal weight), 25.0- 29.9 kg/m2 (overweight), 30.0-39.9 kg/m2 (obese), and ≥40 kg/m2 (extremely obese) (3). Unhealthy diet and physical inactivity have been identified as primary determinants of increased obesity. Various studies have identified more than 500 candidate genes associated with body weight, motor, and functional abilities as well as their role in the devel opment of obesity. Therefore, genetic factors might be important in determining an individual’s sus ceptibility to obesity (4,5). A number of studies have reported a correlation between variations of a DNA sequence in specific genes and the phenotype of obesity (6,7). Genetic variants that influence an individual’s response to diet are actually metabolic pathways of fatty acids and carbohydrates, as well as the fat cells formation and the production of energy The aim of our study was to examine the utility of the existing prediction SNP test possibly related to obe sity and physical activity in 18 healthy volunteers. ABSTRACT 81,9(56,7<2)6$5$-(92 )$&8/7<2)+($/7+678',(6 UNIVERSITY OF SARAJEVO FACULTY OF HEALTH STUDIES 81,9(56,7<2)6$5$-(92 )$&8/7<2)+($/7+678',(6 UNIVERSITY OF SARAJEVO FACULTY OF HEALTH STUDIES Mirela Džehverović, et al. Journal of Health Sciences 2017;7(2):91-98 http://www.jhsci.ba RESULTS abilities of the locomotor system. DNA fragments in chromosome 1 (APOA2 and MTHFR), chro mosome 2 (MCM6 LCT-13910 and MCM6 LCT-22018), chromosome 3 (peroxisome prolifera tors-activated receptor gamma [PPARG]), chromo some 4 (FABP2), chromosome 5 (beta-2-adrenergic receptor [ADRB]2 position 1 and position 2), chro mosome 8 (ADRB3), chromosome 11 (A-actinin-3 [ACTN3]), chromosome 17 (angiotensin-convert ing enzyme [ACE]), and chromosome 19 (FUT2) were amplified using the polymerase chain reaction (PCR). A validated set of reagents (PyroMark PCR Kit, Qiagen) was used for PCR. Amplicon sequences were analyzed using pyrosequencing (PyroMark Q24, Qiagen) which was performed in GENOS Company. Amplified ACE fragments were sepa rated using capillary electrophoresis by 3130 genetic analyzers (Applied Biosystems). The fragment size was determined based on the familiar molecular weight standard using GeneMapper® ID-X Software (Applied Biosystems). abilities of the locomotor system. DNA fragments in chromosome 1 (APOA2 and MTHFR), chro mosome 2 (MCM6 LCT-13910 and MCM6 LCT-22018), chromosome 3 (peroxisome prolifera tors-activated receptor gamma [PPARG]), chromo some 4 (FABP2), chromosome 5 (beta-2-adrenergic receptor [ADRB]2 position 1 and position 2), chro mosome 8 (ADRB3), chromosome 11 (A-actinin-3 [ACTN3]), chromosome 17 (angiotensin-convert ing enzyme [ACE]), and chromosome 19 (FUT2) were amplified using the polymerase chain reaction (PCR). A validated set of reagents (PyroMark PCR Kit, Qiagen) was used for PCR. Amplicon sequences were analyzed using pyrosequencing (PyroMark Q24, Qiagen) which was performed in GENOS Company. Amplified ACE fragments were sepa rated using capillary electrophoresis by 3130 genetic analyzers (Applied Biosystems). The fragment size was determined based on the familiar molecular weight standard using GeneMapper® ID-X Software (Applied Biosystems). The volunteers’ personal genotypes (Table 2) were subjected to the expert evaluation. Personalized training and nutrition plans were obtained for each volunteer. According to the obtained genotypes, only 5 vol unteers had a genetic predisposition for obesity development, while 13 were at moderate risk for the weight increase. The genotypes of 8 volunteers indi cated a genetic predisposition for weight gain due to a diet rich in fats, while the genotypes of 10 volun teers showed moderate risk. Furthermore, 14 vol unteers reported a moderate genetic predisposition for abdominal adipose-tissue accumulation, while it was not the case for the other 4 volunteers whose genotype was not associated with abdominal fat tis sue accumulation. RESULTS Regarding the training program, the personal genotypes of 8 volunteers showed a genetic predisposition for a more efficient physio logical response to training based on strength exer cises. According to the genotypes of 10 volunteers, a physiological response to training should be equally efficient regardless of whether the training is based on strength or endurance. METHODS We conducted the research on a total of 18 healthy volunteers (50% male and 50% female), aged 20-50. The volunteers were assigned into a group with normal BMI (7 volunteers) and group with increased BMI (11 volunteers). Height and weight were measured with participants dressed in light weight clothing and without shoes. BMI was calcu lated with the weight (in kg) divided by the square of height (in m). All the subjects gave their writ ten informed consent for the use of their biological material and data while the ethical approval for this research was obtained from the Ethics Committee of the Institute for Genetic Engineering and Biotechnology, University of Sarajevo (Approval No. 296-1/14 dated May 13, 2015). DNA was extracted from buccal swabs using Chelex® 100, Bio-Rad, according to the manufacturers pro tocol. Using public databases, we selected 13 most common polymorphisms (Table 1) which are related to obesity development, cardiovascular diseases, and metabolic disorders, as well as motor and functional TABLE 1. List of polymorphisms of the genes related to diet and exercise Gene Polymorphism Protein NCBI FABP2 c. 163A>G p.Thr55Ala rs1799883 PPARG c.‑2‑28078C>G p.Pro12Ala rs1801282 APOA2 c.‑323C>T ////// rs5082 ADRB279 c. 79C>G p.Gin27Glu rs1042714 ADRB246 c. 46A>G p.Gly16Arg rs1042713 ADRB3 c. 190T>C p.Trp64Arg rs4994 ACTN3 c. 1858C>T p.Arg620Ter rs1815739 ACE c. 584‑109_584‑108ins ///// rs464994 MTHFR677 c. 665C>T p.Ala222Val rs1801133 MTHFR1298 c. 1409A>C p.Glu470Ala rs1801131 MCM6 (LCT‑13910) c. 1917+326C>T ///// rs4988235 MCM6 (LCT‑22018) c. 1362+117G>A ///// rs182549 FUT2 c. 461G>A p.Trp154Ter rs601338 PPARG: Peroxisome proliferators‑activated receptor gamma; ADRB: Beta‑adrenergic receptor; ACE: Angiotensin‑converting enzyme; ACTN3: A‑actinin‑3 TABLE 1. List of polymorphisms of the genes related to diet and exercise 92 Mirela Džehverović, et al. Journal of Health Sciences 2017;7(2):91-98 Statistical analysis Th l y The Kolmogorov–Smirnov test was applied to estimate the normality of the. To estimate the dif ference between BMI before and after the imple mentation of personalized training and nutrition plan, we used the paired samples t-test. Once the difference in BMI between overweight and normal groups was calculated, the independent samples t-test was performed. The observed sample size was small, therefore, we applied the exact permutation test (n = 10000). Biostatistical calculations were per formed using the paleontological statistics software, version 3.14 (8). A single-locus case-control test (9) based on the exact p-value test was performed for the allele genotypic association between the 13 observed gene polymorphisms and individual BMI status (normal or overweight). When estimating the relationship between the gene polymorphisms and BMI value, a single-locus F-test was used. The same test was applied to estimate the relationship between the gene polymorphisms and the amount of BMI reduction after the implementation of personalized training and nutrition plan. Both the above-men tioned analyses were conducted using Powermarker v3.25 (10). The statistical significance of p < 0.05 was considered significant. Before the 12-month diet and training program, 7 volunteers were in a BMI category with normal weight, 9 were in the overweight category, while the remaining 2 volunteers were in the obese BMI cat egory. All 18 volunteers have followed the recom mended exercise and diet plans. After 12 months, BMI values remained unchanged for 8 volunteers. Furthermore, 10 volunteers reduced the BMI value, and 4 of them even dropped to a lower BMI cate gory (Figure 1). The Kolmogorov–Smirnov test showed that all BMI values within observed groups were in a range of normal distribution (p > 0.05). A statistically sig nificant difference was noticed between the values of BMI before and after the implementation of the personalized training and nutrition plan within the group of volunteers with overweight BMI val ues (paired samples t = 3.382; p = 0.006; exact p = 0.015). This result showed visible BMI reduc tion within the group of the overweighed volunteers following the implementation of the personalized training and nutrition plan. When having included only the volunteers within the normal BMI range, 93 http://www.jhsci.ba TABLE 2. Statistical analysis Th l Body mass index (BMI) values before and after the 12-month program (individuals who dropped to a lower BMI category). risks for obesity. Sedentary lifestyle and increased calories intake are prominent issues that often cause the obesity development and are associated with many metabolic disorders. the result showed no difference between BMI values before and after the implementation of the person alized training and nutrition plan (paired samples t = 2.006; p = 0.092; exact p = 0.25). The difference between the volunteers with normal and increased BMI values was reported before (independent sam ples t = 5.129; p = 0.000; exact p = 0.000) as well as after (independent samples t = 4.915; p = 0.000; exact p = 0.000) the implementation of the person alized training and nutrition plan. Almost one-third of the total number of volunteers reduced BMI from overweighed to normal after applying the personalized diet and workout program (Table 3). There was no statistically significant allelic and gen otype association between the 13 observed poly morphisms and individual BMI status (normal or overweight) (Table 4). No difference was found in the allele and genotype frequency of the observed gene polymorphisms between the male and female volunteers (Table 4). The single-locus F-test showed no association between the gene polymorphisms and BMI values. In addition, no correlation was detected between the gene polymorphisms and amount of BMI reduction prior and after the imple mentation of the personalized training and nutri tion plan within the overweighed group of volun teers (Table 5). Recommendations for the diet and training plan were made according to the genotyping of 18 volunteers from the population of Bosnia and Herzegovina. Different studies show the associa tion between obesity development and diet (3-7). According to the studies, FABP2, PPARG, APOA2, ADBR2, and ADRB3 polymorphisms are related to nutrition and ADRB2, ADRB3, ACE, and ACTN3 polymorphisms are related to exercise. The intestinal fatty acid binding protein encoded by FABP2 is involved in lipid metabolism. Polymorphism c.163A>G (p.Thr55Ala) (NCBI: rs1799883) is related with the increased binding affinity for the long-chain fatty acid, which can cause an increase in BMI and the triglycerides level in the blood (11). A genotype AA of the FABP2 gene is associated with a moderate risk of increased weight gain. Both polymorphisms were detected in all the volunteers, regardless of the BMI category. Statistical analysis Th l The PPARG is a transcription factor that plays a key role in the adipocyte gene expression and differenti ation of adipocytes. Obesity and nutritional factors are crucial in the expression of PPARG in human adipocytes. A lower BMI is associated with poly morphisms c.-2-28078C>G (p.Pro12Ala) (NCBI: rs1801282) of this gene (12,13). However, CC homozygosity was reported in all the participants Statistical analysis Th l Volunteers’ personal genotypes Parameters Gene PY‑113/15 PY‑114/15 PY‑115/15 PY‑116/15 PY‑117/15 PY‑118/15 PY‑119/15 PY‑120/15 PY‑129/15 Genotype Genotype Genotype Genotype Genotype Genotype Genotype Genotype Genotype Nutrition FABP2 GA GG GG AA GG AA GA GA GG PPARG CC CC CC CG CC CC CC CC CG APOA2 TT TT TC TC TC TC TT TC TC ADRB2 79 GG GG GC GC GC CC GC GC CC Nutrition and physical activity ADRB2 46 GG GG GA GA GA AA GA GA GG ADRB3 TT TT TT TT TT TT TT TT TT Physical activity ACTN3 CC GG CC CC CT CT CC CC CT ACE Ins/Ins Ins/Del Ins/Del Del/Del Ins/Del Del/Del Ins/Del Ins/Ins Del/Del Folate metabolism MTHFR 677 CC CT CC CC TT CT CT CT CT MTHFR 1298 AC AC AA AA AA AA AA AA AC Lactose intolerance MCM6 (LCT‑13910) CC CT CC CC CT CT CT TT CT MCM6 (LCT‑22018) GG GA GG GG GA GA GA AA GA Secretor status FUT2 GG GG GG AA GG GG GG GA GA Parameters Genes PY‑121/15 PY‑122/15 PY‑123/15 PY‑124/15 PY‑125/15 PY‑126/15 PY‑127/15 PY‑128/15 PY‑130/15 FABP2 GA GA GA GA GA AA GG GG GG Nutrition PPARG CC CC CC CC CC CC CC CC CC APOA2 TT CC TC TC CC TT TC TC TC ADRB2 79 GC CC GC GG GC GG CC CC CC ADRB2 46 GG GA GA GG GA GG GA GA GA Nutrition and physical activity ADRB3 TT CT TT TT TT TT TT TT TT ACTN3 CC CC CT CT CT CT CC CC CT Folate metabolism ACE Del/Del Ins/Ins Ins/Del Ins/Del Ins/Del Ins/Del Ins/Del Ins/Del Ins/Del MTHFR 677 CC CT CC TT TT CC TT CC CT Lactose intolerance MTHFR 1298 AA AC AA AA AA AC AA AC AA MCM6 (LCT‑13910) CT CT CC CC CC CT CT CT CT Secretor status MCM6 (LCT‑22018) GA GA GA GG GG GA GA GA GA FUT2 GA GA GG GA GA GA GA GG AA PPARG: Peroxisome proliferators‑activated receptor gamma; ADRB: Beta‑adrenergic receptor; ACE: Angiotensin‑converting enzyme; ACTN3: A‑actinin‑3 TABLE 2. Volunteers’ personal genotypes 94 Mirela Džehverović, et al. Journal of Health Sciences 2017;7(2):91-98 http://www.jhsci.ba FIGURE 1. Body mass index (BMI) values before and after the 12-month program (individuals who dropped to a lower BMI category). FIGURE 1. DISCUSSION Obesity is increasing worldwide, and recent advances in the genomics technology enable the prediction of 95 Mirela Džehverović, et al. Journal of Health Sciences 2017;7(2):91-98 http://www.jhsci.ba except for two participants that presented no changes in BMI values throughout the study. of abdominal fat and obesity development as a consequence of consuming food rich in saturated fats (14,15). The volunteers with TC genotype were recommended to reduce the intake of satu rated fatty acids. The ADRB2 gene is expressed in adipocytes and regulates lipid metabolism. Both polymorphic variants of this gene c.79C>G (p.Gin27Glu) (NCBI: rs1042714) and c.46A>G (p.Gly16Arg) (NCBI: rs1042713) may lead to a decrease of lipid metabolism and accumulation of fat tissue (16). An apolipoprotein gene polymorphism c.-323C>T (NCBI: rs5082) is associated with the accumulation TABLE 3. BMI values before (BMI‑B) and after (BMI‑A) the implementation of personalized training and nutrition plan; BMI reduction (BMI‑diff) ( ) Sample ID BMI Sex BMI‑P BMI‑A BMI‑diff 114 O M 25.1 24.49 0.61 115 O F 28.4 26.78 1.62 116 O F 26.56 26.56 0 117 O M 30.6 29.37 1.23 118 O M 27.2 27.2 0 120 O M 28.3 28.3 0 121 O F 34.78 32.99 1.79 125 O F 25.31 22.57 2.74 126 O M 27.1 27.1 0 128 O F 26.8 25.71 1.09 130 O M 26 24.93 1.07 113 N F 20.5 19.84 0.66 119 N F 23.5 22.52 0.98 122 N M 22.7 22.7 0 123 N F 24 22.72 1.28 124 N M 18.7 18.7 0 127 N F 20.7 20.7 0 129 N F 21.7 21.7 0 BMI: Body mass index The ADRB3 gene is predominantly expressed in the adipose tissue and regulates lipid metabolism and thermogenesis. A polymorphism c.190T>C (p.Trp64Arg) (NCBI: rs4994) of the ADRB3 gene may lead to impaired lipid metabolism which is related to overweight and enlarged waist circumference. Moreover, it has been reported that intensive workout decreases BMI in indi viduals with this genotype (17,18). The associa tion between c.190T>C (p.Trp64Arg) polymor phisms and increased BMI is still controversial (19). In addition, ACE is important in blood pressure control. ACE gene insertion is related to a decreased ACE protein activity, therefore, better physical endurance. Deletion in this gene, is however, related to an increased ACE protein activity and the possibility to perform physical TABLE 4. ndex; PPARG: Peroxisome proliferators-activated receptor gamma; ADRB: Beta-adrenergic receptor; ACE BMI: Body mass index; PPARG: Peroxisome proliferators-activated receptor gamma; ADRB: Beta-adrenergic receptor; ACE: A i t i ti ACTN3 A ti i 3 ndex; PPARG: Peroxisome proliferators activated receptor gamma; ADRB: Beta adrenergic receptor; ACE ing enzyme; ACTN3: A-actinin-3 y ; p p g ; g sin-converting enzyme; ACTN3: A-actinin-3 ACKNOWLEDGMENTS Results of this study were obtained from a scientific project supported by the Ministry of Education and Science of the Federation of Bosnia and Herzegovina. DISCUSSION Single-locus case–control test results of the allelic and genotype association between the 13 observed gene polymorphisms, individual BMI status, and gender se–control test results of the allelic and genotype association between the 13 observed gene BMI status, and gender polymorphisms, individual BMI status, and gender Marker BMI status (normal or overweight) Gender distribution Allele exact p Genotype exact p Allele exact p Genotype exact p FABP2 1.000 0.101 0.739 1.000 PPARG 1.000 1.000 0.473 0.461 APOA2 1.000 1.000 1.000 1.000 ADRB279 0.753 0.372 1.000 0.188 ADRB246 0.721 1.000 0.729 0.789 ADRB3 0.370 0.367 0.453 0.434 ACTN3 1.000 1.000 0.411 0.351 ACE 0.493 0.649 0.508 0.657 MTHFR677 0.493 0.691 0.191 0.107 MTHFR1298 0.686 0.617 1.000 1.000 LCT-13910 0.482 0.788 0.162 0.185 LCT-22018 0.735 0.789 0.168 0.262 FUT2 0.705 0.788 0.724 1.000 BMI: Body mass index; PPARG: Peroxisome proliferators-activated receptor gamma; ADRB: Beta-adrenergic receptor; ACE: Angiotensin-converting enzyme; ACTN3: A-actinin-3 96 Mirela Džehverović, et al. Journal of Health Sciences 2017;7(2):91-98 http://www.jhsci.ba TABLE 5. Single‑locus F‑test results of the gene polymorphisms and BMI values association problems is highly recommended, and a genetic analysis of associated polymorphisms can provide valuable information for personalized training and diet programs. 5 S g e ocus es esu s o e ge e polymorphisms and BMI values association Marker BMI BMI‑difference* F‑statistics p F‑statistic p FABP2 0.2394 0.7900 0.0015 0.9986 PPARG 0.2420 0.6295 1.1623 0.3090 APOA2 0.2091 0.8137 3.2132 0.0946 ADRB279 3.5841 0.0534 1.0029 0.4086 ADRB246 0.1480 0.8637 0.6025 0.5705 ADRB3 0.5056 0.4873 NaN NaN ACTN3 0.1193 0.7343 0.0739 0.79193 ACE 0.8668 0.4403 1.0376 0.39751 MTHFR677 0.8585 0.4436 2.6599 0.13013 MTHFR1298 1.2724 0.2760 0.6140 0.45341 LCT‑13910 0.8285 0.4557 1.0951 0.37985 LCT‑22018 0.6089 0.5569 0.7250 0.51363 FUT2 0.1316 0.8777 0.2477 0.78637 *Group of individuals with an increased BMI. 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https://openalex.org/W2264291204	https://europepmc.org/articles/pmc4747545?pdf=render	English	null	Vitamin C Intake and Pancreatic Cancer Risk: A Meta-Analysis of Published Case-Control and Cohort Studies	PloS one	2,016	cc-by	4,916	RESEARCH ARTICLE Yong-Fei Hua1, Gao-Qing Wang1, Wei Jiang1, Jing Huang1, Guo-Chong Chen2, Cai- De Lu1* 1 Department of Hepatobiliary and Pancreatic Surgery, Ningbo Medical Treatment Center Lihuili Hospital, Ningbo, 315000, China, 2 Department of Nutrition and Food Hygiene, School of Public Health, Soochow University, Suzhou, 215123, China * lucaide@nbu.edu.cn Results Our final analyses included 20 observational studies comprising nearly 5 thousand cases of pancreatic cancer. When comparing the highest with the lowest categories of vitamin C intake, the summary odds ratio/relative risk for case-control studies (14 studies), cohort studies (6 studies) and all studies combined was 0.58 (95% CI: 0.52–0.66), 0.93 (95% CI: 0.78–1.11) and 0.66 (95% CI: 0.58–0.75), respectively. The difference in the findings between case-control and cohort studies was statistically significant (P < .001). Possible publication bias was shown in the meta-analysis of case-control studies. Copyright: © 2016 Hua 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. Background Observational studies inconsistently reported the relationship between vitamin C intake and risk of pancreatic cancer. We conducted a meta-analysis of published case-control and cohort studies to quantify the association. Methods Citation: Hua Y-F, Wang G-Q, Jiang W, Huang J, Chen G-C, Lu C-D (2016) Vitamin C Intake and Pancreatic Cancer Risk: A Meta-Analysis of Published Case-Control and Cohort Studies. PLoS ONE 11(2): e0148816. doi:10.1371/journal. pone.0148816 Potentially eligible studies were found on PubMed and EMBASE databases through May 31, 2015. A random-effects model was assigned to compute summary point estimates with corresponding 95% confidence intervals (CIs). Subgroup and meta-regression analyses were also performed to explore sources of heterogeneity. Vitamin C Intake and Pancreatic Cancer Risk: A Meta-Analysis of Published Case-Control and Cohort Studies Yong-Fei Hua1, Gao-Qing Wang1, Wei Jiang1, Jing Huang1, Guo-Chong Chen2, Cai- De Lu1* Introduction Globally, pancreatic cancer represents the 4th leading cause of cancer death. Pancreatic cancer has a poor prognosis and high fatality rate, with a 5-year survival rate of less than 5%[1]. Poten- tial risk factors include chronic pancreatitis, cigarette smoking, family history, and diabetes[1, 2]. Dietary factors may also pay a role in the development of pancreatic cancer, but few certain- ties have been achieved[3]. Oxidative stress is implicated in the pathogenesis of acute and chronic pancreatitis[4]. Both in vitro and in vivo researches demonstrate an important role of inflammation in the initiation and progression of pancreatic cancer[5]. Vitamin C is a strong antioxidant, and is shown to inhibit preneoplastic lesions in animals’ pancreas[6]. In experimental studies, vitamin C has been found to induce apoptosis, enhance immune function, and protect free radicals-induced DNA damage[3, 7]. Thus, vitamin C intake is biologically plausible to protect against pancre- atic cancer. Recently, an expert panel convened by the Word Cancer Research Fund and the American Institute for Cancer Research concluded that the evidence for a beneficial effect of fruit against pancreatic cancer was limited and inconsistent, and the evidence for vegetable and vitamin C was judged as ‘limited-no conclusion’[8].On the contrary, a more recent meta-analysis of 17 observational studies (13 case-control studies and 4 cohort studies) suggested a significant 29.5% reduction in the risk of pancreatic cancer for high-versus-low vitamin C intake[9]. How- ever, results from this meta-analysis could be limited because of including duplicate publica- tions from the same population[10, 11], analyzing data of dietary vitamin C together with those of circulating vitamin C[12], pooling risk estimates expressed as continuous vitamin C intake in the high-versus-low analysis[13], and missing a number of eligible studies[14–19]. Given the inconsistent evidence, we performed a meta-analysis of published observational studies to better quantify the association of vitamin C intake with risk of pancreatic cancer. Case-control studies and prospective cohort studies were separately analyzed throughout the meta-analysis because they are considerably different in many aspects. Literature search and selection A literature search was performed on PubMed and EMBASE databases through May 31, 2015 using the search strategy as follows: (vitamin C OR ascorbate) and (pancreatic cancer OR pan- creatic carcinoma OR cancer of pancreas). No language restrictions were imposed. The refer- ence lists of retrieved full-text publications were also carefully screened to identify any further studies. Studies that fulfilled the following criteria were considered eligible: (i) case-control or cohort study design; (ii) vitamin C intake as the exposure of interest; (iii) pancreatic cancer as the outcome of interest; and (iv) odds ratio (OR) or relative risk (RR) or hazard ratio (HR) esti- mates with corresponding 95% confidence interval (CI) were available. Conclusion There is insufficient evidence to conclude any relationship between vitamin C intake and risk of pancreatic cancer. The strong inverse association observed in case-control studies may be affected by biases (eg, recall and selection biases) that particularly affect case-con- trol studies and/or potential publication bias. Future prospective studies of vitamin C intake and pancreatic cancer are needed. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. 1 / 10 PLOS ONE \| DOI:10.1371/journal.pone.0148816 February 9, 2016 Meta-Analysis of Vitamin C and Pancreatic Cancer Study characteristics A flow chart of study selection is reported in Fig 1. Our final analysis included 14 case-control studies[10, 16–19, 23, 27–34] and 6 cohort studies[14, 15, 35–38]. The 14 case-control studies were published between 1988 and 2013, covering 3,818 pancreatic cancer cases and 10,115 con- trols. Geographic regions of these studies ranged from North America (N = 8), Europe (N = 3) to Asia (N = 3). Most of the case-control studies selected control subjects from general popula- tions (11/14), and recruited both men and women (12/14). Ten of the 14 studies only reported results for dietary vitamin C, 2 studies merely investigated supplementary vitamin C, and 2 studies involved vitamin C of different sources. The 6 prospective cohort studies were pub- lished between 2002 and 2013, with a total of 1,140 cancer events and 278,000 participants. The length of follow-up ranged from 7.1 to 21 years. Four studies only reported results for die- tary vitamin C, and 2 studies contained vitamin C of different sources. Four of the 6 studies included both sexes, 1 study included men only, and the remaining 1 consisted entirely of women. Half of the 6 cohorts were from the US, and the others were conducted in Europe. The characteristics of the included case-control studies and cohort studies are, respectively, summa- rized in S1 and S2 Tables. Data extraction The following data were extracted from each included study by use of a standardized data-col- lection form: the first author’s last name, publication year, country, source of controls (for case-control studies), length of follow-up (for cohort studies), sex of subjects, sample size, source of vitamin C, categorized vitamin C intake and corresponding risk estimates that reflected the greatest degree of adjustment, and variables accounted for in the statistical model. Literature search, study selection and data extraction were carried out independently by two authors (YFH and WJ), with any disagreement resolved by consensus. 2 / 10 PLOS ONE \| DOI:10.1371/journal.pone.0148816 February 9, 2016 Meta-Analysis of Vitamin C and Pancreatic Cancer Statistical analysis In this meta-analysis, the common measure of association was OR in case-control studies, and RR in cohort studies. Results from case-control and cohort studies were separately presented. A random-effects model[20] which considers both within-and between-study variation was assigned to combine study-specific risk estimates. Several studies presented results for different sources of vitamin C intake (total, diet and supplement), and the results for total intake were included in the main analysis. Subgroup analysis were performed according to geographic area, source of control (for case-control studies), sex of subjects, years of follow-up (for cohort stud- ies), number of events, and source of vitamin C. Considering the distinct cutoffs across studies, we conducted a dose-response analysis with the method of Greenland and Longnecker[21] and Orsiniet al.[22]. The method requires that the number of cases and controls (or person-years) and the risk estimates with their variance for at least 3 quantitative exposure categories are known. For every study, the median/mean level of the intake for each category was assigned to each corresponding risk estimate. When the median/mean intake per category was not provided, the midpoint of the upper and lower boundaries in each category was assigned as the average intake. If the highest/lowest category was open-ended, we assumed the width of the interval to be the same as in the closest category. Because of limited number of eligible studies in case-control studies[16, 18, 23]., we only con- ducted the dose-response analysis for prospective cohort studies. Heterogeneity test was performed using Q and I2 statistics [24]. For the Q statistic, level of significance was set at P<0.1; and for the I2, a value of <25%, 25–75% and >75% represents lit- tle/no, moderate, and considerable heterogeneity. Potential publication bias was investigated with both Begg correlation test and Egger regression test[25, 26],. All statistical analyses were performed using STATA software, version 12.0 (STATA Corp., College Station, TX, USA). Meta-analysis of case-control studies The multivariable-adjusted OR of pancreatic cancer from individual case-control studies and all studies combined are reported in Fig 2. All studies showed an inverse association between vitamin C intake and risk of pancreatic cancer, among which 11 reported significant results. 3 / 10 PLOS ONE \| DOI:10.1371/journal.pone.0148816 February 9, 2016 Meta-Analysis of Vitamin C and Pancreatic Cancer Fig 1. Flow chart of literature search and selection. doi:10.1371/journal.pone.0148816.g001 Fig 1. Flow chart of literature search and selection. doi:10.1371/journal.pone.0148816.g001 doi:10.1371/journal.pone.0148816.g001 Results of the meta-analysis conferred a summary OR of 0.58 (95% CI: 0.52–0.66) for the high- est compared with the lowest intake of vitamin C, with no evidence of heterogeneity (Phetero- geneity = 0.57, I2 = 0%). Subgroup analysis stratified by predefined study and population characteristics showed consistent results (Table 1). Source of control was found to be an effect modifier (Pdifference = 0.04), with a more pronounced effect among hospital-based case-control studies (OR = 0.46) than population-based ones (OR = 0.63). Vitamin C from diet (OR = 0.58) also showed a stronger protection than supplement (OR = 0.79), but the between-group differ- ence was statistically nonsignificant (Pdifference = 0.29). Dose-response analysis were not per- formed due to limited number of eligible studies[16, 18,23]. doi:10.1371/journal.pone.0148816.g002 Meta-Analysis of Vitamin C and Pancreatic Cancer Fig 3. Relative risks of pancreatic cancer for the highest compared with the lowest categories of vitamin C intake in cohort studies. d i 10 1371/j l 0148816 003 Fig 3. Relative risks of pancreatic cancer for the highest compared with the lowest categories of vitamin C in of pancreatic cancer for the highest compared with the lowest categories of vitamin C intake in cohort studie Fig 3. Relative risks of pancreatic cancer for the highest compared with the lowest categories of vitamin C intake in cohort studies. doi:10.1371/journal.pone.0148816.g003 doi:10.1371/journal.pone.0148816.g003 analysis was not performed due to limited studies. Dose-response meta-analysis of all cohorts showed a RR of 0.97 (95% CI: 0.89–1.05) for each 100-mg/day increment in vitamin C intake. Publication bias Egger’s test indicated possible publication bias in the meta-analyses of case-control studies (PEgger = 0.07) and all studies (PEgger = 0.06), but notin the analysis of cohort studies (PEgger = 0.61). Begg’s test excluded such a bias (PBegg>0.20). Meta-analysis of all studies A combined analysis of the 20 observational studies (4,958 cancers) yielded a summary OR of 0.66 (95% CI: 0.58–0.75), with moderate heterogeneity among studies (Pheterogeneity = 0.05, I2 = 35.8%). Meta-regression analysis suggested a statistically significant difference in the results between case-control and cohort studies (Pdifference<0.001). PLOS ONE \| DOI:10.1371/journal.pone.0148816 February 9, 2016 Meta-analysis of cohort studies Fig 3 presents multivariable-adjusted risk estimates of pancreatic cancer from each cohort and all cohorts combined. Three of the 6 cohorts (accounting for 36.1% of total cases) reported an inverse association between vitamin C intake and risk of pancreatic cancer (RR ranged between 0.79 and 0.91), but none showed statistical significance. Overall, the summary RR for the highest- versus-lowest intake of vitamin C was 0.93 (95% CI: 0.78–1.11), with no evidence of heterogeneity (Pheterogeneity = 0.95, I2 = 0%). The null association persisted in the subgroup analysis (Table 2). Supplementary vitamin C showed a nonsignificant inverse association with pancreatic cancer (RR = 0.83, 95% CI: 0.64–1.07), but only 2 studies were included in the analysis. Sex-specific 4 / 10 PLOS ONE \| DOI:10.1371/journal.pone.0148816 February 9, 2016 Meta-Analysis of Vitamin C and Pancreatic Cancer Fig 2. Odds ratios of pancreatic cancer for the highest compared with the lowest categories of vitamin C intake in case-control studies. doi:10.1371/journal.pone.0148816.g002 Fig 2. Odds ratios of pancreatic cancer for the highest compared with the lowest categories of vitamin C intake in case-control studies. Fig 2. Odds ratios of pancreatic cancer for the highest compared with the lowest categories of vitamin C intake in case-control studies. doi:10.1371/journal.pone.0148816.g002 Fig 2. Odds ratios of pancreatic cancer for the highest compared with the lowest categories of vitamin C intake in case-control studies. Table 1. Results of stratified analysis for case-control studies. N OR (95% CI) P for heterogeneity I2 (%) P for differences Area North America 8 0.60 (0.51–0.70) 0.36 9.6 0.46 Europe 3 0.54 (0.36–0.83) 0.22 34.7 Asia-paciﬁc 3 0.53 (0.40–0.72) 0.83 0.0 Source of control Population 11 0.63 (0.55–0.73) 0.79 0.0 0.04 Hospital 3 0.46 (0.37–0.59) 0.73 0.0 Sex Men 5 0.61 (0.44–0.86) 0.02 66.7 0.70 Women 5 0.59 (0.47–0.73) 0.90 0.0 No. of cases 200 9 0.58 (0.51–0.66) 0.42 2.3 0.90 <200 5 0.59 (0.45–0.79) 0.53 0.0 Source of vitamin C Diet 12 0.58 (0.49–0.69) 0.11 33.4 0.29 Supplement 4 0.70 (0.60–0.82) 0.57 0.0 doi:10.1371/journal.pone.0148816.t001 Table 1. Results of stratified analysis for case-control studies. PLOS ONE \| DOI:10.1371/journal.pone.0148816 February 9, 2016 5 / 10 Meta-Analysis of Vitamin C and Pancreatic Cancer Table 2. Results of stratified analysis for cohort studies. N RR (95% CI) P for heterogeneity I2 (%) P for differences Area North America 3 0.86 (0.66–1.13) 0.80 0 0.49 Europe 3 0.99 (0.78–1.25) 0.94 0 Length of follow-up 10 years 3 0.98 (0.78–1.24) 0.96 0 0.51 <10 years 3 0.86 (0.65–1.14) 0.76 0 No. of cases 200 2 1.00 (0.78–1.28) 0.97 0 0.49 <200 4 0.87 (0.68–1.12) 0.90 0 Source of vitamin C Diet 5 0.92 (0.73–1.16) 0.97 0 0.56 Supplement 2 0.83 (0.64–1.07) 0.96 0 doi:10.1371/journal.pone.0148816.t002 Table 2. Results of stratified analysis for cohort studies. control studies showed an inverse association. It is more likely that the distinct findings were due to differences in methodologies they applied. Case-control studies relative to cohort studies are more prone to biases such as recall bias and selection bias. For instance, those foods rich in vitamin C (e.g., fruit and vegetables) have been widely considered beneficial for public health and also for cancer prevention. In this condition, patients with pancreatic cancer probably underreported their dietary intakes of these foods, leading to an overestimation of the associa- tion. Further, the temporal sequence between exposure and outcome is difficult to determine in case-control studies. In other words, cases may have altered their dietary intakes after the diagnosis of cancer. In addition, potential selection bias also merits a particular consideration when studying pancreatic cancer, a malignancy characterized by high fatality rates. On the other hand, both case-control and cohort studies are subject to exposure misclassification when investigating dietary factors and health outcomes, because they mostly used food fre- quency questionnaires to collect diet information. For cohort studies that generally made a sin- gle measurement of dietary intakes at baseline, additional misclassification of exposure could occur because participants may change their diet habits during a long-term follow-up period. Circulating vitamin C concentrations represent a better indicator of vitamin C status than dietary intakes. However, current evidence linking circulating vitamin C to pancreatic cancer risk has been limited and inconclusive. Results from the European Prospective Investigation into Cancer and Nutrition (EPIC)-Norfolk study[35] showed a strong inverse association between serum vitamin C and pancreatic cancer during 17 years of follow-up (RR for inter- quartile comparison = 0.42, 95% CI: 0.20–0.91, 76 cancers). Discussion In this meta-analysis of published observational studies that involved nearly 5,000 pancreatic cancers, high vitamin C intake was significantly associated with a 42% reduction in the risk of pancreatic cancer in case-control studies, whereas no association was found in cohort studies. Fruit and vegetables are a rich source of dietary vitamin C, and have long been suspected to prevent several cancers including pancreatic cancer[8]. However, recent cumulative evidence showed that the protection of fruit and vegetables on pancreatic cancer was observed in case- control studies but not in cohort studies[39], which agreed with the findings of this meta- analysis. The considerable differences between results from case-control studies and those from cohort studies may be attributed to several reasons. There was a suggestion of publication bias in case-control studies, which indicated that some small studies or studies with null effects may have been unpublished. However, such a bias was unlikely to fully explain why especially case- 6 / 10 PLOS ONE \| DOI:10.1371/journal.pone.0148816 February 9, 2016 Conversely, a more recent case- control study nested in the whole EPIC study contradicted the previous findings (RR for inter- quartile comparison = 0.91, 95% CI: 0.55–1.51, 442 cancers)[12]. Long-term, double-blind, randomized controlled trials (RCTs) provide the most robust esti- mates of causal effects. However, such RCTs examining the effect vitamin C supplement on pancreatic cancer are also limited. A Cochrane systematic review[40] reported a null effect, but only 1 trial of vitamin C supplementation (combined with vitamin E and beta-carotene) and pancreatic cancer was included. The Physicians’ Health Study (PHS) II randomized trial[41] showed a nonsignificant 14% reduction in pancreatic cancer death (50 deaths) associated with vitamin C supplement after a mean follow-up duration of 8.0 years (RR = 0.86, 95% CI: 0.49– 1.49). Subsequently, a posttrial observation[42] with additional 3.8 years of follow up strength- ened the RR to be 0.50 (95% CI: 0.22, 1.18). This observation might raise the problem of a low 7 / 10 PLOS ONE \| DOI:10.1371/journal.pone.0148816 February 9, 2016 Meta-Analysis of Vitamin C and Pancreatic Cancer statistical power resulting from short durations in these RCTs. Although well-designed RCTs provide the best evidence, the trials are expensive and only the most promising candidate can be tested in such a way. Given these null findings and possibly increased mortality associated with antioxidants supplements in clinical trials[40], it appears imprudent to specifically investi- gate the effects of vitamin C supplement on the prevention of pancreatic cancer in large RCTs before the possible benefits are consistently showed in large prospective observational studies. This meta-analysis comprehensively quantified current evidence from published case-con- trol and cohort studies comprising nearly 5,000 cases of pancreatic cancer and therefore largely strengthened the power of the analyses. However, we also acknowledged that there were several limitations to our study. Our results may have been affected by residual or unmeasured con- founders because higher dietary vitamin C often positively corrected with better lifestyle pat- terns. What is more, as was discussed above, potential methodological flaws inherit in the original studies may bias our findings towards either direction. In addition, since the meta- analysis is on the basis of published literature, our results, in particular those from case-control studies may have been affected by publication bias. In sum, this meta-analysis showed a significant inverse association between vitamin C intake and risk of pancreatic cancer in case-control studies but not in prospective cohort stud- ies. S2 Table. Characteristics of the included cohort studies of vitamin C intake and risk of pan- creatic cancer. (DOCX) S2 Table. Characteristics of the included cohort studies of vitamin C intake and risk of pan- creatic cancer. (DOCX) Author Contributions Conceived and designed the experiments: YFH CDL. Performed the experiments: GQW WJ. Analyzed the data: GQW WJ GCC. Contributed reagents/materials/analysis tools: CDL. Wrote the paper: YFH. Helped to develop the search strategy: GCC. Helped to revise the Introduction and Discussion sections during the first revision of the manuscript: JH. S1 PRISMA Checklist. PRISMA Checklist. (DOCX) S1 Table. Characteristics of the included case-control studies of vitamin C intake and risk of pancreatic cancer. Thus, there is insufficient evidence to conclude any relationship between vitamin C intake and risk of pancreatic cancer. PLOS ONE \| DOI:10.1371/journal.pone.0148816 February 9, 2016 References 1. Michl P, Gress TM. 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https://openalex.org/W3168164173	https://periodicos.univali.br/index.php/rtva/article/download/17628/10083	Portuguese	null	Cicloturismo como promotor do desenvolvimento de áreas rurais: Possibilidades na região do Baixo Parnaíba Maranhense	Turismo: visão e ação/Turismo : visão e ação	2,021	cc-by	9,534	Rodrigo: Graduando em Turismo na Universidade Federal do Maranhão, São Bernardo, Maranhão, Brasil. E-mail: rod20sousa@gmail.com. Orcid: 0000-0003-0706-1415 Karoliny: Mestre em Cultura e Turismo, Universidade Federal do Maranhão, São Bernardo, Maranhão, Brasil. E-mail: karolinydiniz@gmail.com. Orcid: 0000-0001-7059-5087 CICLOTURISMO COMO PROMOTOR DO DESENVOLVIMENTO DE ÁREAS RURAIS: POSSIBILIDADES NA REGIÃO DO BAIXO PARNAÍBA MARANHENSE CICLOTURISMO COMO PROMOTOR DO DESENVOLVIMENTO DE ÁREAS RURAIS: POSSIBILIDADES NA REGIÃO DO BAIXO PARNAÍBA MARANHENSE Licença CC BY: Artigo distribuído sob os termos Creative Commons, permite uso e distribuição irrestrita em qualquer meio desde que o autor credite a fonte original. CYCLOTOURISM AS A PROMOTER OF DEVELOPMENT OF RURAL AREAS: POSSIBILITIES IN THE BAIXO PARNAÍBA MARANHENSE REGION CICLOTURISMO COMO PROMOTOR DEL DESARROLLO DE LAS ZONAS RURALES: POSIBILIDADES EN LA COMARCA DEL BAIXO PARNAÍBA MARANHENSE http://dx.doi.org/10.14210/rtva.v23n2.p329-349 CICLOTURISMO COMO PROMOTOR DEL DESARROLLO DE LAS ZONAS RURALES: POSIBILIDADES EN LA COMARCA DEL BAIXO PARNAÍBA MARANHENSE RODRIGO OLAVO COSTA SOUSA¹ KAROLINY DINIZ CARVALHO¹ ¹ UNIVERSIDADE FEDERAL DO MARANHÃO, SÃO BERNARDO, MARANHÃO, BRASIL. DATA DE SUBMISSÃO: 24/04/2020 – DATA DE ACEITE: 03/03/2021 RESUMO: Uma das modalidades inseridas no âmbito do turismo no espaço rural consiste nos passeios de bicicleta, segmento que permite aos visitantes um modo mais estreito e particular de vivenciar o espaço e que alia recreação, lazer e educação ambiental, gerando práticas de hospitalidade ao longo dos percursos. O presente artigo aborda o cicloturismo como elemento da oferta de turismo rural no território do Baixo Parnaíba Maranhense. Como objetivos específicos, a pesquisa volta-se para compreender a dinâmica do turismo rural, identificar os sentidos e significados do cicloturismo, e verificar como ocorre a prática do cicloturismo no Baixo Parnaíba, seus aspectos favoráveis e limitantes, com vistas a problematizar a viabilidade ou não desta prática como produto turístico potencial. Trata-se de uma pesquisa orientada pelos condicionantes da investigação qualitativa, possuindo um caráter exploratório e descritivo. Na construção do referencial teórico, valeu-se das contribuições de Tulik (2003), Rodrigues (2001) e Kageyama (2008) sobre a dinâmica do turismo rural; as análises acerca do cicloturismo foram realizadas a partir dos estudos de Han et al. (2017), Cavallari (2012) Lima (2015), Carvalho et al. (2013); a relação entre cicloturismo e desenvolvimento local foi tecida a partir dos estudos de Buarque (2002), Vázquez Barquero (2001) e Duarte (2008). Na pesquisa de campo, fez-se uso da técnica de observação direta e sistemática (Cervo, 2007), realizada por meio do acompanhamento de grupos de ciclistas nos roteiros de cicloturismo local. Evidenciou-se práticas de hospitalidade e de acolhimento e a abertura de pequenos negócios de alimentação ao longo das rotas desenvolvidas, sinalizando para o empreendedorismo comunitário, no entanto, a pesquisa aponta para algumas fragilidades, como a ausência de políticas públicas sistemáticas, a fim de organizar e estruturar a oferta de turismo rural, consoante às expectativas e demandas comunitárias. ISSN: 1983-7151 ur., Visão e Ação, v23, n2, p329-349, Mai./Ago. 2021 - Balneário Camboriú, Santa Catarina, Brasil PALAVRAS-CHAVE: Turismo Rural; Cicloturismo; Desenvolvimento Local; Território do Baixo Parnaíba Maranhense. PALAVRAS-CHAVE: Turismo Rural; Cicloturismo; Desenvolvimento Local; Território do Baixo Parnaíba Maranhense. ABSTRACT: One of the modalities in the field of tourism in rural areas is cycling, a segment that gives visitors a more narrow and particular way of experiencing the space, while combining recreation, leisure and environmental education, generating practices of hospitality along the cycling routes. This article focuses on cycling as an element of the rural tourism offer in the Baixo Parnaíba Maranhense region. DATA DE SUBMISSÃO: 24/04/2020 – DATA DE ACEITE: 03/03/2021 The specific objectives of this research are to understand the dynamics of rural tourism, to identify the senses and meanings of cycling tourism, and to examine how cycling occurs in the Baixo Parnaiba and the factors that favor or limit this practice, in order to question the viability of this practice as a potential tourist product. This study uses qualitative research, having an exploratory and descriptive character. In the construction of the theoretical framework, reference was made to the authors Tulik (2003), Rodrigues (2001) and Kageyama (2008) in relation to the dynamics of rural tourism; the studies of Han et al. (2017), Cavallari (2012) Lima (2015), Carvalho et al. (2013) on the analyses of cycling tourism; the studies of Buarque (2002), Vázquez Barquero (2001) and Duarte (2008) on the relationship between cycling and local development. In the field research, the technique of direct and systematic observation was used (Cervo, 2007). This involved monitoring groups of cyclists on the local cycling tours. The were signs of hospitality and practices of welcoming, and the opening of small food businesses along the developed routes, signaling community entrepreneurship. However, the research points to some weaknesses, such as the absence of systematic public policies, that hinder the organization and structuring of the offer of rural tourism in the region, according to the community’s expectations and demands. KEYWORDS: Rural Tourism; Cycling tourism; Local Development; Territory of Baixo Parnaíba Maranhense. RESUMEN: Una de las modalidades insertadas en el campo del turismo en el medio rural consiste en los paseos en bicicleta, segmento que permite al visitante una forma más estrecha y particular de vivir el espacio y que combina recreación, ocio y educación ambiental, generando prácticas de hospitalidad a lo largo de las rutas. Este artículo aborda la bicicleta como elemento de la oferta turística rural en el territorio del Baixo Parnaíba Maranhense. Como objetivos específicos, la investigación tiene como objetivo comprender la dinámica del turismo rural, identificar los significados y significados del cicloturismo, y verificar cómo se da la práctica de la bicicleta en el Baixo Parnaiba, sus aspectos favorables y limitantes, con el fin de cuestionar la viabilidad o no de esta práctica como producto turístico potencial. Es una investigación guiada por las condiciones de la investigación cualitativa, que tiene un carácter exploratorio y descriptivo. INTRODUÇÃO O turismo é um fenômeno complexo que se transforma devido às modificações históricas, socioculturais e às mudanças científicas e tecnológicas, tais como a evolução dos meios de transporte e comunicação e a própria globalização que vem impactando a atividade. É formado por elementos materiais e imateriais, os quais sofrem um processo de agregação econômica, e promove a articulação dos públicos e privados, com o fim de proporcionar aos visitantes/turistas uma experiência de viagem ao mesmo tempo espacial, cultural, educativa e ambiental. A importância adquirida pelo turismo na atualidade reforça a sua aproximação com as áreas rurais, onde os visitantes podem interagir com os elementos dos ecossistemas e com o cotidiano das comunidades, suas diferentes formas de expressão cultural e formas de relacionamento com o meio ambiente, seguindo a perspectiva de sustentabilidade e desenvolvimento local. A associação entre atividades agrícolas e não agrícolas é sinalizada como estratégia de diversificação das atividades locais, fortalecimento e valorizando o patrimônio local. O turismo no meio rural abrange diversas modalidades de consumo de experiências, tais como o turismo rural, o agroturismo, sendo compreendido como uma das alternativas voltadas ao aproveitamento econômico das áreas rurais que agregam valor à produção e ao cotidiano local ao organizar atividades de lazer e hospitalidade relacionadas às vivências rurais (Zimmermann, 2000; Tulik, 2003). Uma das atividades inseridas no âmbito do turismo no espaço rural consiste nos passeios de bicicleta, segmento que permite aos visitantes um modo mais estreito e particular de vivenciar o espaço e que alia recreação, lazer e educação ambiental, gerando práticas de hospitalidade ao longo dos percursos. Os roteiros e trilhas de cicloturismo podem se configurar num modo sustentável de se praticar o turismo no espaço rural, dando visibilidade aos atores sociais, suas histórias e tradições (Soares, 2010; Lima, 2015; Han et al.,2017). Estas são as reflexões que propiciaram a realização deste estudo, delimitando o recorte espacial para os municípios que abrangem a microrregião do Baixo Parnaíba Maranhense. A escolha desses municípios como objeto de estudo deve-se à existência de um diversificado patrimônio ambiental e cultural, e pelo fato das comunidades locais estarem sofrendo um processo de marginalização com o estabelecimento de projetos agroexportadores de soja e eucalipto, os quais produzem impactos negativos nos modos de vida e nas práticas de reprodução material e simbólica local (Costa, 2016). http://dx.doi.org/10.14210/rtva.v23n2.p329-349 http://dx.doi.org/10.14210/rtva.v23n2.p329-349 DATA DE SUBMISSÃO: 24/04/2020 – DATA DE ACEITE: 03/03/2021 En la construcción del marco teórico, Tulik (2003), Rodrigues (2001) y Kageyama (2008) contribuyeron sobre la dinámica del turismo rural; los análisis sobre cicloturismo se realizaron a partir de los estudios de Han et al. (2017), Cavallari (2012) Lima (2015), Carvalho et al. (2013); la relación entre la bicicleta y el desarrollo local se tejió a partir de los estudios de Buarque (2002), Vázquez Barquero (2001) y Duarte (2008). En la investigación de campo se utilizó la técnica de observación directa y sistemática (Cervo, 2007), realizada a través del seguimiento de grupos de ciclistas en los recorridos ciclistas locales. Se evidenciaron prácticas de hospitalidad y acogida y apertura de pequeños negocios alimentarios a lo largo de las rutas desarrolladas, señalando el emprendimiento comunitario, sin embargo, la investigación apunta a algunas debilidades, como la ausencia de políticas públicas sistemáticas, con el fin de organizar y estructurar la oferta de turismo rural de acuerdo con las expectativas y demandas de la comunidad. PALABRAS CLAVE: Turismo Rural; Cicloturismo; Desarrollo local; Territorio del Baixo Parnaíba Maranhense. WWW.UNIVALI.BR/PERÍODICOS TURISMO RURAL E CICLOTURISMO: DIÁLOGOS CONCEITUAIS O turismo rural é uma atividade que vem se desenvolvendo desde o século XX (Brasil, 2010) e pode ser entendido como o segmento que valoriza os aspectos naturais e culturais de localidades rurais. Embora o conceito de turismo rural não seja consensual, posto que apresenta diferentes contornos teóricos, como o Turismo no Espaço Rural, Agroturismo e o Turismo Rural na Agricultura Familiar (TRAF), de um modo geral, e segundo o Ministério do Turismo (Brasil, 2010), o turismo rural abrange as atividades turísticas que são desenvolvidas no meio rural com o objetivo de agregar valor aos produtos e serviços, à produção agropecuária e promover o patrimônio cultural e natural das comunidades. Trata-se de um conceito amplo e que leva em consideração aspectos relacionados aos territórios, aos recursos naturais e culturais e às formas de aproveitamento das áreas rurais pelo e para o turismo. Tal imprecisão conceitual é analisada por Tulik (2010), ao indicar a existência de nuances diferenciadoras entre o turismo em áreas rurais e o turismo rural. Enquanto aquele se refere ao conjunto de atividades desenvolvidas no espaço rural, este pressupõe o ambiente rural como motivo principal da atratividade turística, ou seja, as especificidades do rural são fatores determinantes na caracterização deste segmento de mercado. Rodrigues (2001) aborda essa questão de modo semelhante, entendendo que o turismo rural estaria relacionado aos elementos que conferem à paisagem a sua fisionomia nitidamente rural: as produções agrárias, a paisagem e os ecossistemas ricos em biodiversidade. Outros autores que auxiliam na compreensão do turismo rural são Matias e Sardinha (2002), os quais identificam características inerentes a esta modalidade de turismo, com destaque para as vivências dos turistas no cotidiano e no modo de vida rural, para o contato direto e a interação com a natureza. Compartilham ainda a visão de que o referido segmento é praticado em escala reduzida, com baixo impacto ambiental. Assim como os demais segmentos de turismo dito alternativos, o turismo rural propicia um contato mais próximo dos visitantes com o cotidiano do meio rural: paisagens campestres, produções agrícolas, práticas de sociabilidade, patrimônio material e simbólico (Schetino, 2006; Oliveira, 2018). O turismo pode se converter num fator de ressignificação das paisagens e das comunidades rurais, fortalecendo suas particularidades e ampliando as opções de práticas econômicas e de convivência intercultural, com o estreitamento das relações entre o rural e o urbano. INTRODUÇÃO Nesse cenário de conflitos, a atividade turística é projetada como fator de promoção cultural, manutenção das famílias nas zonas rurais, dinamização da renda familiar e aproveitamento sustentável dos recursos naturais e dos bens simbólicos representativos do patrimônio destas comunidades. Assim, busca-se neste artigo, tecer algumas considerações sobre o cicloturismo como elemento da oferta de turismo rural no território do Baixo Parnaíba Maranhense, passível de articular os atores sociais locais (comunidade, gestores públicos e privados) em torno de práticas solidárias de organização do espaço rural com vistas à estruturação de produtos turísticos sustentáveis. Como objetivos específicos, a pesquisa volta-se para compreender a dinâmica do turismo rural, identificar os sentidos e significados do cicloturismo, e verificar como ocorre a prática do cicloturismo no Baixo Parnaíba, seus aspectos favoráveis e ISSN: 1983-7151 Tur., Visão e Ação, v23, n2, p329-349, Mai./Ago. 2021 - Balneário Camboriú, Santa Catarina, Bras limitantes, com o intuito de problematizar a viabilidade, ou não, desta prática como produto turístico potencial. TURISMO RURAL E CICLOTURISMO: DIÁLOGOS CONCEITUAIS Kageyama (2008) expressa bem essas mudanças, apontando a hibridez do espaço rural com a diversidade de ocupações, serviços, atividades produtivas, em estreita associação com as iniciativas ligadas ao ócio, ao lazer e ao turismo. Tal como estas reflexões teóricas permitem compreender, o espaço rural deixa de ser percebido como local de produção, de abastecimento das cidades e passa a ser visto como espaço polissêmico e agregador de múltiplos setores econômicos. Nesse contexto, o turismo intensifica o processo de novas ruralidades ao propiciar a revalorização do rural por meio de produtos, equipamentos e serviços. WWW.UNIVALI.BR/PERÍODICOS http://dx.doi.org/10.14210/rtva.v23n2.p329-349 A atividade turística ressignifica o rural, torna funcional os espaços cotidianos e de trabalho, transformando-os em espaços de lazer e de consumo cultural. As paisagens, tradições, visões de mundo, religiosidade e demais expressões culturais, e subjetividades, são alçadas a elementos diferenciadores do cotidiano. Busca-se uma associação entre atividades agrícolas e não agrícolas que estabelecem novas relações e dinâmicas, por meio de ações de turistificação do espaço rural: trilhas ecológicas, contato dos agricultores rurais com turistas nacionais e internacionais, visitas às propriedades rurais, roteiros gastronômicos, são algumas das possibilidades que o turismo acena para as zonas rurais, contribuindo para a multifuncionalidade e pluriatividade do meio rural. No processo turístico de valorização das paisagens, destaca-se também os seus componentes culturais. O patrimônio material e simbólico das comunidades rurais são fatores que atraem os visitantes na perspectiva de intercâmbio e interpretação cultural. Dessa forma, o turismo rural também se caracteriza pelo interesse na valorização do patrimônio cultural e natural como elementos da oferta turística. Os empreendedores locais tendem a conferir originalidade aos seus produtos e serviços, contemplando manifestações socioculturais e práticas tradicionais – artesanato, música, arquitetura No processo turístico de valorização das paisagens, destaca-se também os seus componentes culturais. O patrimônio material e simbólico das comunidades rurais são fatores que atraem os visitantes na perspectiva de intercâmbio e interpretação cultural. Dessa forma, o turismo rural também se caracteriza pelo interesse na valorização do patrimônio cultural e natural como elementos da oferta turística. Os empreendedores locais tendem a conferir originalidade aos seus produtos e serviços, contemplando manifestações socioculturais e práticas tradicionais – artesanato, música, arquitetura – além de primar pela conservação do ambiente natural, da paisagem e da cultura (Carvalho & Moesch, 2013). Dentre os impactos positivos do turismo rural podemos destacar: as vivências socioeducativas e uma maior interação entre turistas e comunidades locais, conservação do patrimônio ambiental, valorização da herança cultural, dos simbolismos e dos saberes e fazeres tradicionais, aumento da autoestima dos moradores, agregação de valor às atividades econômicas, mediante a organização de bens, produtos e serviços ligados aos setores de turismo e hospitalidade. A dinâmica do turismo no meio rural ocasiona processos de transformação, induzindo o desenvolvimento em bases comunitárias (Figura 1). – além de primar pela conservação do ambiente natural, da paisagem e da cultura (Carvalho & Moesch, 2013). Dentre os impactos positivos do turismo rural podemos destacar: as vivências socioeducativas e uma maior interação entre turistas e comunidades locais, conservação do patrimônio ambiental, valorização da herança cultural, dos simbolismos e dos saberes e fazeres tradicionais, aumento da autoestima dos moradores, agregação de valor às atividades econômicas, mediante a organização de bens, produtos e serviços ligados aos setores de turismo e hospitalidade. A dinâmica do turismo no meio rural ocasiona processos de transformação, induzindo o desenvolvimento em bases comunitárias (Figura 1). – além de primar pela conservação do ambiente natural, da paisagem e da cultura (Carvalho & Moesch, 2013). Dentre os impactos positivos do turismo rural podemos destacar: as vivências socioeducativas e uma maior interação entre turistas e comunidades locais, conservação do patrimônio ambiental, valorização da herança cultural, dos simbolismos e dos saberes e fazeres tradicionais, aumento da autoestima dos moradores, agregação de valor às atividades econômicas, mediante a organização de bens, produtos e serviços ligados aos setores de turismo e hospitalidade. A dinâmica do turismo no meio rural ocasiona processos de transformação, induzindo o desenvolvimento em bases comunitárias (Figura 1). Figura 1- Exemplos de oferta do Turismo Rural Figura 1 Exemplos de oferta do Turismo Rural Fonte: Guia da Chapada Diamantina, 2020. Fonte: Guia da Chapada Diamantina, 2020. ISSN: 1983-7151 Tur., Visão e Ação, v23, n2, p329-349, Mai./Ago. 2021 - Balneário Camboriú, Santa Catarina, Brasil Tur., Visão e Ação, v23, n2, p329-349, Mai./Ago. 2021 - Balneário Camboriú, Santa Catarina, Brasil ur., Visão e Ação, v23, n2, p329-349, Mai./Ago. 2021 - Balneário Camboriú, Santa Catarina, Brasil No cenário de novas ruralidades e de novos paradigmas em torno da mobilidade sustentável (Maneze, 2018), o cicloturismo apresenta-se como uma prática turística que possibilita vivenciar os espaços urbanos/naturais de modo sustentável, na qual o contato com a dinâmica sociocultural ocorre num tempo e no espaço particulares. Caracteriza-se pelo deslocamento motivado pela busca do contato com o diferente, com as paisagens e modos de vida específicos, num percurso pela memória, patrimônio e imaginário dos lugares (Lima, 2015); aliado ao movimento físico, os ciclistas promovem a circulação de bens culturais, ideias e representações dos locais visitados. Na visão de Schetino (2006, p. 192) o cicloturismo compreende: […] Qualquer viagem ou passeio turístico no qual se utilize a bicicleta como meio de transporte. http://dx.doi.org/10.14210/rtva.v23n2.p329-349 importantes ao desvelar as motivações dos visitantes, destacando-se a busca pela aventura, emoção e por experiências diferenciadoras. importantes ao desvelar as motivações dos visitantes, destacando-se a busca pela aventura, emoção e por experiências diferenciadoras. De modo complementar, Pedrini (2013, p. 32) aponta as motivações que direcionam o comportamento dos cicloturistas: aventura (percorrem caminhos que não conhecem), competição (estão em busca de superação, de quebrar marcas pessoais), vertigem (estão correndo riscos ao atingir altas velocidades nas descidas, muitas vezes em lugares que o celular não pega e sem socorro por perto) e fantasia (ao conviver com habitantes locais, ao incorporar o simples, pois tudo que carregam é peso extra). Com base nesses estudos, observa-se que a estruturação de roteiros, rotas e circuitos cicloturísticos no meio rural pode se constituir em fator de valorização dos espaços onde estão inseridos, conferindo visibilidade aos patrimônios ambientais e urbanos, fortalecendo as comunidades em prol de um desenvolvimento econômico balizado nas expectativas locais. Ruschel (2008, p. 70) ressalta também que o uso da bicicleta possibilita a inclusão social no âmbito do turismo, posto que permite ao ciclista desenvolver a sociabilidade, conhecer aspectos diferentes dos seu cotidiano e inserir-se em atividades culturais na busca por conhecimento e educação. Nessa direção, Lima (2015) frisa que os cicloturistas, ao percorrerem os caminhos rurais, produzem também sentidos e significados aos locais; fragmentos da memória e das narrativas orais, as paisagens adquirem significância à medida que os cicloturistas abrem caminhos para a valorização do espaço material e simbólico dos espaços visitados. Maneze (2018) acrescenta ainda que os passeios de bicicleta conferem autonomia e protagonismo aos viajantes, provocando transformações nas subjetividades, favorecendo laços afetivos com o meio ambiente. Assim, ao reforçar os laços sociais, o sentido do lugar (Pires, 2001; Yazigi, 2002), a atividade e o turismo, de um modo geral, possibilitam ações de mobilização, sinergia, autonomia e articulação dos atores locais em prol de novas perspectivas de desenvolvimento local. Nesse norte, Duarte (2008, p. 25) assinala que o cicloturismo figura como elemento indutor do desenvolvimento socioeconômico, em virtude de mobilizar os atores sociais em iniciativas sustentáveis. Segundo o autor, este processo pode ocorrer de maneiras distintas em cada país, seja por meio do Estado, seja pelo envolvimento e participação de instituições que detêm um forte compromisso com a sustentabilidade e de empresas que atuam de forma participativa e corresponsável no planejamento e gestão dos seus produtos e serviços. Essa definição contempla não só as grandes viagens solitárias ou em grupos contidas em livros e relatos como também a nova e crescente demanda do mercado, da utilização de bicicletas em pequenos passeios e roteiros turísticos. No cicloturismo os turistas/visitantes podem deslocar-se por períodos inferiores a 24 horas, caracterizando o ciclolazer, ou realizar longos percursos fora do seu local de moradia com vistas ao consumo de diversas experiências – contemplação, lazer, recreação, contato cultural, aventura –, sendo a bicicleta elemento indispensável nas cicloviagens. Han et al. (2017) pontuam seis variáveis que caracterizam o cicloturismo: 1) É um deslocamento distante do local de residência; 2) A duração pode variar de um a vários dias; 3) É uma prática não competitiva; 4) A viagem é a principal motivação; 5) É desenvolvido dentro de um contexto ativo e desportivo e 6) Representa uma forma de lazer e entretenimento. A partir desse entendimento, o cicloturismo pressupõe um contato mais estreito entre visitantes/turistas e comunidades, por meio do intercâmbio de saberes e práticas de hospitalidade presentes ao longo dos percursos e no aumento da percepção ambiental, uma vez que a bicicleta, como modal de transporte, aguça os sentidos: audição, tato, visão, paladar, olfato são potencializados para desvelar a complexidade do meio ambiente, propiciando novas maneiras de perceber, estranhar e experienciar a paisagem, mesmo aquelas consideradas familiares (Oliveira, 2018). Embora haja uma aproximação com as atividades de lazer, o cicloturismo não equivale necessariamente a uma prática esportiva e não deve ser apreendido meramente como uma atividade física realizada ao ar livre. Nesse patamar, Olinto e Asprino (2014, s/p) esclarecem que no cicloturismo a busca pela quebra de recordes ou grandes velocidades é substituída pelo desafio, pela recreação e pelo conhecimento. Roldan (2000, p. 22) segue em sentido semelhante, argumentando que o prazer do cicloturista está na experiência, no significado emocional do viajar, independentemente da duração do percurso ou do destino escolhido. Pedrini, Flores e Cavalcante (2012) abordam a segmentação de mercado no turismo, observando que o cicloturismo aproxima-se dos segmentos de turismo de aventura e do ecoturismo. Eles entendem o cicloturismo como uma prática responsável e capaz de ocasionar benefícios sociais e movimentar setores importantes da economia rural. Para tanto, os autores analisaram o perfil dos cicloturistas do Circuito Vale Europeu, localizado no Estado de Santa Catarina, trazendo para o debate contribuições WWW.UNIVALI.BR/PERÍODICOS A diversificação das atividades produtivas é um dos fatores que justificam a mobilização do poder público, iniciativa privada e comunidades locais em torno de projetos de turismo local, em que a atividade detém vínculos com as identidades culturais e os aspectos simbólicos das populações que residem nos locais onde os passeios de bicicleta são promovidos. Os roteiros de cicloturismo tendem a contribuir, portanto, na diversificação das economias regionais, valorização da herança cultural, conservação do patrimônio, além de incentivar fluxos turísticos no período de baixa temporada (Carvalho et al., 2013). Apesar das possibilidades que os segmentos de turismo em áreas rurais têm em favorecer novas perspectivas de desenvolvimento social e econômico, não podemos ur., Visão e Ação, v23, n2, p329-349, Mai./Ago. 2021 - Balneário Camboriú, Santa Catarina, Brasil desconsiderar os efeitos negativos que podem surgir das relações entre turismo, meio ambiente e comunidades, tais como a degradação ambiental, a mercantilização do patrimônio cultural, alteração nos valores e estilos de vida local, prostituição e aumento da violência. A atividade turística, quando não acompanhada de um planejamento sistemático, pode ocasionar impactos negativos de diversas ordens, como, por exemplo, conflitos sociais, degradação da natureza, perda da demanda, dentre outros, inviabilizando, dessa forma, o ciclo de vida do destino (Dreher, 2003). A implantação de determinado segmento turístico implica ações de avaliação e monitoramento constante das atividades desenvolvidas, mediante instrumentos de planejamento e gestão. A relação contraditória entre turismo e áreas rurais enseja a adoção de instrumentos de planejamento e gestão – participação da comunidade por meio de fóruns, instâncias de governança e demais espaços de diálogo, zoneamento ambiental, elaboração de planos de manejo, determinação da capacidade de carga ecológica – com vistas a minimizar os impactos negativos desta atividade. Ressalta-se a necessidade de ordenamento territorial e planejamento dos espaços rurais, com vista a minimizar os impactos negativos, acompanhar a dinâmica da atividade e ampliar os seus benefícios para as comunidades. A gestão e organização das ofertas de turismo no meio rural devem levar em consideração as energias e sinergias comunitárias, as quais podem se articular em vínculos solidários, a fim de ofertar atrações turísticas baseadas nas especificidades locais, em termos de história, oralidade, religiosidade, gastronomia, e nas práticas de lazer e trabalho cotidianos. Desse modo, o turismo ressignifica as culturas na perspectiva de viabilização de projetos de transformação local (Vázquez-Barquero, 2001). METODOLOGIA Trata-se de uma pesquisa orientada pelos condicionantes da investigação qualitativa, possuindo um caráter exploratório e descritivo, uma vez que se propôs a compreender as características do fenômeno cicloturismo no contexto de sua produção. A pesquisa qualitativa possui como foco a apreensão dos significados do objeto de estudo por meio de técnicas variadas de interpretação, tais como entrevistas e observação participante, valendo-se em grande medida do método indutivo em suas abordagens. De acordo com Silva e Menezes (2001, p. 20), na pesquisa qualitativa: [...] Há uma relação dinâmica entre o mundo real e o sujeito, isto é, um vínculo indissociável entre o mundo objetivo e a subjetividade do sujeito que não pode ser traduzido em números. A interpretação dos fenômenos e a atribuição de significados são básicas no processo de pesquisa qualitativa. Não requer o uso de métodos e técnicas estatísticas. O ambiente natural é a fonte direta para coleta de dados e o pesquisador é o instrumento-chave. É descritiva. Os pesquisadores tendem a analisar seus dados indutivamente. O processo e seu significado são os focos principais de abordagem. Nas pesquisas qualitativas, a ênfase ocorre nos significados das práticas sociais, valores, percepções e visões de mundo dos diferentes atores, não se detendo, portanto, nos aspectos quantificáveis ou numéricos. Diversas técnicas de coletas de dados podem e devem ser utilizadas no desenvolvimento das pesquisas qualitativas, em especial, as entrevistas, o grupo focal, a observação participante. Assim, o pesquisador pode utilizar instrumentos que se complementam e possibilitam maior aprofundamento do objeto de estudo; nesse sentido, a triangulação metodológica torna-se uma proposta apropriada nos estudos descritivos e exploratórios, uma vez que torna possível uma compreensão holística acerca do fenômeno analisado (Lakatos & Marconi, 2006). Em face dos objetivos propostos, a pesquisa foi realizada em dois momentos: a pesquisa bibliográfica e a pesquisa de campo. Na pesquisa bibliográfica, inicialmente foi realizado o levantamento sistemático das publicações sobre a temática cicloturismo nos principais bancos de dados – Scielo, portal de periódicos da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) e Biblioteca Digital de Teses e Dissertações – e nos periódicos internacionais, a fim de compor um quadro teórico- conceitual sobre as relações entre cicloturismo e turismo rural. Desse modo, alcançou-se o entendimento sobre cicloturismo e turismo rural por meio das contribuições de Tulik (2003), Rodrigues (2001) e Kageyama (2008); as análises acerca do cicloturismo foram realizadas a partir dos estudos de Han et al. http://dx.doi.org/10.14210/rtva.v23n2.p329-349 como a plantação de soja e eucalipto. Estas contribuem para um processo de exclusão das populações locais e disputas pela legitimação de terras. Por meio do cicloturismo, assistem-se às transformações recentes nos povoados rurais, cujos resultados podem ocasionar, em longo prazo, novas (re)configurações no referido território, na busca de novas oportunidades econômicas e sociais para as comunidades. Considera-se na presente discussão o conceito de desenvolvimento local de Buarque (2002), entendido como um processo endógeno que possui o objetivo de promover o dinamismo econômico e a melhoria da qualidade de vida da população. Dialogando com este pensamento, na visão de Martins (2002), o desenvolvimento local deve ser entendido como aquele que satisfaz as necessidades humanas, sendo que um de seus maiores desafios consiste em criar as condições para o pleno exercício do protagonismo comunitário, tendo em vista que o cenário contemporâneo se caracteriza por algumas fragilidades, como a ausência da cidadania, da identificação sociocultural e territorial e do sentido de vizinhança. Diante da argumentação desenvolvida, o enquadramento teórico proposto adequa-se à realidade cicloturística dos municípios do Baixo Parnaíba Maranhense, onde a bicicleta é utilizada para a realização de percursos cotidianos, idas ao trabalho ou ao comércio, para a atividade de lazer ou desportiva ou como elemento de suporte para a visitação de locais de interesse turístico, sendo categorizado por Segovia (2017) como ciclolazer. Diante do exposto, as reflexões enunciadas voltam-se para este território, aqui entendido como espaço físico e simbólico, com práticas tradicionais convivendo dialeticamente com atividades globalizadas de produção e consumo dos espaços. Atualmente, o Baixo Parnaíba Maranhense vem passando por desafios decorrentes da implantação de atividades agrícolas que comprometem o meio ambiente local, tais WWW.UNIVALI.BR/PERÍODICOS METODOLOGIA (2017), Cavallari (2012) Lima (2015), Carvalho et al. (2013) e Pedrini, Flores e Cavalcante (2012). ISSN: 1983-7151 Tur., Visão e Ação, v23, n2, p329-349, Mai./Ago. 2021 - Balneário Camboriú, Santa Catarina, Bras A relação entre cicloturismo e desenvolvimento local foi tecida a partir dos estudos de Buarque (2002), Vázquez Barquero (2001) e Duarte (2008). Na pesquisa de campo, fez-se uso da técnica de observação direta e sistemática, ou seja, uma “observação estruturada, planejada ou controlada, tendo como característica básica o planejamento prévio e a utilização de anotações e de controle tempo” (Cervo, 2007, p. 31), realizada por meio do acompanhamento de grupos de ciclistas nos roteiros de cicloturismo. Ressalta-se que um dos pesquisadores faz parte do grupo Pedal Livre, um coletivo de ciclistas do município de São Bernardo que promove rotas de cicloturismo pela região do Baixo Parnaíba, além de eventos competitivos e beneficentes em parceria com o poder público local. A participação neste grupo facilitou a entrada no campo, com o estabelecimento de contatos prévios com outros grupos de ciclismo existentes e, consequentemente, a coleta de informações sobre a relação entre os cicloturistas e o patrimônio rural ao longo das rotas desenvolvidas. O acompanhamento dos grupos ocorreu nos meses de agosto a novembro de 2019, aos finais de semana, nos turnos matutino e vespertino, períodos em que os roteiros de cicloturismo são realizados com maior frequência. Realizou-se também a observação participante de grupos de ciclistas amadores e profissionais ao longo dos eventos beneficentes e competitivos nas zonas rurais dos municípios de São Bernardo, Magalhães de Almeida, Tutóia e Santana do Maranhão. Nesses momentos, utilizaram-se roteiros de observação e anotações de campo, a fim de identificar as relações entre cicloturistas, meio ambiente e comunidades locais, a dinâmica do cicloturismo nas modalidades recreativa e competitiva, e as possíveis contribuições do cicloturismo para o desenvolvimento social e econômico das localidades abrangidas pelos roteiros. A matriz de observação (Quadro 1) sintetiza as principais categorias e os aspectos que orientaram as anotações de campo, entendendo que o observador participante é consciente da sua influência nos resultados e no significado do fenômeno observado. Buscou-se integrar a experiência vivenciada pelos atores sociais – os cicloturistas – dando ênfase ao significado, ao agenciamento e à reflexão (Alcântara, 2008). Quadro 1- Matriz de observação do fenômeno cicloturístico. Quadro 1- Matriz de observação do fenômeno cicloturístico. METODOLOGIA Dimensões Evidências Significados da experiência Significados do fenômeno cicloturístico: prática esportiva e de lazer, intercâmbio e trocas culturais, aprendizado, interação com o cotidiano e o patrimônio das comunidades visitadas. Percepções sobre uso da bicicleta (transporte, autonomia, liberdade). Hospitalidade Situações de acolhimento aos cicloturistas, interações dos visitantes com os moradores dos povoados. Conservação ambiental Conservação dos atrativos naturais; identificação de elementos do patrimônio rural que agregam valor às rotas cicloturísticas. Sustentabilidade econômica Focos de empreendedorismo comunitário, existência de serviços de apoio (restaurantes, meios de hospedagem, aluguel de bicicleta, oficinas/borracharia nas rotas e trajetos percorridos; existência de parcerias público-privadas. Fonte: Elaborado pelos autores (2019). Fonte: Elaborado pelos autores (2019). WWW.UNIVALI.BR/PERÍODICOS http://dx.doi.org/10.14210/rtva.v23n2.p329-349 Posteriormente, as etapas de organização, análise e tratamento das informações coletadas no campo seguiram as orientações de Minayo, Deslandes e Gomes (2007), interpretando-as com base no referencial teórico adotado pela pesquisa. A arregimentação de fontes primárias e secundárias possibilitou a compreensão dos aspectos físicos e simbólicos do território do Baixo Parnaíba Maranhense, bem como da dinâmica do cicloturismo nos povoados ali existentes, tendo em vista que esta modalidade de turismo rural pode se converter em estratégia de manutenção das famílias, valorização da paisagem local e de dinamização econômica. A PRÁTICA DO CICLOTURISMO E A VALORIZAÇÃO DO TERRITÓRIO DO BAIXO PARNAÍBA MARANHENSE O território denominado Baixo Parnaíba Maranhense (Figura 2) consiste numa “[...] ampla extensão de terras, com cobertura florestal característica do Cerrado ou integrando também áreas de transição entre Cerrado e Caatinga, envolvendo distintos municípios [...], objeto de distintas classificações, obedecendo a diferentes nomenclaturas” (Oliveira, 2015, p. 66, grifo do autor). Atualmente, o território do Baixo Parnaíba Maranhense é formado por 13 municípios: Araióses, Tutóia, Santana do Maranhão, Água Doce do Maranhão, Santa Quitéria do Maranhão, São Bernardo, Magalhães de Almeida, Anapurus, Mata Roma, Chapadinha, São Benedito do Rio Preto, Urbano Santos e Belágua. Figura 2- Mapa de localização do Território do Baixo Parnaíba Maranhense. Figura 2- Mapa de localização do Território do Baixo Parnaíba Maranhense. Fonte: Brasil, 2005. Fonte: Brasil, 2005. ISSN: 1983-7151 Tur., Visão e Ação, v23, n2, p329-349, Mai./Ago. 2021 - Balneário Camboriú, Santa Catarina, Brasil Tur., Visão e Ação, v23, n2, p329-349, Mai./Ago. 2021 - Balneário Camboriú, Santa Catarina, Brasil ur., Visão e Ação, v23, n2, p329-349, Mai./Ago. 2021 - Balneário Camboriú, Santa Catarina, Brasil Nesse território, predominam feições eminentemente rurais, com a presença de povoados que possuem características materiais e simbólicas que expressam elementos de ruralidade. O termo povoado é assumido na pesquisa como a unidade territorial, delimitada por fronteiras consensualmente acatadas por seus moradores e reconhecida pelos vizinhos, onde estão situadas as moradias e demais edificações (casas de forno, casas de festa, igrejas, escolas, estabelecimentos comerciais, postos de saúde, quando existentes, e outros) e as áreas de trabalho. Em algumas situações, as áreas de trabalho das famílias de um povoado podem se encontrar em outras localidades (Paula Andrade, 2008). De acordo com o Ministério do Turismo (Brasil, 2010, p. 17) “a ruralidade contempla as características mais gerais do meio rural: a produção territorializada de qualidade, a paisagem, a biodiversidade, a cultura e certo modo de vida, identificados pela atividade agrícola, a lógica familiar, a cultura comunitária, a identificação com os ciclos da natureza”. http://dx.doi.org/10.14210/rtva.v23n2.p329-349 como espaços de confluência e constantes diálogos, enfatizando o sentido de novo rural ou novas ruralidades. Tal característica é evidenciada no território do Baixo Parnaíba Maranhense, uma vez que o meio rural apresenta um diálogo permanente com a área urbana, seja no sentido econômico, por meio do comércio de bens e produtos oriundos da zona rural, seja no sentido da população rural utilizar serviços, estar empregada ou ainda estudar nas sedes municipais, revelando as inter-relações entre o rural e o urbano. como espaços de confluência e constantes diálogos, enfatizando o sentido de novo rural ou novas ruralidades. Tal característica é evidenciada no território do Baixo Parnaíba Maranhense, uma vez que o meio rural apresenta um diálogo permanente com a área urbana, seja no sentido econômico, por meio do comércio de bens e produtos oriundos da zona rural, seja no sentido da população rural utilizar serviços, estar empregada ou ainda estudar nas sedes municipais, revelando as inter-relações entre o rural e o urbano. Mesmo nas sedes municipais que apresentam feições mais urbanizadas, as relações cotidianas são marcadas por elementos rurais, como as relações de compadrio, confiança e solidariedade comunitária. No município de São Bernardo, por exemplo, evidenciam-se práticas de agricultura urbana nos quintais de alguns moradores que trouxeram esse hábito da zona rural e cultivam frutas como banana, acerola, alface, manga, mamão; hortaliças como pimentão, pimenta-de-cheiro e criam pequenos animais (galinha, pato, porco), revelando, assim, a continuidade de práticas do meio rural no perímetro urbano (Oliveira & Leal, 2013). Nas festas em homenagem aos santos padroeiros, as trocas materiais e simbólicas entre os devotos, romeiros, e demais atores que transitam nos espaços festivos tradicionais contribuem para reforçar esse intercâmbio; a dinâmica dos festejos religiosos “[...] produzem e dão corpo a um repertório simbólico e cultural que conforma as experiências populares do sagrado, proporcionando trocas e sociabilidades importantes à constituição das identidades locais e da memória coletiva no Baixo Parnaíba Maranhense” (Sousa, Pinto & Júnior, 2018, p. 510). A PRÁTICA DO CICLOTURISMO E A VALORIZAÇÃO DO TERRITÓRIO DO BAIXO PARNAÍBA MARANHENSE Paisagens naturais conservadas, relações sociais marcadas por laços de coesão e solidariedade comunitária, as vivências cotidianas entre os moradores e os espaços naturais, a organização das suas práticas econômicas, com destaque para os saberes e fazeres artesanais, como a produção de farinha e cachaça artesanal (Figura 3) são elementos que significam este território, entendido como espaço de vivência, trabalho, conflitos e lazer “[...] um lugar de vida, isto é, lugar onde se vive (particularidades do modo de vida e referência “identitária”) e lugar de onde se vê e se vive o mundo [...]”(Wanderley, 2001, p. 32). Figura 3- Paisagens rurais do Baixo Parnaíba Maranhense. Fonte: Marques, 2019. Fonte: Marques, 2019. Fonte: Marques, 2019. Na contemporaneidade, a dicotomia entre os meios rural e urbano é suplantada por uma visão dinâmica que os entende não como entes separados, mas WWW.UNIVALI.BR/PERÍODICOS Guardadas as especificidades da dinâmica socioeconômica e cultural dos municípios que integram o Baixo Parnaíba Maranhense, atualmente, o território é cenário de tensões e conflitos agrários, bem como de estratégias de resistência do campesinato local, face ao avanço de um modelo agroexportador de soja e eucalipto que se estabeleceu nos municípios a partir da década de 1980, que tem promovido reconfigurações e impactado as relações entre os atores locais e os seus espaços socioambientais, “[...] essas comunidades continuam na busca do desenvolvimento do bem viver dos povos em meio ao enfrentamento político, aos conflitos e às relações com a natureza” (Costa, 2016, p. 29, grifo do autor). A atividade turística e excursionista vem sendo utilizada como estratégia de valorização do território por meio de políticas públicas endereçadas à organização de ofertas e serviços de hospitalidade, dotação de infraestrutura e promoção dos municípios como destinos nos segmentos de turismo ecológico, sol e praia e turismo de aventura. No mapa de regionalização do turismo do Estado do Maranhão, os municípios de Água Doce do Maranhão, Araióses, Paulino Neves e Tutóia compõem o polo turístico Delta das Américas, e já figuram como destinos turísticos em expansão. Nesse norte, assiste-se também ao movimento de grupos de ciclistas que percorrem o território do Baixo Parnaíba na busca por experiências de lazer, aventura, e contato com as paisagens e comunidades. Esses grupos se organizam de forma solidária, promovendo passeios e eventos esportivos e atividades beneficentes, em parceria com os gestores públicos municipais. Ao mesmo tempo, incentivam o uso da bicicleta como forma sustentável do consumo turísticos dos lugares e promoção do bem-estar e qualidade de vida. ISSN: 1983-7151 Tur., Visão e Ação, v23, n2, p329-349, Mai./Ago. 2021 - Balneário Camboriú, Santa Catarina, Brasil Tur., Visão e Ação, v23, n2, p329-349, Mai./Ago. 2021 - Balneário Camboriú, Santa Catarina, Brasil Tur., Visão e Ação, v23, n2, p329-349, Mai./Ago. 2021 - Balneário Camboriú, Santa Catarina, Bras Dentre os motivos que levam à prática de cicloturismo destacam-se as características geomorfológicas e às condições climáticas e hidrográficas que permeiam o Baixo Parnaíba Maranhense, somadas ao diversificado patrimônio cultural existente no meio rural. No que concerne às características geomorfológicas, e pelo fato do Estado do Maranhão estar situado numa área de transição, encontramos diversos tipos de vegetação, como o cerrado, a caatinga, e a floresta estacional perenifólia; já os ecossistemas de manguezais, dunas, praias, ilhas e ilhotas são encontrados nos municípios de Tutóia e Araióses. Nessa área predominam solos do tipo Latossolo Amarelo e Podzólico Vermelho Amarelo Concrecionário (Bandeira, 2013). Em relação aos aspectos climáticos, o clima característico dos municípios do Baixo Parnaíba é o subúmido, com exceção dos municípios de São Bernardo, Magalhães de Almeida e parte de Araióses, Santana do Maranhão e Santa Quitéria do Maranhão, nos quais ocorre o clima subúmido seco (Brasil, 2005). Ao reunir tais condições, os municípios do Baixo Parnaíba tornam-se áreas propícias para o desenvolvimento de roteiros cicloturísticos. Os roteiros e eventos de cicloturismo direcionam-se às áreas rurais, em especial aos povoados, onde as características dos ecossistemas e o ritmo cadenciado de vida dos moradores compõem uma atmosfera compatível com as atividades de lazer e aventura ao ar livre. Nesses locais, é comum a organização de eventos que atraem um público significativo de visitantes, dentre ciclistas amadores, profissionais, além de espectadores, os quais movimentam e promovem o meio rural como oferta turística do Baixo Parnaíba Maranhense (Figura 4). Figura 4- Roteiros de Cicloturismo nas zonas rurais . Fonte: Pesquisa de campo (2019). Fonte: Pesquisa de campo (2019). Os cicloturistas normalmente realizam percursos de um dia, caracterizando a modalidade de ciclolazer, ou percorrem longos percursos, abrangendo rotas intermunicipais. Ao longo dos roteiros e das rotas cicloturísticas, os visitantes percorrem estradas de terra, envoltas por dunas e/ou manguezais, e entram em contato com paisagens campestres, plantações, festejos tradicionais, produção artesanal, gastronomia, remanescentes dos antigos engenhos de cana-de-açúcar, e demais elementos do patrimônio histórico e cultural (Figuras 5 e 6). WWW.UNIVALI.BR/PERÍODICOS http://dx.doi.org/10.14210/rtva.v23n2.p329-349 http://dx.doi.org/10.14210/rtva.v23n2.p329-349 Figura 5- Paisagem Rural do município de São Bernardo, Maranhão Figura 6- Produção de farinha artesanal no povoado Cajueiro em São Bernardo F t P i d (2019) Figura 6- Produção de farinha artesanal no povoado Cajueiro em São Bernardo Figura 5- Paisagem Rural do município de São Bernardo, Maranhão Fonte: Pesquisa de campo (2019). Fonte: Pesquisa de campo (2019). Os eventos esportivos tendem a fomentar ações de/para o cicloturismo, dinamizando também a economia local por meio da venda de acessórios, equipamentos, alimentos e bebidas, além de material de apoio ao desenvolvimento desta atividade. Rotas cicloturísticas, como a Rota Caldo de Cana, Trilha Racha Coco e Rally do Sol impulsionam a valorização dos povoados do Baixo Parnaíba Maranhense, ao tempo em que atraem um público significativo de participantes e espectadores. Além da proximidade com o patrimônio geológico, ambiental e cultural dos municípios, os cicloturistas interagem com os moradores, sendo comuns as atividades de acolhimento dos moradores das áreas rurais nas suas residências, oferta de serviços de apoio, como reparos das bicicletas e dos equipamentos utilizados, ou de informações sobre os atrativos existentes na região. Tal como mostrou Schetino (2006), as situações de intercâmbio e de trocas culturais entre os cicloturistas e os moradores do Baixo Parnaíba caracterizam o cicloturismo como oportunidade para a vivência crítica e criativa do lazer. De acordo com o autor, o locais de percurso ganham novos usos e semânticas com a presença destes visitantes, os quais recebem em troca generosidade e hospitalidade. De modo complementar, não raro, observa-se a abertura de pequenos negócios de alimentação ao longo das rotas desenvolvidas. Em algumas, como a rota Caldo de Cana, localizada no povoado São Raimundo, são vendidos gêneros alimentícios como sucos, vitaminas, caldo de cana, sinalizando para o empreendedorismo comunitário. Em alguns municípios pode-se encontrar uma cadeia produtiva da bicicleta em formação, com lojas especializadas na venda de acessórios e equipamentos para bicicletas, o que estimula a economia local (Figura 7). ISSN: 1983-7151 Tur., Visão e Ação, v23, n2, p329-349, Mai./Ago. 2021 - Balneário Camboriú, Santa Catarina, Brasil ur., Visão e Ação, v23, n2, p329-349, Mai./Ago. 2021 - Balneário Camboriú, Santa Catarina, Brasil Figura 7- Práticas de hospitalidade no meio rural. g p Fonte: Pesquisa de campo (2019). Fonte: Pesquisa de campo (2019). No contexto da multifuncionalidade e pluriatividade do meio rural, destacamos a importância de valorização de atividades que concorram para elevar a qualidade de vida dos moradores, facilitando o acesso a serviços de infraestrutura básica e de serviços. Como demonstra Souza (2019), o cicloturismo traduz oportunidades de enriquecimento da vida cultural de áreas urbanas e rurais, uma vez que os cicloturistas aproveitam a infraestrutura disponível ao longo das rotas, impulsionam negócios comunitários, distribuindo a renda de modo mais uniforme se comparado ao outros perfis de turistas. http://dx.doi.org/10.14210/rtva.v23n2.p329-349 a articulação institucional com vistas a incentivar o turismo/excursionismo em áreas rurais, bem como a ausência de roteiros oficiais de cicloturismo de forma integrada. Nesse horizonte, Souza (2019) pontua que as iniciativas de cicloturismo no contexto brasileiro são gestadas, em sua maioria, pela iniciativa privada, não ocorrendo um envolvimento mais contundente da gestão pública estadual e federal no estímulo à atividade mediante a inserção de planos de desenvolvimento de rotas e circuitos, como acontece em outros países. Além destes entraves, destacam-se as fragilidades no planejamento turístico municipal, com a ausência de ações estratégicas de diversificação da oferta turística, ações de marketing, estruturação de um calendário de eventos, instrumentos necessários para a organização e gestão da atividade turística. Outro entrave diz respeito à baixa qualidade na prestação de serviços nos setores de turismo e hospitalidade, o que pode comprometer a qualidade da experiência turística na região e, consequentemente, a imagem e o posicionamento mercadológico dos destinos. Fonte: Pesquisa de campo (2019). Soares (2010) complementa essa afirmação, quando observa que os passeios de bicicleta valorizam também as especificidades gastronômicas das regiões, face a necessidade dos cicloturistas em consumir a culinária tradicional dos destinos, estimulando, portanto, o turismo gastronômico e a produção associada ao turismo. Os roteiros de cicloturismo ampliam a permanência dos visitantes nas comunidades, estimulando o protagonismo comunitário no âmbito da atividade turística, além disso, associado a um planejamento responsável, pode se configurar numa fonte de renda complementar para as famílias e incentivar o desenvolvimento territorial (Gonçalves, 2018). No entanto, algumas ameaças à consolidação deste cenário favorável ao turismo precisam ser consideradas: inicialmente, ressaltamos as condições de acesso à região do Baixo Parnaíba Maranhense, o que dificulta a mobilidade dos turistas e, consequentemente, o acesso aos atrativos. Especificamente no segmento cicloturismo, constata-se a ausência de políticas públicas voltadas à mobilidade sustentável, ao incentivo ao uso da bicicleta, WWW.UNIVALI.BR/PERÍODICOS CONSIDERAÇÕES FINAIS O turismo figura na contemporaneidade como um fenômeno social complexo e heterogêneo que articula as especificidades dos locais na estruturação de produtos e serviços para satisfazer as necessidades de lazer, evasão, cultura e busca por conhecimentos, por parte de diferentes atores sociais. Ao mesmo tempo, a atividade é pensada como estratégia de valorização dos aspetos naturais e dos bens simbólicos das comunidades, as quais percebem o turismo como fator de promoção cultural e do desenvolvimento econômico e social. Essa realidade é percebida nas zonas rurais, onde o turismo ressignifica o meio rural e intensifica os diálogos entre as esferas local e global, posto que o patrimônio rural passa a ser entendido como locus de atração de visitantes, especificamente no âmbito do Turismo Rural. Caracterizado como um segmento sustentável, o turismo rural vem sendo incentivado em inúmeras localidades, cujas repercussões materizalizam- se não somente na dinamização econômica dos espaços rurais, mas em situações de intercâmbio e de circulação de saberes entre visitantes e visitados. Dentre as possbilidades da oferta de turismo rural, os passeios de bicicleta, caracterizados como ciclolazer e cicloturismo aproximam os visitantes dos locais visitados, ampliam a percepção ambiental ao dar ênfase às paisagens e as relações sociais que são gestadas durante os percursos. O cicloturismo desenvolvido em áreas rurais é pensado como uma mobilidade turística sustentável e também como alternativa de consumo dos espaços rurais de modo a aproximar os turistas e as comunidades, suas memórias, histórias e tradições. O cicloturismo no meio rural promove os patrimônios ambientais e das identidades culturais, ao mesmo tempo em que dinamiza as atividades não agrícolas com o estímulo à construção de novas ruralidades. Já ao movimento de vivenciar as localidades, o cicloturismo propicia o estreitamento dos laços de afetividade entre os moradores e seus locais de vivência e convivência. Desse modo, o cicloturismo reforça ISSN: 1983-7151 Tur., Visão e Ação, v23, n2, p329-349, Mai./Ago. 2021 - Balneário Camboriú, Santa Catarina, Bras o turismo como fator de desenvolvimento socioeconômico no meio rural Estes foram os fios condutores da pesquisa ora realizada, tendo como objeto privilegiado de estudo os municípios que integram a região do Baixo Parnaíba Maranhense. A expansão das atividades turísticas locais vem possibilitando novas formas de vivenciar os povoados, antes distantes dos lugares centralizados da práxis turística. http://dx.doi.org/10.14210/rtva.v23n2.p329-349 no planejamento da atividade turística, de forma a gerar benefícios para as comunidades e novas perspectivas de desenvolvimento includente e qualidade de vida. no planejamento da atividade turística, de forma a gerar benefícios para as comunidades e novas perspectivas de desenvolvimento includente e qualidade de vida. CONSIDERAÇÕES FINAIS Por meio de atividades espontâneas de grupos de ciclistas, ocorrem processos de apropriação e valorização turística dos povoados rurais; roteiros e trilhas de cicloturismo revigoram os patrimônios desses locais, e geram focos de empreendedorismo comunitário, realização de eventos, os quais movimentam a economia e produzem novos sentidos ao meio rural do Baixo Parnaíba. A expansão das atividades cicloturísticas fomenta novas formas de vivenciar os povoados, antes distantes dos lugares centralizados da dinâmica do turismo no Baixo Parnaíba. Dentre os benefícios verificados, destacam-se os processos de apropriação e valorização turística dos povoados rurais. Os roteiros cicloturísticos incentivam não apenas o fluxo de visitantes, mas promovem novos sentidos ao meio rural, enriquecendo a vida cotidiana dos moradores e o estreitamento dos seus laços com o patrimônio local. Os passeios favorecem comportamentos pró-ambientais, maior conexão e apego emocional em relação às localidades percorridas, traduzindo situações de aprendizado, afeto e experiências. Ao longo da pesquisa realizada, constatou-se a viabilidade de desenvolver o cicloturismo como oferta do turismo rural nos municípios do Baixo Parnaíba Maranhense, face às condições geomorfológicas e climáticas, patrimônio ambiental conservado, e aos elementos do patrimônio cultural – paisagens campestres, plantações, festejos tradicionais, produção artesanal, gastronomia, remanescentes dos antigos engenhos de cana-de-açúcar, dentre outros bens simbólicos. Enquanto estratégia de desenvolvimento, o reconhecimento das potencialidades locais pelos cicloturistas impulsiona novas dinâmicas que vêm promovendo uma re-localização dos lugares de vida e de suas funções, explicitando uma valorização das zonas rurais pelo turismo. Sinaliza, em longo prazo, para a promoção do turismo rural na região. No entanto, a pesquisa ressaltou também a necessidade de se estabelecer políticas públicas direcionadas ao turismo rural e ao cicloturismo, tendo em vista que a maioria dos municípios carecem de estruturas básicas e de serviços, além de ações sistemáticas de formatação e comercialização de produtos rurais, a fim de inserir os municípios na dinâmica turística. O processo de elaboração de roteiros cicloturísticos pressupõe a alocação de infraestrutura, em especial a sinalização indicativa, turística e interpretativa, pontos de apoio ao cicloturismo e serviços específicos, como o aluguel de bicicleta. Para tanto, o envolvimento dos gestores públicos, empresariado e comunidade local, constitui fator primordial para que a região desenvolva a prática do cicloturismo em atenção às normativas deste segmento. Ressalva-se que os interesses e demandas locais, bem como a dinâmica das relações e os laços de afetividade estabelecidos com o meio rural devem ser considerados WWW.UNIVALI.BR/PERÍODICOS REFERÊNCIAS Alcântara, D. De (2008). Abordagem experiencial e revitalização dos centros históricos: os casos do Corredor Cultural no Rio de Janeiro e do Gaslamp Quarter em San Diego. Rio de Janeiro: UFRJ/ FAU, 2008. Bandeira, I. C. N. (2013). Geodiversidade do Estado do Maranhão. Teresina: CPRM. Brasil. Ministério do Turismo. (2010). Turismo rural: orientações básicas. Brasília: Ministério do Turismo. Brasil. Ministério de Desenvolvimento Agrário. SDT – Secretaria de Desenvolvimento Territorial Território Baixo Parnaíba. (2005). Plano territorial de desenvolvimento rural sustentável. São Luís. Buarque, S. C. (2002). Construindo o desenvolvimento local sustentável. Metodologia de planejamento. Rio de Janeiro: Garamond. Carvalho, T. J. L., Ramos, J. L. & Sydow, E. (2013). O cicloturismo como fator de desenvolvimento da atividade turística nas cidades de Araguaína e Nova Olinda (TO). Anais do IX Congresso Nacional de Ecoturismo e do V Encontro Interdisciplinar de Turismo em Unidades de Conservação. Revista Brasileira de Ecoturismo, São Paulo, 6(4), pp. 63-82. Carvalho, M.S. & Moesch, M.M. (2013). Turismo como fenômeno social e suas implicações no espaço rural. Revista Brasileira de Ecoturismo. São Paulo, 6 (2), pp. 442-457. Costa, S. B. da. (2016) Chapadas e lutas: resistência camponesa no Baixo Parnaíba Maranhense na rota do agronegócio silvicultor – conflitos territoriais e “usos” da natureza. [Tese de Doutorado, Universidade Federal de Pernambuco]. Cavallari, G. (2012). Manual de mountain bike & cicloturismo. 1ª ed. São Paulo:Ed. Kalapalo. Cervo, A. L. (2007). Metodologia científica. São Paulo: Pearson Prentice Hall. Dreher, M.T. (2003) Planejamento do turismo em áreas não urbanas: envolvendo a comunidade. Anais do Congresso Internacional sobre Turismo Rural e desenvolvimento sustentável. Joinville. pp. 241- 249. Recuperado em: http://www.ufrgs.br/pgdr/arquivos/623.pdf. Acesso em 29/03/2020. Duarte, J. C. de R. D. (2008). Cicloturistas e suas percepções ambientais: um estudo na Estrada Real. Director Dr. Nelson Antônio Quadros Vieira Filho. (Tesina degrado inédita). Centro universitário UNA. Gonçalves, M. L. R. (2018) O cicloturismo como uma ferramenta estratégica para o desenvolvimento territorial de Nova Veneza – SC. [Dissertação de Mestrado, Universidade do Estado de Santa Catarina]. Guia da Chapada Diamantina. Recuperado em: http://www.guiachapadadiamantina.com.br/ Acesso em 23 de fevereiro de 2020. Han, H., Meng, B. & Kim, W. (2017). Bike-traveling as a growing phenomenon: Role of attributes, value, satisfaction, desire, and gender in developing loyalty. Tourism Management, 59, pp. 91-103. ISSN: 1983-7151 Kageyama, A. A. (2008) Desenvolvimento rural: conceitos e aplicação ao caso brasileiro. Porto Alegre: Editora da UFRGS. Lakatos, E. M., Marconi, M. A. (2006). Técnicas de pesquisa. REFERÊNCIAS 6. ed. São Paulo: Atlas. Lima, B. L. (2015). Estrada Geral do Sertão: potenciais turísticos de um caminho quase esquecido. [Dissertação de Mestrado, Universidade de Brasília]. Maneze, C. A.L. (2018) A transformação humana nas viagens: encontro de si e busca de ser. [Dissertação de Mestrado, Universidade de Brasília]. Marques, S. K. (2019). Visita Técnica `nos municípios do Baixo Parnaíba Maranhense. Universidade Federal do Maranhão, São Bernardo. Matias, A., Sardinha, R. Avanços em Economia e Gestão do Turismo. Lisboa: Colecção Sociedade e Organizações/48, 2002. Minayo, M. C.S., Deslandes, S.F. & Gomes, R. (2007). Pesquisa social: teoria, método e criatividade. Petrópolis: Vozes. Martins, S. R. O. (2002). Desenvolvimento Local: questões conceituais e metodológicas. Interações, Campo Grande, 3(5}, pp. 13-20. Olinto, A., Asprino, R. (2014). Cicloturismo no Brasil. Recuperado em: <http://www.olinto.com.br/index. php/guia-livro-dvd-viagem-bicicleta/mantiqueira/>. Acesso em 23 de fevereiro de 2020. Oliveira, R. R. de. (2018). Devagar quase parando: o uso da bicicleta como ferramenta para o estudo da paisagem. In: Oliveira, R. R. de, Ruiz, A. E. (Orgs.) Geografia histórica do café [recurso eletrônico]. Rio de Janeiro: Ed. PUC-Rio, pp. 37-60. Oliveira, L. R., Leal, M. N. (2013). Quintais produtivos (Home Garden) no município de São Bernardo/ MA. Anais do 14º. Encontro de Geógrafos da América Latina- Lima, pp. 1-18. Oliveira, D. M. V. (2015). Das varedas das encostas aos variantes da firma no Baixo Parnaíba Maranhense: as (re) organizações territoriais como forma de manutenção camponesa diante da ação de uma grande empresa – Suzano celulose. Rio de janeiro: Niterói. Silva, E. L., Menezes, E. M. (2001) Metodologia da Pesquisa e Elaboração de Dissertação. Florianópolis: UFSC. Paula Andrade, M. de. (2008). Os gaúchos descobrem o Brasil: projetos agropecuários contra a agricultura camponesa. São Luís: EDUFMA. Pedrini, L., Flores, L. C. da S. & Cavalcante, L. de S. (2012). Cicloturistas no circuito do Vale Europeu: perfil e percepção ambiental. Anais do IX Seminário da Associação Nacional Pesquisa e Pós- Graduação em Turismo. Universidade Anhembi Morumbi, São Paulo. Pedrini, L. (2013). Cicloturismo no Circuito do Vale Europeu Catarinense: Um estudo do comportamento do cliente. [Dissertação de Mestrado, Universidade do Vale do Itajaí ]. Pires, P. dos S. (2001) Caracterização e análise visual da paisagem rural com enfoque turístico: uma contribuição metodológica. Turismo-Visão e Ação, 4 (8), pp. 83-98. WWW.UNIVALI.BR/PERÍODICOS Rodrigues, A. B. (Org.). (2001). Turismo Rural: práticas e perspectivas. São Paulo: Contexto (Coleção Turismo Contexto). Roldan, T. R. R. (2000). Cicloturismo: planejamento e treinamento. [ Monografia, Universidade Estadual de Campinas]. Ruschel, D. (2008). A bicicleta como meio de transporte sustentável no turismo. [Monografia, Pontifícia Universidade Católica do Rio Grande do Sul]. Schetino, A. M. (2006). Cicloturismo como vivência crítica e criativa de lazer. [Monografia, Universidade Federal de Minas Gerais]. Segovia, Y. N. S. (2017). O desenvolvimento do cicloturismo na perspectiva da gestão urbana. [Dissertação de Mestrado, Universidade Católica do Paraná]. Soares, A. (Org.). (2010) Circuitos de Cicloturismo: manual de incentivo e orientação para os municípios brasileiros. Recuperado em: http://www.clubedecicloturismo.com.br/arquivos/ Manual-Circuitos-Cicloturismo.pdf Acesso em 29 de abril de 2020. Sousa, R., Pinto, E. R. & Júnior, C. C. de M. (2018) “Agosto em festa se enfeita”: religiosidade, práticas devocionais e representações simbólicas no Festejo de São Bernardo – MA. In: Revista Interdisciplinar em Cultura e Sociedade (RICS). São Luís, 4, pp. 495-516. Souza, F. H. P. de. (2019) O ciclismo como incremento do turismo em Fortaleza: propostas de cicloturismo. [Dissertação de Mestrado, Universidade Estadual do Ceará). Turismo Rural. São Paulo: Aleph (Coleção ABC do Turismo). Tulik, O. (2003). Turismo Rural. São Paulo: Aleph (Coleção ABC do Turismo). Vázquez Barquero, A. (2001) Desenvolvimento endógeno em tempos de globalização. Porto Alegre: Editora UFRGS, 278 p. Zimmermann, A. (2003). Planejamento e organização do turismo rural no Brasil. In: Almeida, J. A. A., Froehich, J. M.& Riedl, M. (Orgs.). Turismo rural e desenvolvimento sustentável. Campinas: Papirus. (Coleção Turismo). 3a. edição, pp. 127-163. Wanderley, M. N. B. (2001 A ruralidade no Brasil moderno: por um pacto social pelo desenvolvimento rural. Giarraca, N. (Comp.) ¿Una nueva ruralidade en América Latina? Buenos Aires: CLACSO/ ASDI, pp. 31-44. Disponível em: http://bibliotecavirtual.clacso.org.ar/ar/libros/rural/ wanderley. pdf. Recuperado em 27 de janeiro de 2020. Yazigi, E. (Org). (2002). Turismo e Paisagem. São Paulo: Contexto. CONTRIBUIÇÃO DOS AUTORES Rodrigo Olavo Costa Sousa: Concepção teórica do estudo; pesquisa bibliográfica, pesquisa de campo, sistematização dos resultados da pesquisa e revisão textual. Karoliny Diniz Carvalho: Orientadora; revisão textual. ISSN: 1983-7151
https://openalex.org/W2292998423	https://pubs.aip.org/aip/adv/article-pdf/doi/10.1063/1.4943673/12990451/035105_1_online.pdf	English	null	Material/element-dependent fluorescence-yield modes on soft X-ray absorption spectroscopy of cathode materials for Li-ion batteries	AIP advances	2,016	cc-by	4,980	RESEARCH ARTICLE \| MARCH 07 2016 Material/element-dependent fluorescence-yield modes on soft X-ray absorption spectroscopy of cathode materials for Li-ion batteries RESEARCH ARTICLE \| MARCH 07 2016 Daisuke Asakura; Eiji Hosono; Yusuke Nanba; Haoshen Zhou; Jun Okabayashi; Chunmei Ban; Per-Anders Glans; Jinghua Guo; Takashi Mizokawa; Gang Chen; Andrew J. Achkar; David G. Hawthron Thomas Z. Regier; Hiroki Wadati AIP Advances 6, 035105 (2016) https://doi.org/10.1063/1.4943673 Material/element-dependent fluorescence-yield modes on soft X-ray absorption spectroscopy of cathode materials for Li-ion batteries Daisuke Asakura,1,a Eiji Hosono,1 Yusuke Nanba,1,b Haoshen Zhou,1 2 3 4 4 5 Daisuke Asakura,1,a Eiji Hosono,1 Yusuke Nanba,1,b Haoshen Zhou,1 O 2 C 3 G 4 G 4 5 Takashi Mizokawa,6,c Gang Chen,7 Andrew J. Achkar,8 David G. Hawth Thomas Z. Regier,9 and Hiroki Wadati10 1Energy Technology Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8568, Japan Thomas Z. Regier,9 and Hiroki Wadati10 1Energy Technology Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8568, Japan 2Research Center for Spectrochemistry, The University of Tokyo, Bunkyo-ku, Tokyo, 113-0033, Japan 2Research Center for Spectrochemistry, The University of Tokyo, Bunkyo-ku, Tokyo, 113-0033, Japan 3 3The Chemical and Materials Science Center, National Renewable Energy Laboratory, Golden, Colorado, 80401, USA 3The Chemical and Materials Science Center, National Renewable Energy Laboratory, Golden, Colorado, 80401, USA 4Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, California, 94720, USA 5 4Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, California, 94720, USA 5 5Department of Chemistry and Biochemistry, University of California, Santa Cruz, California, 95064, USA. 5Department of Chemistry and Biochemistry, University of California, Santa Cruz, California, 95064, USA. 6 6Department of Complexity Science and Engineering, The University of Tokyo, Kashiwa, Chiba, 277-8561, Japan 6Department of Complexity Science and Engineering, The University of Tokyo, Kashiwa, Chiba, 277-8561, Japan p 7College of Physics/State Key Laboratory of Superhard Materials, Jilin University, p 7College of Physics/State Key Laboratory of Superhard Materials, Jilin University, 7College of Physics/State Key Laboratory of Superha Changchun, 130012, People’s Republic of China Changchun, 130012, People’s Republic of China 8Department of Physics and Astronomy, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada 8Department of Physics and Astronomy, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada 9Canadian Light Source, University of Saskatchewan, Saskatoon, 9Canadian Light Source, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 0X4, Canada 24 October 2024 05:21:04 10Institute for Solid State Physics, The University of Tokyo, Kashiwa, Chiba, 277-8581, Japan (Received 1 December 2015; accepted 25 February 2016; published online 7 March 2016) (Received 1 December 2015; accepted 25 February 2016; published online 7 March 2016) We evaluate the utilities of fluorescence-yield (FY) modes in soft X-ray absorption spectroscopy (XAS) of several cathode materials for Li-ion batteries. 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AIP Advances 6, 035105 (2016) AIP Advances 6, 035105 (2016) In LIB research, TM K-edge X-ray absorption spectroscopy in hard X-ray region is widely used to investigate the electronic structures of cathode materials.1,2 The redox reactions of TMs can be clarified by the edge/peak shift in the TM K-edge absorption spectra. However, the TM K-edge X-ray absorption spectroscopy is less sensitive to the 3d electronic states of TMs than the TM L2,3-edge (i.e. the 2p-3d transition) X-ray absorption spectroscopy, because the 1s-4p dipole transition is dominant at the TM K edges. To obtain the detailed information of the 3d orbitals, i.e. electronic-structure parameters such as charge-transfer energy, soft X-ray absorption spectroscopy (XAS) covering the TM L2,3 edges (i.e. the 2p-3d transition) has been widely used for various fields including solid state physics.3,4 Total-electron yield (TEY) is the most conventional detection mode for the XAS experiments in the soft X-ray region, while the probing depth is approximately 5 nm at most. The surface sensitivity of the TEY mode is frequently inappropriate for investigating the redox reaction in the cathode materials. In contrast, total-fluorescence-yield (total FY: TFY) XAS can probe the bulk electronic structures as deep as ∼100 nm from the sample surface. However, the TM L3-edge TFY XAS spectra tend to be distorted by self-absorption and saturation effects.5 These effects are depen- dent on the density, particle size (for powder) and thickness (for thin film) of the sample and the measurement geometry such as normal/grazing incidence. In addition, the distortion in the line shapes of TM L3-edge TFY XAS is enhanced in case where the target sample does not contain heavy elements such as rare earth.6 Thus, powdered Li-TM oxides and related compounds used for cathode materials of LIB are frequently affected by these effects.7 The distortion is significant when the O Kα fluorescent emission is strongly dependent on the incoming photon energies at the TM L2,3 regions. To reduce the distortion in spectral line shapes, partial-fluorescence-yield (PFY) and inverse partial-fluorescence-yield (IPFY) modes have been utilized in previous works,6–14 although the mechanisms of PFY and IPFY are still under debate.6,8,9,15–19 The PFY detection mode using a silicon-drift detector (SDD) can select a specific emission, e.g. TM Lα,β, suppressing the influence of the O Kα emission. Material/element-dependent fluorescence-yield modes on soft X-ray absorption spectroscopy of cathode materials for Li-ion batteries In the case of total-FY (TFY) XAS for LiNi0.5Mn1.5O4, the line shape of the Mn L3-edge XAS was largely distorted by the self-absorption and saturation effects, while the distortions were less pronounced at the Ni L3 edge. The distortions were suppressed for the inverse-partial-FY (IPFY) spectra. We found that, in the cathode materials, the IPFY XAS is highly effective for the Cr, Mn, and Fe L edges and the TFY and PFY modes are useful enough for the Ni L edge which is far from the O K edge. C 2016 Au- thor(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). [http://dx.doi.org/10.1063/1.4943673] Electronic-structure analyses for the electrode materials of Li-ion batteries (LIBs) are essential to understand the charge-discharge properties, which is capable of leading to improvements of the battery performance. The 3d transition metals (TMs) are the major elements in the cathode materials for LIBs, such as TM oxides and phosphates. The TMs in the LIBs cathode materials experience the oxidation and reduction during charging/discharging process (i.e. Li-ion extrac- tion/insertion). The redox chemistry determines the electrochemical properties of the electrode materials. Thus, element-selective electronic-structure analyses are indispensable for the applica- tion of LIBs. © Author(s) 2016. 6, 035105-1 2158-3226/2016/6(3)/035105/8 035105-2 Asakura et al. 035105-2 035105-2 Asakura et al. The TFY spectra AIP Advances 6, 035105 (2016) 035105-3 Asakura et al. FIG. 1. (a) The Fe L2,3-edge XAS spectra for LiFePO4 taken by TEY, TFY, PFY and IPFY detection modes. Calculated spectrum for Fe2+ HS state is also shown. (b) Emitted-photon-energy (hνout) profiles for several incident photon energies (hνin). The corresponding incident energies are indicated in the inset: TEY spectrum. FIG. 1. (a) The Fe L2,3-edge XAS spectra for LiFePO4 taken by TEY, TFY, PFY and IPFY detection modes. Calculated spectrum for Fe2+ HS state is also shown. (b) Emitted-photon-energy (hνout) profiles for several incident photon energies (hνin). The corresponding incident energies are indicated in the inset: TEY spectrum. were measured with a channel plate detector. The IPFY spectra were measured using an SDD with an energy width of ∼120 eV. The energy resolution of incident photon energies was set to 5000. All the XAS measurements were performed at room temperature. Configuration-interaction full-multiplet (CIFM) calculations were adopted for the analysis of the L2,3-edge XAS spectra.25–28 Ligand-to-metal charge transfer (LMCT) was taken into account in the calculation. 24 October 2024 05:21:04 g g Figure 1(a) shows the Fe L2,3-edge TEY, TFY, PFY, and IPFY XAS spectra for LiFePO4. The main peak at 708.5 eV in the TEY spectrum is assigned to Fe2+ high-spin (HS) state from the multiplet calculation while the peak at 710.5 eV is slightly larger than the calculated Fe2+ HS spec- trum. Most likely, a small fraction of Fe3+ HS should coexist according to previous reports.29,30 The TFY spectrum exhibits a dip-like structure at 706.5 eV and less pronounced L3 peaks at 708.4 and 710.5 eV. The L3 peak intensity is similar to that in the L2 edge. In the PFY spectrum corresponding to the Fe Lα,β emission, the dip-like structure at 706.5 eV was not observed although the L3/L2 intensity ratio is similar to that in the TFY spectrum and smaller than that in TEY. On the other hand, the IPFY spectrum which is the inverse of the O Kα emission shows the TEY-like intensity ratio of L3/L2. Furthermore, the profile of the IPFY spectrum is almost the same as that of TEY, suggesting the bulk and surface electronic structures are identical. These results indicate that the saturation and self-absorption effects are emphasized at the Fe L edge of this material. IPFY can be the most appropriate method to detect the bulk Fe 3d states. 035105-2 Asakura et al. The distortions in the line shape of L3-edge XAS can be further reduced by use of IPFY detection that is the inversed intensity of O Kα emission.6–12 24 October 2024 05:21:04 y In this study, we select LiFePO4 and LiNi0.5Mn1.5O4 as model examples to investigate those effects in TFY mode and utilities of PFY and IPFY modes. LiFePO4 is a promising cathode material because of the low-cost constituent element and the highly stable charge-discharge properties.20 In the olivine-type crystal structure, the Fe atoms are located in the FeO6 octahedrons and should be divalent. Spinel-type LiNi0.5Mn1.5O4 is an attractive cathode material in terms of the high voltage of ∼4.8 V vs. Li/Li+.21 The Ni and Mn atoms also form the (Ni,Mn)O6 octahedrons with Oh symmetry. The formal valences of Ni and Mn for the initial state are considered as 2+ and 4+, respectively. In addition, we performed the Cr L-edge XAS for Li0.35Mn4+0.65Cr3+0.35O222 to judge the utility of FY modes at Cr L edges. g We demonstrate TFY, PFY and IPFY XAS for those as-synthesized typical cathode materials in order to discuss appropriate use of the FY modes for each material/element. In this study, we focus on trends in the TMs of the cathode materials as observed in the FY XAS spectra. LiFePO4 was synthesized with a solid-state reaction method.23 LiNi0.5Mn1.5O4 was synthe- sized by an electrospinning method.24 We confirmed the successful syntheses of the cathode materials using powder X-ray diffraction and electrochemical experiments. The fabrication pro- cess of Li0.35Mn0.65Cr0.35O2 has been described elsewhere.22 We also prepared a charged sam- ple of LiNi0.5Mn1.5O4. The electrochemical experiments were performed by the same procedures described in Ref. 24. For LiFePO4 and LiNi0.5Mn1.5O4, TEY, PFY, and IPFY XAS measurements were performed at BL-7A of the Photon Factory. The energy resolution of incident photon energies for the XAS measurements was E/∆E ∼1500. An SDD was used for the PFY and IPFY detection. The energy width of SDD profile for fixed incident energies is approximately 100 eV. TFY XAS measurements were carried out at BL7.0.1 and BL8.0.1 of the Advanced Light Source with an energy resolution of E/∆E ∼2000 for the incident photon energies. A photodiode was used for the detection of TFY system. For Li0.35Mn0.65Cr0.35O2, TEY, TFY PFY, and IPFY XAS experiments were performed at 11ID-1 Spherical Grating Monochromator (SGM) of the Canadian Light Source. 035105-2 Asakura et al. The results are consistent with the data collected by Yang et al.7 Figure 1(b) shows the emitted-photon-energy (hνout) profiles taken at three incident photon energies (hνin). Each profile was measured for 1 min. Although the energy width of the SDD profile is about 100 eV, the O Kα and Fe Lα,β emissions can be distinguished. The O Kα and Fe Lα,β emis- sions were not observed for hνin = 520 eV because 520 eV is lower than the O K edge absorption threshold. For hνin = 699 eV (below the Fe L3 edge), only the O Kα emission is detected. The Fe Lα,β emission (for 600 < hνout < 800 eV) is enhanced for hνin = 708.5 eV corresponding to the Fe L3 edge, while O Kα emission (for 420 < hνout < 600 eV) is considerably reduced. The ∼40% reduction of O Kα emission causes the apparent reduction of Fe L3 peak in the TFY spectrum. In addition, compared with the profile for hνin = 708.5 eV, the Fe Lα,β and O Kα emission intensities were reduced and unchanged for hνin = 734 eV, respectively. This is consistent with the Fe L2 TFY and PFY spectra which show high intensities. As Fig. 2(a) shows, the Mn L2,3-edge TEY spectrum is attributed to a Mn4+ state. However, the TFY spectrum exhibits a considerable deformation at the Mn L3 edge, suggesting that the self-absorption and saturation effects strongly affect the Mn L3-edge TFY spectrum. The IPFY 035105-4 Asakura et al. AIP Advances 6, 035105 (2016) FIG. 2. (a) The Mn L2,3-edge and (b) Ni L2,3-edge XAS spectra for LiNi0.5Mn1.5O4 taken by TEY, TFY, PFY and IPFY detection modes. Calculated spectra are also shown. (c) Emitted-photon-energy (hνout) profiles for several incident photon energies (hνin). The corresponding incident energies are indicated in the insets: TEY spectra. 24 October 2024 05:21:04 24 October 2024 05:21:04 FIG. 2. (a) The Mn L2,3-edge and (b) Ni L2,3-edge XAS spectra for LiNi0.5Mn1.5O4 taken by TEY, TFY, PFY and IPFY detection modes. Calculated spectra are also shown. (c) Emitted-photon-energy (hνout) profiles for several incident photon energies (hνin). The corresponding incident energies are indicated in the insets: TEY spectra. spectrum displays a clear Mn L3-edge structure although the L3/L2 intensity ratio is slightly smaller than that in TEY. The IPFY spectrum is also assigned to the Mn4+ state. 035105-2 Asakura et al. In the case of Ni L2,3 edges, the TFY and PFY detection modes should be more appropriate than IPFY mode in terms of the S/N ratio. Most likely, Co L2,3 edges for Li-TM oxides (and phosphates) including Co would be positioned as the intermediate character between Fe and Ni L2,3 edges. As Fig. 2(c) indicates, the hνin dependence of O Kα emission weakens as hνin increases. To further verify this tendency, we also performed the Cr L-edge XAS for Li0.35Mn0.65Cr0.35O2 (Fig. 3(a)). The TEY spectrum is attributed to the Cr3+ state. On the other hand, not only the L3 edge, but also the L2 edge is reversed with negative sign in the TFY spectrum, which is more problematic than the case of Mn L-edges (Fig. 3(b)). The Cr L edges (∼575 to ∼595 eV) are so close to the O K edge (∼525 to ∼560 eV) that the Cr Lα,β emission cannot be separated from the O Kα emission within the energy width of SDD (∼100 eV) (Fig. 3(c)). The Cr L-edge IPFY spectrum in Fig. 3(a) is composed by extracting the structure below 570 eV in Fig. 3(c) as the OKα emission. However, the S/N ratio and L3/L2 intensity ratio are not ideal. If the energy resolution of the detector is improved, for example by using a high-resolution soft X-ray emission spectrometer,31,32 then the separation of Cr Lα,β emissions from O Kα emission should be facilitated. 24 October 2024 05:21:04 β Figure 4 summarizes the utilities of TFY, PFY and IPFY for each TM L edge of Li-TM oxides/phosphates. Note that the tendency is limited to powdered Li-TM oxides/phosphates with particle sizes between 10 nm and 10 µm generally used as cathode materials of LIB. The IPFY mode can be useful for Cr, Mn, and Fe L edges for those materials. For L edges with higher energies, TFY and PFY are more suitable than IPFY. In addition, TFY and PFY should be available for Ti and V without any problems because these early TMs’ L edges are located below the O K edge. Appropriate choice of the FY modes is inevitable to study the bulk electronic structures of Li-TM oxides and related materials. As Fig. 5 shows, the bulk electronic structure is often different from the surface electronic structure in particular for charged (or discharged) samples. In the case of LiNi0.5Mn1.5O4 (Fig. 035105-2 Asakura et al. The similarity between TEY and IPFY spectra indicates that the surface and bulk electronic structures are almost identical. p The Ni L2,3-edge TEY spectrum exhibits the Ni2+ HS multiplet structure (Fig. 2(b)). In contrast to the Mn L edge, the distortions of the Ni L3 edge TFY and PFY spectra are suppressed. Although the L3/L2 intensity ratios of TFY and PFY are slightly smaller than that for TEY, the TFY and PFY spectra are available for the analysis using the multiplet calculations. The IPFY spectrum is also similar to the TFY and PFY spectra. However, the signal-to-noise (S/N) ratio of IPFY spectrum is lower than those of TFY and PFY spectra. Thus, the IPFY detection mode is less advantageous for the analysis of Ni L2,3 edges of this material. Figure 2(c) shows the hνout profiles of emissions taken at several hνin. The O Kα, Mn Lα,β and Ni Lα,β emissions can be observed. The Mn Lα,β emissions (for 600 < hνout < 720 eV) are 035105-5 Asakura et al. Asakura et al. AIP Advances 6, 035105 (2016) enhanced at hνin = 644 eV. However, the degree of enhancement is lower than the degree of reduc- tion of the O Kα emission, leading to the deformation at the Mn L3 edge in the TFY spectrum. On the other hand, at the Ni L3 edge, the degree of reduction of the O Kα emission becomes small and the Ni Lα,β emission (for 750 < hνout < 970 eV) increases considerably, such that the deformations at the Ni L3 edge in the TFY and PFY spectra are less pronounced. The Mn L3-edge TFY XAS spectrum for LiNi0.5Mn1.5O4 exhibits the large distortions in XAS line shapes, suggesting that the XAS line shapes of Mn L3 edge in Li-TM oxides (and phosphates) should be very sensitive to the self-absorption and saturation effects, while these effects depend on the density and composition of sample and experimental geometries. This is consistent with the Mn L3 edge/Lα,β emission (∼640 to ∼660 eV) being very near O K edge/Kα emission (∼525 to ∼560 eV) in photon energy. The Fe L3 edge XAS is also sensitive, although the influence of self-absorption and saturation effects should be smaller than that in the MnL3 edge. Considering the present results, the IPFY detection mode can become powerful tools for the Mn and Fe L2,3-edge measurements. 035105-2 Asakura et al. 5), the degree of oxidation of Ni due to the charge reaction is different between the surface and bulk. The Ni L-edge TFY XAS spectrum for the charged state shows enhanced peaks at 856 and 873 eV, indicating partial oxidation reaction of Ni2+ →Ni3+33 in the bulk region. On the other hand, in the surface-sensitive TEY mode, the line shape attributed to the Ni2+ state is almost maintained for the charged state, suggesting existence of redox-inactive Ni and/or some side reactions such as NiO formation near the surface. Thus, a combined use of TEY and FY is essential for the electronic-structure analyses of the electrode materials. In conclusion, we demonstrated TEY, TFY, PFY and IPFY XAS studies for powdered several Li-TM oxides and a phosphate which are cathode materials for LIBs. The utilities of FY modes strongly depend on the element (edge) and material (composition). The FY modes should be care- fully chosen for each material. The distortion of Ni L3-edge TFY XAS line shapes is not large whereas Cr, Mn and Fe L3-edge TFY XAS are greatly affected. We conclude that, in the FY XAS studies of the cathode materials, the IPFY XAS is most appropriate for the Cr, Mn, and Fe L edges and the TFY and PFY XAS modes are available enough for the Ni L edge which is far from the O K edge. This consideration will contribute to TM L-edge FY XAS studies of charged/discharged samples for novel electrode materials. AIP Advances 6, 035105 (2016) 035105-6 Asakura et al. FIG. 3. (a) The Cr L2,3-edge and (b) Mn L2,3-edge XAS spectra for Li0.35Mn0.65Cr0.35O2. (c) Emitted-photon-energy (hνout) profiles for several incident photon energies (hνin). The Cr Lα,β emission should be around 580 eV, which cannot be separated from the O Kα emission in the present energy resolution. 24 October 2024 05:21:04 24 October 2024 05:21:04 FIG. 3. (a) The Cr L2,3-edge and (b) Mn L2,3-edge XAS spectra for Li0.35Mn0.65Cr0.35O2. (c) Emitted-photon-energy (hνout) profiles for several incident photon energies (hνin). The Cr Lα,β emission should be around 580 eV, which cannot be separated from the O Kα emission in the present energy resolution. FIG. 4. A scheme of utilities of TFY, PFY and IPFY for each TM L edge of powdered Li-TM oxides/phosphates (Ti, V, Co and Cu L edges are not investigated here). The color scales of the arrows indicate the utilities of each XAS mode. 035105-2 Asakura et al. FIG. 4. A scheme of utilities of TFY, PFY and IPFY for each TM L edge of powdered Li-TM oxides/phosphates (Ti, V, C and Cu L edges are not investigated here). The color scales of the arrows indicate the utilities of each XAS mode. AIP Advances 6, 035105 (2016) 035105-7 Asakura et al. FIG. 5. The Ni L-edge XAS spectra for the pristine and charged LiNi0.5Mn1.5O4 recorded by (a) the TEY and (b) TFY modes. The spectra were normalized at 854.2 eV to emphasize the differences at 856 and 873 eV in (b). FIG. 5. The Ni L-edge XAS spectra for the pristine and charged LiNi0.5Mn1.5O4 recorded by (a) the TEY and (b) TFY modes. The spectra were normalized at 854.2 eV to emphasize the differences at 856 and 873 eV in (b). ACKNOWLEDGEMENTS This work was performed under the approvals of the Photon Factory Program Advisory Com- mittee (Proposal Nos. 2012G107, 2013G169 and 2014G100) and the Advanced Light Source (Proposal No. 04907R). The Advanced Light Source is supported by the Director, Office of Sci- ence, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. Research described in this paper was partly performed at the Canadian Light Source, which is supported by CFI, NSERC, the University of Saskatchewan, the Government of Saskatchewan, WD, NRC, and CIHR. The synthesis work at NREL was supported by the NREL LDRD Program. This work was also conducted based on the Japan-U.S. cooperation project for research and standardization of Clean Energy Technologies. 24 October 2024 05:21:04 3 F. de Groot and A. Kotani, Core Level Spectroscopy of Solids (Advances in Condensed Matter Science) (CRC Press Taylor & Francis Group, Boca Raton, FL, 2008). 5 S. Eisebitt, T. Boske, J.-E. Rubensson, and W. Eberhardt, Phys. Rev. B 47, 14103 (1993). H. Wadati, A. J. Achkar, D. G. Hawthorn, T. Z. Regier, M. P. Singh, K. D. Truong, P. Fournier, G. Chen, T. Mizokawa, and G. A. Sawatzky, Appl. Phys. Lett. 100, 193906 (2012). 7 S. Yang, D. Wang, G. Liang, Y. M. Yiu, J. Wang, L. Liu, X. Sun, and T.-K. Sham, Energy Environ. Sic. 5, 7007 (2012). 8 A. J. Achkar, T. Z. 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https://openalex.org/W4235561908	https://zenodo.org/records/1509214/files/article.pdf	English	null	Reviews	Analyst (London. 1877. Online)/Analyst	1,922	public-domain	4,108	REVIEWS REVIEWS Published on 01 January 1922. Downloaded on 30/10/2014 04:18:29. View Article Online / Journal Homepage / Table of Contents for this issue View Article Online / Journal Homepage / Table of Contents for this issue 137 View Article Online / Journal Homepage / Table of Contents for this issue View Article Online / Journal Homepage / Table of Contents for this issue Reviews. A TEXT BOOK OF ,hALYSIS OF ISORG-4NIC SUBSTASCES. By S. A. KAI-, D.SC. Pp. viii+80. London: Gurney & Jackson. 1921. Price 7s. 6d. net. Yet another book on Quantitative Analysis, but one which can be thoroughly recommended for classwork with beginners. Not too big (eighty pages only), nor overloaded with unnecessary detail, it deals adequately with the commoner metallic and non-metallic radicals, and clearly sets out the usual ,methods of inorganic analysis. The book opens with a few pages of general instruction, followed by an explanation of the grouping of radicals for analytical purposes, with some useful preliminary exercises. The remaining sixty pages are devoted to systematic analysis of the ordinary type, with the addition of special paragraphs on alloys, minerals, glasses, etc. The main text is fully interspersed with notes, so that the student should have no difficulty in understanding thoroughly the reason for each step, and in avoiding many pitfalls that beset the beginner’s path; in this way much time is likely to be saved, and the student will not be so dependent on the teacher as is frequently the case. More attention is directed to the preliminary examination of substances than many would consider desirable, and no less than six types of dry test are described. On the other hand only three organic acids receive notice ; subsequent editions would probably be improved by the inc1usio:i of one or two more. The book contains a short index, and some blank pages for additional notes; furthermore it is well printed in clear type-a point of importance in a manual for laboratory work. &4. F. KITCHISG. &4. F. KITCHISG SOME MICRO-CHENICAL TESTS FOR ALK.4LOIDS. CHARLES H. STEPIIESSOS. 1’1). 110, with 27 plates. London: Charles Griffin & Co. 1921. Price 21s. net. There are but few books which deal with the identification of alkaloids, and the most important of them is written in German, namely, “Beitrage XZLY nzikro- chemischen Analjw Einiger Alknloide zrnd Drogen,” Alide Grutterink, 1910. The SOME MICRO-CHENICAL TESTS FOR ALK.4LOIDS. CHARLES H. STEPIIESSOS. 1’1). 110, with 27 plates. London: Charles Griffin & Co. 1921. Price 21s. net. There are but few books which deal with the identification of alkaloids, and the most important of them is written in German, namely, “Beitrage XZLY nzikro- chemischen Analjw Einiger Alknloide zrnd Drogen,” Alide Grutterink, 1910. Reviews. The View Article Online 138 REVIEWS present volume will prove helpful to all interested in the subject, the more so in that it contains the result of much original work. In it will be found a description of the crystalline precipitates which alkaloids give with such reagents as gold, plat hum, palladium, mercury and zinc chlorides, p hospho t ungs tic, phosphomo - lybdic and picric acids, etc. The micro-chemical appearance of the crystals is described, as well as the best conditions for their formation, and the value of the book is greatly enhanced by a series of beautiful micro-photographs, some 162 in all. present volume will prove helpful to all interested in the subject, the more so in that it contains the result of much original work. In it will be found a description of the crystalline precipitates which alkaloids give with such reagents as gold, plat hum, palladium, mercury and zinc chlorides, p hospho t ungs tic, phosphomo - lybdic and picric acids, etc. The micro-chemical appearance of the crystals is described, as well as the best conditions for their formation, and the value of the book is greatly enhanced by a series of beautiful micro-photographs, some 162 in all. It is very difficult in the case of many alkaloids to identify them when only small amounts are available. Apart from physiological tests, it is customary to rely upon colour reactions and upon the crystalline and amorphous precipitates resulting from the addition of various reagents, but, especially where the material tested is likely to be contaminated with impurities, such chemical tests can, as a rule, only provide contributory evidence of identity, and, unfortunately, extracted alkaloids, as obtained in the course of analysis, are generally impure. Published on 01 January 1922. Downloaded on 30/10/2014 04:18:29. This book, described on the title page as a “Text Book of Micro-organisms General and Applied,” is written by no less than 25 contributors, mostly Pro- fessors of American Universities and Colleges, but representing also France and Germany, each with special knowledge of his subject, under the general editor- ship of Professor Marshall. p Since the publication of the first edition in 1911 (reprinted with corrections in 1912) this book has been considerably enlarged and improved, and now contains 992 pages excluding the index. It is divided into three distinct parts:-1. Morpho- logical and Cultural ; 2. Physiological ; and 3. Applied. General principles and the key to part 3 are thus dealt with in the first two parts, and part 3, the most interesting and generally useful, is unencumbered with preliminary detail. g g y y Part 1.-The cytology of the living cell generally, and of moulds, yeasts, bacteria and protozoa in particular, is admirably dealt with by Guilliermond and Todd with excellent illustrations. In this part, also, the distinctive features of the principal types of moulds and yeasts are described, and the recent classification of bacteria adopted by the Society of American Bacteriologists is given. Part II. opens with a discussion of the bearing of phenomena in the realm of physical chemistry upon microbial activity, and a brief account is given of ionisa- tion and dissociation, leading up to the true definition of acidity, neutrality and alkalinity, and its determination by means of the hydrogen electrode; surface tension; adsorption; osmosis; and colloidal phenomena. It is to be regretted that a table showing the colour changes of indicators and another showing solutions ,of known H ion concentration, together with their application to the adjustment *of culture media, are not included. The rest of Part 11. deals with chemical studies of the contents of microbial cells; nutrition and metabolism-enzymes and their action, products of metabol- ism, etc. ; physical influences-osmosis, desiccation, temperature, light, electricity, etc. ; chemical influences ; and mutual influences-symbiosis, etc. Published on 01 January 1922. Downloaded on 30/10/2014 04:18:29. y g y p In the hands of those with much experience of alkaloids the information given in this book will prove of great value. There is, however, a danger lest the un- initiated may place too great reliance upon the superficial appearance of the crystals obtained. The appearance and characteristics of groups of crystals vary greatly according to the degree of purity, concentration and temperature of the liquid in which they form, so that, apart from actual goniometric measurements, crystalline form is apt to prove deceptive. The reviewer places greater reliance upon determinations of melting point of such crystalline deposits, and it is surprising how minute an amount may suffice for this purpose in careful The book contains a record of a very large amount of experimental work which has not been published elsewhere. The precipitates of some 51 different alkaloids are described, and in most cases the reactions with 35 different reagents have been tried. It is consequently a notable contribution to the subject of which it treats. The question whether an amorphous or crystalline precipitate is obtained cannot always be regarded as conclusive evidence. For instance, in the famous Crippen murder case it was stated that hyoscine salts gave an amorphous precipitate with bromine water, and in the evidence much reliance was placed upon this test, but it has subsequently been shown that, even at dilutions of 1 in 1000, the precipitate obtained on the addition of bromine water to hyoscine salts rapidly changes from the amorphous to the crystalline state. A distinction is made between amorphous and crystalline precipitates. In addition to the description of crystalline precipitates, a full account is given of the melting points and chemical examination of the various alkaloids employed in the investigations. The book is excellently printed and the plates are well produced. p FRANCIS H. CARR. View Article Online View Article Online 139 REVIEWS MICROBIOLOGY. Edited by CHARLES E. MARSHALL. 3rd Edition. Pp. xxviii + 1043. London: J. & A. Churchill. 1921. Price 21s. net. MICROBIOLOGY. Edited by CHARLES E. MARSHALL. 3rd Edition. Pp. xxviii + 1043. London: J. & A. Churchill. 1921. Price 21s. net. Published on 01 January 1922. Downloaded on 30/10/2014 04:18:29. Artificial culture media are dealt with in a single short paragraph in Part I., and not under the heading “ nutrition.” The author has intentionally eliminated laboratory features as far as possible, but, in excluding a discussion upon culture media with special reference to those which have been introduced in recent years (with ex- amples of suitable media for the isolation and study of special types of micro- .organisms), this elimination has been carried too far. Part III., which occupies the major portion of this volume, is made up of 10 “Divisions,” as follows:-Microbiology (1) of the Air, (2) of Water and Sewage, (3) of the Soil, (4) of Milk and Milk Products, (5) of Foods, (6) of Alcoholic Fermenta- tion and Derived Products, (7) of Special Industries, (8) of Diseases of Man and Domestic Animals, (9) of Diseases of Insects, and (10) of Diseases of Plants. The scope is, of course, enormous, covering as it does almost the whole field of bacteriological science, and it would be unreasonable to expect to find a full treat- .merit of all the subjects dealt with. The contributors have, however, given a View Article Online 140 REVIEWS very able presentation of the general principles and more important features of their subjects, and a considerable amount of,recent work is included. very able presentation of the general principles and more important features of their subjects, and a considerable amount of,recent work is included. j Nothing has been added since 1912, no reference is made, as regards water, to the recent work of Clark, Lubs, and Levene on the differentiation of the colon-aerogenes group, sterilisation by hypochlorite and chlorine is barely mentioned, and the books recommended for a fuller presentation of the subject do not include the standard works of Houston and Thresh, nor the latest book, “Food and Water,” of Savage. The activated sludge process of sewage purification is not mentioned at all. The section on Water and Sewage is rather disappointing. The section dealing’with the Microbiology of Soil was well revised in 1917, and then included a useful discussion of the function of the genus Actinomyces and Protozoa; now further new matter on the colloidal nature of the soil, soil reaction and H ion concentration, and the function of the Moulds in ammonification has been added. Published on 01 January 1922. Downloaded on 30/10/2014 04:18:29. A description of the morphology and cultural characteristics of only a few of the soil micro-organisms is given, whereas the usefulness of this book would have been considerably enhanced had a concise but up-to-date description of the distinctive features of such organisms as B. butyricus, B. megatheriztm and varieties, B. mesentericus, B. mycoides, B. protezts and varieties, B. putrificus, B, Welchii, B. sporogenes, and other micro-organisms less well known, been included either in this section or elsewhere. Published on 01 January 1922. Downloaded on 30/10/2014 04:18:29. The section on Milk and Milk Products might have been brought more up to date by the incorporation of some of the work of Orla Jensen on theLactic Acid Bacteria (published in English 1919) and by co-ordinating his work with the work of others, e.g. the identity of S t y . cremoris (Jensen) the predominant type of the so-called “ pure cultures ’’ for butter-making and Str. Zacticus (Kruse) should have been pointed out, and the fermentation tests given by which this streptococcus may be distinguished from Str. Zactis (Jensen) and other streptococci. The function of his Tetracoccus Ziquefaciens in producing flavour in certain classes of cheese might have been alluded to, and a description given of suitable media for the isolation and study of micro-organisms of importance in dairy processes, such as whey agar and calcium lactate broth, fat-agar emulsion, etc. g Under the head- ing “ Controlling Factors in Successful Canning,” instructive graphs showing the influence of temperature, number of spores, and speed of rotation upon steril. ising time are given by Bigelow. This section includes also Compressed Yeast, Bread, Vegetables preserved by fermentaion, Silage fermentation, etc. A good account of recent work upon the distribution in nature and occurrence in canned vegetables and other foods of B. botulinus has been given. In this section the sub- ject of Microbial Food Poisoning is dealt with. As in most German literature, no distinction is made between B. paratyphosus B, B. aertryche, and B. suipestifer, and one finds reference only to B. paratyphosus, whereas the last two are distinguish- able from the first by absorption of agglutinin tests and have quite a distinct etiological connection, the symptoms of paratyphoid fever and of microbial food poisoning being altogether different. No reference is made to the work of Savage, The section on the Microbiology of Foods is full of interest. Published on 01 January 1922. Downloaded on 30/10/2014 04:18:29. View Article Online View Article Online 141 REVIEWS and his recent work “ Food Poisoning and Food Infections ” (1920) is not included amongst the references at the end of the chapter. American authors have an unfortunate tendency to overlook English literature. and his recent work “ Food Poisoning and Food Infections ” (1920) is not included amongst the references at the end of the chapter. American authors have an unfortunate tendency to overlook English literature. y g The section dealing with the Microbiology of Alcohol and Derived Products (Div. VI.) is ably contributed by Bioletti ,and includes Wine, Beer, Cider and Perry, Hydromel or Mead, Distilled Alcohol and Acetic Fermentation. y y The Microbiology of Special Industries (Div. VIII.) is quite new, and includes Acetone and Acetic Acid, Lactic Acid, Citric Acid, White Lead, Leather, Indigo and Retting. A little more space might have been devoted to this interesting section, and a fuller description given of the micro-organisms concerned. Published on 01 January 1922. Downloaded on 30/10/2014 04:18:29. View Article Online 142 REVIEWS general arrangement, as well as the main bulk of the text, being unchanged, and the actual volume being increased only from 103 to 113 pages. general arrangement, as well as the main bulk of the text, being unchanged, and the actual volume being increased only from 103 to 113 pages. g y p g The manner in which the subject matter is dealt with is attractive, and the ok constitutes a connected and readable account of the diverse processes by which g y p g The manner in which the subject matter is dealt with is attractive, and the book constitutes a connected and readable account of the diverse processes by which the fixation of nitrogen from the air is effected, and also of the fundamental principles underlying these processes. Theory, indeed, occupies a predominant position throughout the book, which is consequently of especial value to the more advanced chemical student. In view of the great developments made in the fixation of nitrogen since 1914, expansion of the treatment of certain portions of the subject would seem desirable. Moreover, the bibliography, although moderately complete, contains compara- tively few references to the large amount of work published during the past few years. Published on 01 January 1922. Downloaded on 30/10/2014 04:18:29. y The author is one of the few writers of chemical text-books who possess the ability to express themselves in fluent and correct English, and he has exercised care to avoid typographical errors. yp g p It is unfortunate that it has been found necessary to double the original price of the book, but, even in this respect, the latter compares favourably with many scientific publications of the present day. T. H. POPE. ably with man T. H. POPE SYNTHETIC TANNINS. By G. GRASSER Translated by F. G. A. ENNA Pp. viii. +143. London: Crosby Lockwood. 1922. Price 12s. net. To the chemist this book is a valuable and useful collection of work on syn- thetic tannins, but is of very little use to the purely practical leather man from a works’ standpoint. Published on 01 January 1922. Downloaded on 30/10/2014 04:18:29. p g g “Division VIII.” on the Microbiology of Diseases of Man and Domestic Animals covers 246 pages, and includes chapters on Channels of Infection, Immu- nity and Susceptibility, Manufacture of Vaccines, Manufacture of Anti-sera, etc, Control of Infectious Diseases (giving American Public Health Regulations), and Microbial Diseases of Man and Domestic Animals. The last chapter of this section gives a description of some 90 diseases and of their causative organisms (when known) with cultural characters ; these descriptions are necessarily brief, but exceptionally good. The fermentation tests for B. pullorurn should have been given and contrasted with those for B. sanguinarium (not given), and the use of bile salt agar for its isolation might have been indicated. Gordon and Hine’s legumin-trypagar for the cultivation of the meningococcus and gonococcus should certainly have been given, and Matthew’s trypsinised blood agar for B. influenzae. A table showing the cultural reactions of the Typhoid-Paratyphoid-Dysentery group would be a useful addition to this section. g p Diseases of Plants some 20 and 30 diseases respectively are described with their causative organisms. These two chapters are exceedingly interesting, and provide in a concise form information with which one is least familiar, and at a loss to find elsewhere. Under Nosema Disease of Bees, brief reference should have been made to the Isle of Wight Bee Disease, so long associated with Nosema a+ and the work of John Rennie, who showed that Tarsonema Woodi was the causative par asit e . In the last two sections (9) Microbial Diseases of Insects and (10) Microbial p Notwithstanding the criticisms which this review contains, Professor Marshall’s book is certainly one of great value, and will be found very serviceable not merely to students, as his modest claims in the preface almost imply, but to many advanced workers also; for, if something is lacking in detail, it is abundantlymade up for in scope. The book is singularly free from printers’ errors, the type is excellent and the paper good. In spite of its 1043 pages it is a convenient and handy volume. y D. R. WOOD FIXATION OF ATMOSPHERIC NITROGEN. By JOSEPH KNOX, D.Sc. 2nd Edition. This book is little more than a reprint of the 1st edition, published in 1914, the London: Gurney & Jackson. 1921. Price 4s. net. Published on 01 January 1922. Downloaded on 30/10/2014 04:18:29. After a comprehensive chapter on the synthesis of vegetable tannins, in- cluding the recent work of Fischer and Frendenberg, the synthesis of tanning matters, as distinct from vegetable tannins, is dealt with from the following standpoints :-( 1) Aromatic Sulphonic Acids ; (2) Condensation of Phenols ; (3) Condensation of Napthalene derivatives ; (4) Condensation of Anthracene ; and and (5) Di-and-tri-phenylmethane groups. A very interesting table is given, showing the relative behaviour of a large number of organic substances towards gelatin hide powder and pelt respectively. The tanning effects of mixtures and natural products, e.g. phenolsulphonic acid and formaldehyde, are briefly outlined. The usual chapter on tanning analysis follows, but is amplified by the addition of useful results on the analysis of synthetic tannins (of great importance to the leather trade chemist). Details are given of the manufacture of a number of the more important synthetic tannins. With regard to their industrial production, ‘it is pointed out that “ but few synthetic tannins are to-day of practical and commercial interest.” The analysis of leathers containing Neradol C is briefly dealt with. E. D. MERRY. View Article Online View Article Online 143 REVIEWS THE POPULAR CHEMICAL DICTIONARY. By C. T. KINGZETT, F.I.C. Pp. viii +536, London: Ballisre, Tyndall and Cox. with various figures in the text. 1922. Price 31s. net. The first edition of this work was reviewed in the ANALYST (1920, p. 158), when certain inaccuracies were pointed out; most of these have been rectified in the present enlarged edition. Although the inclusion of the word “Popular” in the title tends to disarm acute criticism, it must be confessed that there is still much room for improvement. On the whole, inorganic materials appear to be accurately described, but unfortu- nately the same cannot be said of organic materials, particularly so-called raw materials. As an example, the article on rubber is a good instance, for, apart from various inaccuracies, it appears to contain little that is necessary in a popular treatise and much that is unnecessary. Although the botanical names of certain rubber-yielding plants are quoted (some incorrectly), there is no clear statement that the vast majority of the world’s supply is derived from cultivated Para rubber trees (Hevea brasiliensis), introduced into Ceylon, Malay, etc., while part of the paragraph on the vulcanisation of rubber is quite obsolete. The information on vegetable oils is often inaccurate. Published on 01 January 1922. Downloaded on 30/10/2014 04:18:29. For example, one frequently finds the statement that an oil is “soluble in alcohol,” whereas unfortunately this is only accurate for castor oil. Cotton seed oil is mentioned as being used as a lubricant, for which purpose it is certainly not well suited; candle-nut oil (Alewitis moluccana for Aleurites, a mistake surviving from the first edition) is not stated to be a drying oil. Palm oil is said to be obtained by crushing the fermented fruits ; native oil-makers certainly do frequently allow fruit to ferment, but the practice is not always employed and is unnecessary, and, indeed, highly undesirable. Again, the specific gravity of West African palm oil is given as 0.92-0.93, and this is followed by a statement from a recent publication quoting 0.8586 and 04636 for West African and South American oils respectively. Apparently the influence of temperature on specific gravity determinations has been forgotten; in fact, only rarely is any temperature quoted with specific gravity figures. Published on 01 January 1922. Downloaded on 30/10/2014 04:18:29. There are a great many similar inaccuracies in the descriptions of raw products, but the above instances are sufficient to shake one’s faith in all references to such materials, and it is to be hoped that these will be carefully revised in the next edition. RUSSELL G. PELLY. y RUSSELL G. PELLY KELLY’S DIRECTORY OF THE CHEMICAL INDUSTRIES. 15th Edition. Pp. xxxi +856. London: Kelly’s Directories, Ltd. 1921. Price 30s. This useful work of reference was first published in 1869 under the title of The Directory of Chemists and Druggists, and contained 488 pages. In 1919 it was renamed the Directory of Chemical Industries, and contained 842 pages. In the present edition a large number of additions and alterations has been made, and the size has increased to 856 pages, and the book now covers every industry and trade directly or indirectly connected with the production and sale of chemical products in Great Britain and Ireland. Some years ago Mr. E. G. Clayton pointed View Article Online View Article Online 144 PUBLICATIONS RECEIVED out in his Memoir of Dr. A . H . Hassall (p. 40) that in the year 1850 there were no names of analytical chemists in the Post Ofice Directory. In 1854 there were eleven, in 1855 twenty, in 1857 twenty-four, and by 1877 the number had risen to fifty- four. The present publishers have informed the writer that the number of entries under the heading “Analysts” in the Post Ofice Directory was 25 in 1878, 24 in 1888, and 23 in 1922, so that, so far as this was an index, the number of practising analytical chemists in London had kept fairly constant during the last 45 years. In this Chemical Directory, however, there are 198 entries under the heading of Analytical Chemists. The work shows evidence of the same careful compilation as the other directories issued by this firm, and the cut edges of the book are coloured differently to facilitate reference. If we might suggest an improvement, however, it would be a great advantage if the names of the various section headings were printed at the top of each page. EDITOR. Published on 01 January 1922. Downloaded on 30/10/2014 04:18:29. g EDITOR
https://openalex.org/W2757678756	https://trepo.tuni.fi/bitstream/10024/102122/1/the_story_of_journalist_organizations_in_Czechoslovakia.pdf	English	null	The Story of Journalist Organizations in Czechoslovakia	Media and communication	2,017	cc-by	7,697	Issue Issue This article is part of the issue “Histories of Collaboration and Dissent: Journalists’ Associations Squeezed by Political System Changes”, edited by Epp Lauk (University of Jyväskylä, Finland) and Kaarle Nordenstreng (University of Tampere, Finland). © 2017 by the authors; licensee Cogitatio (Lisbon, Portugal). This article is licensed under a Creative Commons Attribu- tion 4.0 International License (CC BY). * Corresponding author Submitted: 15 May 2017 \| Accepted: 23 August 2017 \| Published: 27 September 2017 Abstract This article reviews the political history of Czechoslovakia as a vital part of the Soviet-dominated “Communist bloc” and its repercussions for the journalist associations based in the country. Following an eventful history since 1918, Czechoslovakia changed in 1948 from a liberal democracy into a Communist regime. This had significant consequences for journalists and their national union and also for the International Organization of Journalists (IOJ), which had just established its head- quarters in Prague. The second historical event to shake the political system was the “Prague Spring” of 1968 and its aftermath among journalists and their unions. The third landmark was the “Velvet Revolution” of 1989, which played a significant part in the fall of Communism in Central and Eastern Europe and led to the closing of the old Union of Journalists in 1990, followed by the founding of a new Syndicate which refused to serve as the host of the IOJ. This led to a gradual disintegration and the closing down of what in the 1980s was the world’s largest non-governmental organization in the media field. The Story of Journalist Organizations in Czechoslovakia Markéta Ševčíková 1,* and Kaarle Nordenstreng 2 Markéta Ševčíková 1,* and Kaarle Nordenstreng 2 1 Independent Researcher, Prague, Czech Republic; E-Mail: sevcikova.m@post.cz 2 Faculty of Communication Sciences, University of Tampere, 33014 Tampere, Finland; E-Mail: kaarle.nordenstreng@uta.fi Keywords y Cold War; communism; Czechoslovakia; International Organization of Journalists; journalism; union of jo 1. Introduction The starting point of this article is a story about the rise and fall of the IOJ—an international non-governmental organization of journalists based in Czechoslovakia, which had become an icon of the changing political land- scape of the world since 1946. The same story has been told in an anthology of the history of the international movement of journalists (Nordernstreng, Björk, Beyers- dorf, Høyer, & Lauk, 2016) and also in a monograph on the IOJ (Nordenstreng, in press), but this was done with- out covering the broader context of the political history of Czechoslovakia. The present review aims to fill the gap by first describing the key political turns in the history of Czechoslovakia since World War I (Emmert, 2012). Media and Communication (ISSN: 2183–2439) 2017, Volume 5, Issue 3, Pages 95–102 DOI: 10.17645/mac.v5i3.1042 Article 2.1. Four Periods between 1918 and 1948 An extensive print media developed, the press being primarily parti- san, with a minority non-party press, tabloids and mag- azines, and the new media of radio and film were evolv- ing. Partial censorship existed and was carried out by the Ministry of Interior. Publishers had to submit mandatory copies to district authorities or police headquarters, who had the right to withhold prints prior to their release, to allow the issue only after a change or deletion of the text or even confiscate the press (Končelík, Večeřa, & Orság, 2010). Nevertheless, cultural openness and toler- ance prevailed. vakia) and 30 September 1938 (signing of the Munich Agreement). In 1918 Czechoslovakia ceased to be part of the Austro–Hungarian Empire and became an indepen- dent republic with a democratic polity based on the Con- stitution. The country consisted of Bohemia, Moravia, Silesia, Slovakia and Subcarpathian Ruthenia. The Repub- lic was characteristically multi-ethnic and there were ini- tially large social, cultural and economic differences be- tween its individual regions. The internal unity of the new state was challenged by the German ethnic minor- ity living in the border regions and wishing to become part of Germany, and the leftist labour movement, which founded the Communist Party of Czechoslovakia (hence- forward the CPC) in May 1924. In the short term, how- ever, Czechoslovakia became a mature democratic re- public with a broad spectrum of political parties, and the republic advanced in leaps and bounds in the fields of industry, trade, education and lifestyle. An extensive print media developed, the press being primarily parti- san, with a minority non-party press, tabloids and mag- azines, and the new media of radio and film were evolv- ing. Partial censorship existed and was carried out by the Ministry of Interior. Publishers had to submit mandatory copies to district authorities or police headquarters, who had the right to withhold prints prior to their release, to allow the issue only after a change or deletion of the text or even confiscate the press (Končelík, Večeřa, & Orság, 2010). Nevertheless, cultural openness and toler- ance prevailed. Understandably, the Czech people did not accept the occupation and resistance to it soon emerged. These activities culminated in the assassination of the Reich- sprotektor. Meanwhile, the government-in-exile, led by Edvard Beneš, was established in London. 1 The political system of the Second Republic was simplified and there were only two main political parties: (1) an “official” right-wing Party of Na- tional Unity (Strana národní jednoty) gathering together the Republican Party of Farmers and Peasants, Czechoslovak Traders’ Party, National Fascist Community, Czechoslovak Christian Social Party, National People’s Party, and (2) in “loyal” opposition, the left-wing National Labour Party (Národní strana práce). tional Unity (Strana národní jednoty) gathering together the Republican Party of Farmers and Peasants, Czechoslovak Traders Party, National Fascist Community, Czechoslovak Christian Social Party, National People’s Party, and (2) in “loyal” opposition, the left-wing National Labour Party (Národní strana práce). 2 The Prague uprising was an attempt by the Czech resistance to liberate the city of Prague from German occupying forces. It lasted from 5 May until 8 May 1945, ending in a ceasefire between the Czech resistance and the German army, which decided to leave Prague on the same day. Next morning, the Red Army entered the nearly liberated city. 3 See an educational portal for teachers, students and pupils available on the internet http://www.moderni-dejiny.cz/clanek/kosicky-vladni-program-5- 4-1945 4 The National Committee (1945–1990; NC) was a part of the Czechoslovak state administration with formally elected bodies. The NC was divided ac- cording to the place of operation—from the top level of the country, within regions, districts and towns to municipalities. It performed as an “extended arm” of the CPC for the economy and whole society. 2.1. Four Periods between 1918 and 1948 2.1. Four Periods between 1918 and 1948 If we wish to understand the events which took place in post-war Czechoslovakia, particularly in February 1948, when the Communist Party took over political power in the country, we need to go back another thirty years. Czechoslovak history divides the period between 1918 and 1948 into four main phases: the First Republic, the Second Republic, the Protectorate of Bohemia and Moravia and the Third Republic. The period of the First Republic is defined by two dates: 28 October 1918 (birth of independent Czechoslo- Media and Communication, 2017, Volume 5, Issue 3, Pages 95–102 95 many effectively occupied the entire country. The Czech president formally remained Head of the Protectorate, along with the Protectorate’s Czech government. Never- theless, the real power—with a brutal and totalitarian Nazi regime—was held by a Reichsprotektor, who was hand-picked by Adolf Hitler. This means that the Ger- mans had the Czech economy, industrial and agricultural production, transport, education and culture fully under their control. The media were controlled by a Nazi infor- mation monopoly and subjected to severe censorship. In addition to the official media, illegal and media in-exile were quickly established. The Czech population was sub- jected to a programme of Germanisation. Citizens of Jew- ish origin were deprived of their civil rights, persecuted in various ways, including the confiscation of property, and, starting in the autumn of 1941, were gradually phys- ically eliminated. vakia) and 30 September 1938 (signing of the Munich Agreement). In 1918 Czechoslovakia ceased to be part of the Austro–Hungarian Empire and became an indepen- dent republic with a democratic polity based on the Con- stitution. The country consisted of Bohemia, Moravia, Silesia, Slovakia and Subcarpathian Ruthenia. The Repub- lic was characteristically multi-ethnic and there were ini- tially large social, cultural and economic differences be- tween its individual regions. The internal unity of the new state was challenged by the German ethnic minor- ity living in the border regions and wishing to become part of Germany, and the leftist labour movement, which founded the Communist Party of Czechoslovakia (hence- forward the CPC) in May 1924. In the short term, how- ever, Czechoslovakia became a mature democratic re- public with a broad spectrum of political parties, and the republic advanced in leaps and bounds in the fields of industry, trade, education and lifestyle. 4 The National Committee (1945–1990; NC) was a part of the Czechoslovak state administration with formally elected bodies. The NC was divided ac- cording to the place of operation—from the top level of the country, within regions, districts and towns to municipalities. It performed as an “extended arm” of the CPC for the economy and whole society. 2.1. Four Periods between 1918 and 1948 Any expres- sions of anti-Nazi feeling were brutally suppressed by the occupiers—punished by death, imprisonment or deten- tion in a concentration camp. Nevertheless, a number of Czechs cooperated—collaborated—with the occupy- ing regime during the Protectorate. However, the major- ity of the population continued to oppose the Germans (Emmert, 2012) and, on 5 May 1945, resistance to the occupation developed into an armed Prague uprising,2 which ended on 8 May 1945 with the defeat of Germany. The next period of the Second Republic began with the signing of the Munich Agreement and the creation of the Protectorate of Bohemia and Moravia on 30 Septem- ber 1938, lasting only until 15 March 1939. In this short period, Czechoslovakia lost extensive border territories, Slovakia, and Subcarpathian Ruthenia. Politically, the country was subverted and the democratic principles of the First Republic, organization and freedom were de- stroyed. The Party of National Unity and the National Labour Party became the only two political parties.1 Czechoslovak society perceived the Munich Agreement as a defeat which threw it into disillusion and moral crisis. These facts were also reflected in the media, with the dis- appearance of many periodicals and the intensification of censorship. Finally, the fourth period from the May 1945 to Febru- ary 1948 is called the Third Republic. The basic document establishing the direction of Czechoslovakia after World War II was the Košice Government Programme,3 which introduced the first post-war Czechoslovak Government heavily influenced by the USSR—the winner of the war. It was drawn up by the Moscow-based leadership of the CPC and proclaimed the confiscation of property belong- ing to Germans, Hungarians and Czech collaborators, the punishment of collaborators, an equal relationship be- tween Czechs and Slovaks, the establishment of National Committees4 and fundamental changes in the economic and social spheres, as well as the banning of the activi- ties of right-wing political parties. Accordingly, it not only dealt with the punishment of war criminals, traitors and collaborators, but also led to the rejection of the First Republic’s political and economic system. Some Czech The third period between 16 March 1939 and 8 May 1945 is known as the Protectorate of Bohemia and Moravia. The territory of the Protectorate included prac- tically all of the present-day Czech Republic, so Nazi Ger- Media and Communication, 2017, Volume 5, Issue 3, Pages 95–102 96 members of a 26-member government, on 20 February 1948. 2.1. Four Periods between 1918 and 1948 In the post-war atmosphere the liberalism and hu- manism which characterized the First Republic were per- ceived by the majority of society as the root of all evil, which was to blame for the hardships of war, human degradation and the total collapse of modern society. The central theme of the time was socialism, which was also complemented by nationalism and Slavonic patrio- tism. In May 1945 the Ministry of Information issued a Decree forbidding the publication of any printed mate- rial, with the exception of selected dailies issued by Na- tional Front parties. Further permission to publish was only granted to those organisations which could prove that this was in the public interest. At this stage the CPC brought society under totali- tarian control. The economy was based on the princi- ple of central planning, and companies with more than 50 employees were nationalised. Under pressure from Moscow, the CPC’s Central Committee began to take a hard line against any criticism of the Communist system or in favour of liberal thinking. There were a number of fabricated political trials, forced labour camps were cre- ated and the strict supervision of the censor covered not only the field of culture, including the press, but also the life of the Church. A counter-reaction to the situation was a wave of emigration, during which a large community of post-February 1948 emigrants founded publications in exile. May 1951 saw the launch of Radio Free Europe with regular broadcasts from Munich. In the Third Republic immediately after the war, the Czechoslovak press was made up of three ideological- political types: (1) the Communist press and periodi- cals with Communist sympathies, (2) a democratic left- wing press and (3) the non-Socialist press. Censorship was not exercised, although the press laws dating back to the First Republic provided for it. But the situation changed in autumn 1947, when the Communists began to perceive censorship as an indispensable instrument of power. In December 1947 the Ministry of Informa- tion created the Surveillance Department, which aimed to monitor objectionable texts dealing with the national economic plan, incitement against the Slavonic peoples as well as deviation from the basic line in domestic and foreign policy and matters connected to the defence of the state (Kaplan & Tomášek, 1994). 2.1. Four Periods between 1918 and 1948 The CPC and groups sympathetic to it within indi- vidual parties began to establish the National Front Ac- tion Committees (NFAC), whose aim was to “cleanse” the government and public life of all non-Communist politi- cal forces. Two days later, the People’s Militia also came into being, armed units consisting mostly of workers di- rectly controlled by the CPC. Their main aim was the defence of industrial enterprises and the intimidation of their political (anti-Socialist) opponents. The conflict continued on 24 February 1948 with an hour-long gen- eral strike in support of the CPC’s requirements, and es- calated on 25 February, when President Edvard Beneš agreed to make changes in the government as suggested by Klement Gottwald. historians view this part of Czechoslovak history in two ways: either as a short period of freedom squeezed in be- tween two dictatorships (Nazi and Communist) or as the trappings of democracy leading to the Communist snare. (Drápala, 2000) In May 1946 Czechoslovakia held its first post-war parliamentary elections. A limited number of parties par- ticipated in the election. While the CPC won in the Czech lands with more than 40% of the vote, the Democratic Party won in Slovakia with 60% of the vote. In June 1946, the pre-war leader Edvard Beneš was confirmed as Presi- dent of the Republic, and he appointed a government led byCPC chairman Klement Gottwald. InOctober1946, the Czechoslovak National Assembly approved an economic plan for the reconstruction of post-war Czechoslovakia. In 1947 the USSR began to create the Eastern bloc by arrangements such as a peace treaty concluded with Hungary and a friendship and mutual assistance treaty concluded with Poland. At this time Czechoslovakia fol- lowed Moscow’s “advice” to withdraw from the Marshall Plan and to sign a trade agreement with the USSR. This is how Communists came to power in Czechoslo- vakia—widely known as the “Communist coup”. In early May, a new Constitution was approved, and the defini- tive influence of the CPC in the country was formally con- firmed on 30 May 1948 by the parliamentary elections. All the parties stood on a single list of candidates led by the CPC and 89% of the electorate voted for them. 70% of members of the National Assembly were Communists, and the remaining members from other parties were pro- Communist. Finally, in June 1948, Klement Gottwald be- came the new President. 2.1. Four Periods between 1918 and 1948 After February 1948 the CPC began to introduce cen- sorship which would apply not only to the periodical and non-periodical press, radio, film and the early days of TV, but also to exhibitions, libraries, local records, posters, advertising leaflets, badges, labels and the like. In this way, the media became closely intertwined with the sys- tem of political power, while the limits of journalistic freedom were defined by the National Front and self- censorship (Končelík et al., 2010). The system of censor- ship policed the flow of information both at home and abroad. There was a ban on importing foreign newspa- pers and magazines, press agencies were shut down, as were cultural centres and information offices with their headquarters in the West. The state security routinely monitored correspondence sent from and to capitalist countries and so on. In the coming years, everything that did not suit the official ideas of the socialist press grad- 2.2. The Communist Takeover of 1948 A dramatic government crisis started in Prague on 13 February 1948 when personnel changes in favour of the Communists were made in the National Security Corps. The response to the ever-increasing influence of the CPC was the resignation of the non-Communist ministers, 12 97 Media and Communication, 2017, Volume 5, Issue 3, Pages 95–102 and replaced by Alexander Dubček, a reform Communist from Slovakia. ually disappeared from the newspapers, while the cen- sor intervened not only in matters of content, but also of form, and tried to influence production, print run and scope. In this way, the Communist regime gained a total information monopoly. The motto of Dubček´s new political programme6 was “Socialism with a human face”. Although the Cze- choslovak economy should still be based on state or co-operative ownership, non-Communist parties, broad public community and free elections should also be in- volved in political power. Admission to leading positions and universities should no longer be contingent upon CPC membership. The reforms also abolished censorship and an informal alliance developed between Dubček´s leadership and journalists. Indeed, the media played a crucial role in the Prague Spring (Končelík et al., 2010). Freedom of speech flourished and different associations like the scout movement were restored. The Prague Spring changes were supported throughout society and had a considerable international response. However, in the early 1950s, a more moderate cul- tural policy was promoted. The reason for this was not only the fact the Soviet ideas of socialist art had proved problematic, but it was also a response to the growing conflict within the CPC itself (Knapík, 2006). The concept of the “new course”5 opened up the possibility of liberal- isation in Czechoslovakia’s cultural sphere between 1953 and 1956. After the death of Josef Stalin in 1953 and the criticism by Nikita Khrushchev in 1956, the regime began to look more kindly on some still-rejected person- alities, less strict aesthetic criteria appeared, and even some channels for contact with the West were intro- duced. A new artistic generation began to take shape that did not feel itself as bound to the dogmatic doctrines of the late 1940s and early 1950s. While the Cold War continued as an overall frame, it was possible to hope for the promotion of liberalistic tendencies. 5 The “new course” (nový kurs) was proclaimed by the leadership of the CPC after the deaths of Stalin and Gottwald in 1953. Its result was a more be- nign approach to the middle classes; forced collectivisation in villages was temporarily halted, auxiliary technical battalions were disbanded, political trials were discontinued and the process of revising their outcomes began, etc. See http://www.edejiny.cz/obdobi-destalinizace-v-ceskoslovensku-1953- %E2%80%93-1960 6 There were two main documents determining the Prague Spring: (1) Dubček´s CPC Action programme posted on 5 April 1968, related to the politics and reform steps of CPC, and (2) the “Two thousand words” document written by Ludvík Vaculík together with leading Czech scientists, published on 27 July 1968. It was about the activation of the Czechoslovak public against the ever-evident pressure of the Soviet leadership against the reforms in the country. The manifesto was signed by hundreds of public figures and more than 120,000 citizens, which led to the persecution of many people in the period of normalization in the 1970s. 5 The “new course” (nový kurs) was proclaimed by the leadership of the CPC after the deaths of Stalin and Gottwald in 1953. Its result was a more be- nign approach to the middle classes; forced collectivisation in villages was temporarily halted, auxiliary technical battalions were disbanded, political trials were discontinued and the process of revising their outcomes began, etc. See http://www.edejiny.cz/obdobi-destalinizace-v-ceskoslovensku-1953- %E2%80%93-1960 6 There were two main documents determining the Prague Spring: (1) Dubček´s CPC Action programme posted on 5 April 1968, related to the politics 2.2. The Communist Takeover of 1948 However, the Soviet Communist Party, together with its sister parties in other Socialist countries, were afraid of this revival process, and this fear led to the occupa- tion of Czechoslovakia by Warsaw Pact military forces on 21 August 1968. From September 1968 the so-called Moscow Protocol was implemented in Czechoslovakia. The Czechoslovak population of course protested against the occupation. The most radical act against the restora- tion of the Communist dictatorship was that of Jan Palach, a student of the Charles University, who burned himself to death on 16 January 1969 at the Prague’s Wenceslas Square. In April 1969 Gustáv Husák became the first secretary of the Communist Party. Alexander Dubček and his closest associates were soon removed from public life and replaced by Soviet puppets. Every- one who participated in the Prague Spring or disagreed with the occupation was punished. Censorship was rein- stated and thousands of books and films were banned. Many Czechs and Slovaks left the country. The pressure for reforms in the CPC and other as- pects of everyday life increased after the mid-1960s. As the reform supporters became increasingly recognized in the CPC and public life, the liberal orientation in the country and in the CPC gradually won the support of the majority for a revised form of Communism. 6 There were two main documents determining the Prague Spring: (1) Dubček´s CPC Action programme posted on 5 April 1968, related to the politics and reform steps of CPC, and (2) the “Two thousand words” document written by Ludvík Vaculík together with leading Czech scientists, published on 27 July 1968. It was about the activation of the Czechoslovak public against the ever-evident pressure of the Soviet leadership against the reforms in the country. The manifesto was signed by hundreds of public figures and more than 120,000 citizens, which led to the persecution of many people in the period of normalization in the 1970s. Media and Communication, 2017, Volume 5, Issue 3, Pages 95–102 March 1946. On that occasion it was also agreed that pro- fessional matters should in future fall under the remit of the Revolutionary Trade Union Movement.9 The CJU rep- resented a community of altogether about 800 journal- ists (Cebe, 2012). In 1947, following the Act on the Status of Editors and Journalists’ Unions, two journalists’ unions were formed, the Czech union (formerly CJU) and the Slo- vak union, while above them an umbrella organization was established, the Central Union of Czechoslovak Jour- nalists (CUCSJ). In May 1947 the government passed the Act on the Status of Editors and Journalists’ Unions.10 In fact, the CJU and CUCSJ were more a tool of the regime than a provider of benefits to journalists. 2.4. The Velvet Revolution of 1989 The resistance to the Communist regime began to in- crease in the late 1980s, in connection with Mikhail Gor- bachev’s rise to power in the USSR and policy of per- estroika and glasnost. The CPC claimed to agree with this Soviet reform policy, but this was rather lip service. Demonstrations of disagreement with the totalitarian regime proliferated, but were also severely suppressed by the security forces. The most important actions took place on (1) 28 Oc- tober 1988, the 70th anniversary of the founding of the Czechoslovak Republic, (2) 16 January 1989, the 20th an- niversary of Jan Palach’s suicide, and (3) 29 June 1989, the publication of “A Few Sentences”, a call by dissident leaders for the release of political prisoners, open and free discussion, including thorny historical issues, and the end of censorship.7 The crucial event which precip- itated the Velvet Revolution, happened on 17 Novem- ber 1989 in the centre of Prague, when security po- lice blocked and violently broke up a student procession to commemorate the 50th anniversary of the Interna- tional Day of Students. The fundamental response to this armed intervention was the founding of the Civic Forum, headed by Václav Havel, to begin a dialogue with the Communist leaders. Massive demonstrations took place throughout Czechoslovakia in support of the Civic Forum, and university students went on strike. On this basis, the practice of journalism was tied to compulsory membership of the CJU, which had dire consequences for many journalists in the post-February cleansing. After February 1948, there were dynamic po- litical and social changes. 9 The Revolutionary Trade Union Movement Act was passed on 16 May 1946. In 1947, the decision was taken that the CJU should also fulfil the function of a trade union. Divisional membership was voluntary; however, the CJU pointed out to its members that the CJU would not represent them in profes- sional issues if they were not members of the Revolutionary Trade Union Movement. From 1 January 1948, the Agreement on the Entry of Journalists into the Revolutionary Trade Union Movement came into force and after that date, the majority of journalists joined a trade union. 10 Act No. 101/1974 Coll. It stipulated, among other things, who could become an editor, laid down the rights and responsibilities of editors, including the right of the Union to take disciplinary action against its members. Even in the CJU action com- mittees were founded and only those journalists who were willing to work with the newly-established regime remained members of the Union. The others were ex- cluded from their professional organization and thereby lost all chances of working in the media. The NFAC forced more than one hundred experienced journalists to leave and consequently affected the Czechoslovak media for decades. (Cebe, 2012) The new Communist Constitution of May 1948 had two main consequences for journalists and their union. Firstly, although the new Constitution guaranteed free- dom of expression and prohibited censorship, the CPC consistently monitored all media production, making in- dependent media in practice impossible. The journalist became a mere instrument lacking any autonomy, pro- viding information on only politically-approved interpre- tations of events. The economy was only to be portrayed as flourishing, accompanied by the people’s efforts to build it up. In the same way, both sports and scientific achievements were to be ascribed to the merits of the new Communist regime. The most significant instrument for controlling the media was, of course, the staffing pol- icy, meaning that journalists were in constant fear of los- ing their jobs. (Končelík et al., 2010) On 24 November 1989, the CPC’s secretary gen- eral Ladislav Adamec and his entire central commit- tee withdrew. The name of the state was changed to Czecho-Slovak Federative Republic and the head of the Federal assembly became Prague Spring leader Alexan- der Dubček. On 29 December 1989 Václav Havel was elected as the new President. During 1990 Czechoslo- vakia became a federal republic and the transformation of Czechoslovak society and its economic, social and political life from Communist totalitarianism to liberal democracy could begin after long 40 years. For more, see http://old.ustrcr.cz/en/milestones in recent czech history 1938 1989 8 The succession of changing union formations from 1946 to 1990: 1946–1947: Czech Journalists Union (CJU); 1947–1948: Czech Journalists Union (CJU), Slovak Journalists Union (SJU), Central Union of Czechoslovak Journalists (CUCSJ); 1948–1968: National Union of Czechoslovak Journalists (UCSJ), Union of Slovak Journalists (USJ); 1968–1972: Unions of Czech (UCJ) and Slovak (USJ) Journalists, Headquarters; 1972–1990: Czech Union of Journalists (CUJ), Slovak Union of Journalists (SUJ), National Czechoslovak Union of Journalists (NCSUJ), 1990: The Syndicate of Journalists of the Czech Republic. (Ševčíková, 2015). Media and Communication, 2017, Volume 5, Issue 3, Pages 95–102 7 For more, see http://old.ustrcr.cz/en/milestones-in-recent-czech-history-1938-1989 2.3. The Prague Spring of 1968 In 1968 the world was in turmoil, with the war in Viet- nam, the civil rights movement in the USA and student unrest throughout Europe, especially in France. It was a unique year in post-war history, as 1848 had been a hun- dred years earlier. And the “Prague Spring” became its focal point. The Prague Spring was consequently brought to a drastic end and replaced by a sweeping political pro- cess called “Normalization” lasting for 20 years. Officially Czechoslovakia was a faithful member of the Eastern bloc, and the CPC bureaucratic apparatus was severely controlling all aspects of life. Yet under the surface there existed an underground culture, intellectual dis- sent, samizdat and an exile literature. The CPC bureau- cratic apparatus could no longer effectively control all areas of society. The most significant manifestation of the anti-Communist resistance was the publication of Charter 77 with its civil movement established in January 1977, led by writer and later Czechoslovak President Vá- clav Havel and the university professor Jan Patočka. For a short period, from January to August 1968, there was a huge development among politicians, in- tellectuals, students and ordinary public life spheres in Czechoslovakia. The social and political criticism of the system had been gradual and fundamental and was re- flected in economy, central management, culture and media. At the end of 1967 a crisis in the CPC itself deepened and the party split into two groups: (1) con- servatives, represented by Antonín Novotný, Czechoslo- vak president and CPC first secretary, and (2) reform Communists, who wanted to democratize the old poli- tics. In January 1968 the big break happened, when An- tonín Novotný was removed as first secretary of the CPC Media and Communication, 2017, Volume 5, Issue 3, Pages 95–102 98 3. The Journalists and Their Union 1946–1990 The constitutional statutes and principal reso- lutions were approved unanimously, likewise the leader- ship elected, with Archibald Kenyon of the UK continuing as President and Vice Presidents coming from the USA, USSR, France and Denmark. Hronek was unanimously elected to the combined office of Secretary General and Treasurer. But the site of the headquarters had to be put to a vote, the majority supporting Prague, while a minor- ity led by British and Americans voted for London. The new organizational model continued until Au- gust 1968. Both the Headquarters and UCJ were under the control of the Commission for the Preparation of the II Congress of the UCJ, which soon overturned all the de- cisions of the previous leadership and set up a cleansing commission on the basis of which 479 journalists were excluded from the Union and therefore also from their profession between September 1968 to March 1970, fol- lowed in 1972 by another 800 journalists. As of 1971 the whole union had a complete overhaul in the line of normalization. In May 1972 a congress was convened at which the Czech Union of Journalists (CUJ, 1972–1990), the Slovak Union of Journalists (SUJ, 1972– 1990) and the National Czechoslovak Union of Journalists (NCSUJ, 1972–1990) were established. In less than a year after the Prague congress, by early 1948, the Cold War had broken out and a wave of po- litical system changes in Central and Eastern Europe led to a division of Europe into East and West, separated by the “iron curtain”. The IOJ was part of this battleground, becoming embroiled in Cold-War politics (Nordenstreng et al., 2016). The post-war unity of professional journal- ists was quickly replaced by distrust and antagonism be- tween the capitalist West and the socialist East, exac- erbated by political mobilization in both camps. Efforts by the Czech Secretary General Hronek and the British President Kenyon to hold the IOJ together were over- ruled by orders from Cold-War strategists in London and Washington, on the one hand, and the Soviet Communist Party hardliners in Moscow, on the other (Nordenstreng, in press). The big breakup of the NCSUJ took place at the turn of 1989 and 1990, due to the political events of the Vel- vet Revolution and the end of the Communist regime. Im- mediately after 17 November 1989, a new management of the CUJ, including the Bureau and its secretariat, was elected. 3. The Journalists and Their Union 1946–1990 After World War II, there were naturally significant changes in journalism as a profession. Before 1946 jour- nalists were represented by the protectorate National Union of Journalists (NUJ; see more on this in Cebe, 2017, in this issue). The Czech Journalists’ Union (hencefor- ward the CJU)8 came into being at a general meeting in Secondly, a new, integrated Union of Czechoslovak Journalists (UCSJ; 1948–1972), headquartered in Prague, was founded and, within it, the National Union of Slovak Journalists, headquartered in Bratislava. The state, rather than the UCSJ, made the decisions regarding its focus, organizational structure or the exercise of the journal- Media and Communication, 2017, Volume 5, Issue 3, Pages 95–102 99 just after the post-war parliamentary elections, which brought the Communists to power as the leading polit- ical force (for details of the first IOJ congresses, see Nor- denstreng & Kubka, 1988). ism as a profession and membership, meaning that the organization toed the Communist party line throughout its existence. From 1948, the CJU and UCSJ also served as the gate- way to international organizations, above all to the IOJ. In July 1968, following the events of the Prague Spring, the UCSJ held a landmark congress. A new statute and sev- eral resolutions were adopted in support of a new model of Socialism (“with a human face”), democracy and free- dom of the press, speech and information. The former UCSJ was abolished and new Unions of Czech (UCJ, 1968– 1972) and Slovak (USJ, 1968–1972) Journalists, includ- ing an umbrella organization (Headquarters, 1968–1972) were formed. The provisional constitution was finalised and ap- proved at the second IOJ congress held in Prague in June 1947 and attended by 208 delegates from 21 countries, the UN and UNESCO. The CJU, led by Jiří Hronek, was re- sponsible for organizing the congress and its patron was President Edvard Beneš, who spoke at the opening cere- mony, as did Jan Masaryk (Czechoslovak Minister of For- eign Affairs) and Klement Gottwald (Prime Minister and CPC Chairman). The same positive atmosphere prevailed at the sec- ond congress in Prague as one year earlier in Copen- hagen, although some tension about the impending di- vision of the world could already be sensed. While the host union was in the middle of structural and politi- cal changes, the congress went smoothly in impressive settings. 3. The Journalists and Their Union 1946–1990 At the same time, the new Bureau of the CUJ convened a general meeting on 16 December 1989, at which it was decided to create a new journalist organiza- tion, with the form of a syndicate. The old Union (CUJ) was dissolved at the extraordinary congress of the CUJ on 6 January 1990, and the successor organization be- came the Syndicate of Journalists of the Czech Republic, as a voluntary, independent, non-political, professionally united union of Czech and Moravian journalists. (Prouza, 1990) The new Syndicate refused to join the IOJ and in- stead became member of the IFJ. The IOJ headquarters in Prague chose the Soviet side of the political divide as swiftly as the Czechoslovak Union was moved to the Socialist camp. These two pro- cesses were parallel and obviously reflections of a com- mon overall shift in global relations of power. 11 The Czechoslovak delegation to the Congress was represented by CJU Chairman Otakar Wünsch, Jiří Hronek, K. F. Zieris (CJU secretary) and Emil Štefan (USJ). delegation to the Congress was represented by CJU Chairman Otakar Wünsch, Jiří Hronek, K. F. Zieris (CJU secretary) and Emil 4.1. Founding and Split In 1948–1949 the IOJ lost its West European and North American members, except for left-leaning groups of journalists there (mainly in France and Finland). The third IOJ congress in Helsinki in 1950 confirmed the change by changing the statutes in line with the Commu- nist approach, the bulk of the membership in the Social- ist countries of Central–Eastern Europe and newly inde- The IOJ was founded at the International Congress of Journalists in June 1946 in Copenhagen, the capital of Denmark, which had only recently been liberated from Nazi occupation. The congress was attended by 165 dele- gates11 from 21 countries. The post-war atmosphere was one of joy and optimism. In Czechoslovakia, this came 100 Media and Communication, 2017, Volume 5, Issue 3, Pages 95–102 4.3. The Velvet Revolution pendent countries of the Third World, including Commu- nist China. The Cold War world order began to crumble in late 1989, when first the Berlin Wall fell and then Prague was shaken by the Velvet Revolution, followed by the fall of Communism in Eastern Europe. Global geopolitics was suddenly changed and the IOJ was challenged: its head- quarters and operations in Prague came under sharp at- tack from the rising political forces in Czechoslovakia, and its local member Union—the legal base for its seat in Prague—was closed down and replaced by a new Syn- dicate which did not want to affiliate with the IOJ but joined the IFJ. Moreover, other strong member unions in former Socialist countries, notably the USSR, began to lose political and material ground. The IOJ’s financial re- sources were rapidly dwindling and activities in training, publication, etc. were gradually discontinued. The IOJ had become one of the “democratic inter- national organizations” closely associated with the USSR. The Western journalists’ associations established a new International Federation of Journalists (IFJ) in Brussels in 1952—as part of the “Free West” countering the “Com- munist East”. The Cold War politics led to the loss of the IOJ’s affiliation with the UN and UNESCO, but it gained new members in the developing countries of Africa, Asia and Latin America. In 1966, its membership amounted to 130,000 journalists in 108 countries, while the IFJ had 55,000 members in 31 countries. 4.2. The Prague Spring The IOJ continued to grow despite turmoil in its political environment caused by the Prague Spring in 1968 and its aftermath. The IOJ had its premises in the building of the Czechoslovak Union UCSJ, and in August 1968 the whole building was occupied by Warsaw pact forces. However, the IOJ was soon allowed to return to its premises and continued more or less as before, while the reformist Czechoslovak Union was effectively replaced by a new CJU (see above). Nevertheless, in 1991 the IOJ held its 11th congress in Harare in fairly good shape, but by its 12th congress in Amman in 1995 it was crippled by shrinking finances and quarrelling leadership. After this, member unions one af- ter another decided to join the IFJ, while most of them also remained nominal members of the IOJ. By the end of the 1990s the IOJ had in fact disappeared from the scene, whereas the IFJ had grown into an organization also representing the bulk of the earlier IOJ membership. Finally, in 2016, the last two IOJ presidents pronounced the IOJ effectively dead (Nordenstreng, in press). Unlike in 1948, the IOJ did not dramatically follow the system change implemented in the national union. Actu- ally the IOJ activities, including the monthly publication The Democratic Journalist, had since the 1950s remained quite true to the “Moscow line”, with no symptoms of the Prague Spring. By the 1970s the IOJ was firmly con- solidated on the basis established in the late 1940s, now increasingly dominated by an “anti-imperialist” orienta- tion reflecting the concerns of the developing countries and meeting the Soviet interests. The changing climate of international relations was also instrumental to rein- stating IOJ’s affiliation with the UN and UNESCO. 5. Conclusion In summary: (1) the system change in 1948 from lib- eral democracy to Communism in Czechoslovakia imme- diately involved journalists with their national associa- tion, and the IOJ followed suit driven by the same devel- opments among all the Soviet-dominated Eastern Euro- pean member associations, while the Western members withdrew without an organizational fight; (2) the system instability of 1968 with an attempt to liberalize Commu- nism hit the national association hard but did not extend to the IOJ, which continued largely unchanged; (3) the system change in 1989 caused an immediate upheaval in the national association and the gradual disintegration of the IOJ, again as a result of changes throughout the Soviet block—a reverse process of 1948. Financial support for more and more activities around the world came not only from the nominal mem- bership fees but from public “solidarity lotteries” in East European Socialist countries. Moreover, a peculiar source for financing was invented by new Secretary Gen- eral Jiří Kubka together with the Hungarian Treasurer Norbert Siklósi: the IOJ was permitted, with the blessing of the Communist Party, to establish commercial com- panies in translation and conference services, publish- ing, etc. This privilege made the IOJ quite rich, facili- tating a large secretariat with a publishing house, train- ing schools in European Socialist counties, regional cen- tres on all developing continents and large conferences around the world. The story of Czechoslovak journalist organizations and the IOJ adds further evidence to the overall lesson presented in Nordenstreng et al. (2016, p. 180): inter- national journalists’ associations are invariably bound by their political environments and to believe that journal- ists and their associations can ever be completely apolit- ical is a naïve illusion. However, the international move- ment is not deterministically driven by politics alone; it is also driven by professional interests with more or less autonomy. The heyday of the IOJ was in the late 1980s, when its membership reached 300,000. By this time its polit- ical line had become much more open and broad, par- ticularly by its active engagement in East–West détente from 1973 on, leading gradually to contacts and even cooperation with the IFJ in the 1980s (Nordenstreng et al., 2016). 5. Conclusion This article has mostly concerned the political history of Czechoslovakia, which already suggests another gen- eral conclusion: in order to understand the relationship Media and Communication, 2017, Volume 5, Issue 3, Pages 95–102 101 Kaplan, K., & Tomášek, D. (1994). O cenzuře v Československu v letech 1945–1956: studie [A study of censorship in Czechoslovakia, 1945–1956]. Prague: Institute of Contemporary History of the Academy of Sciences of the Czech Republic. which the media and journalists, including their associa- tions, have to political system changes, it is vital to have a profound knowledge of the history of the country in question. All too often are changes in media and journal- ists studied with a superficial understanding of the his- torical context. Knapík, J. (2006). V zajetí moci: kulturní politika, její systém a aktéři 1948–1956 [In capture of power: Cultural policy, its system and actors 1948–1956]. Prague: Libri. References Cebe, J. (2012). Novinářská organizace v letech 1945– 1948 [Journalists organization 1945–1948] (Unpub- lished Doctoral dissertation). Charles University, Prague, Czech Republic. Nordenstreng, K., & Kubka, J. (1988). Useful recollec- tions. Excursion into the history of the Interna- tional Movement of Journalists. Part II. Prague: IOJ. Retrieved from https://books.google.fi/books?id=Bq 3SAwAAQBAJ&redir_esc=y Cebe, J. (2017). Cleansing among Czech journalists after World War II and a comparison with the situation in France and the Netherlands. Media and Communica- tion, 5(3), 70–78. Prouza, J. (1990). Mimořádný sjezd Českého svazu nov- inářů, Praha 6. ledna 1990, Palác kultury ve 13 hod. Stenografický záznam [Extraordinary Congress of the CUJ, Prague, 6 January 1990, Palace of Culture, 1 P.M., stenographic record] Praha: ČSN. Drápala, M. (2000). Na ztracené vartě Západu: antolo- gie české nesocialistické publicistiky z let 1945–1948 [The lost guard of the West: An anthology of Czech non-Socialist journalism from 1945–1948]. Prague: Prostor. Ševčíková, M. (2015). Mezinárodní organizace novinářů (1946–1995) [International Organization of Jour- nalists (1946–1995)] (Unpublished Doctoral Thesis). Charles University, Prague, Czech Republic. Emmert, F. (2012). Průvodce českými dějinami 20. století [A guide to Czech history of the 20th century]. Brno: Clio. Acknowledgments The authors wish to thank Isabel Stainsby and Virginia Mattila for their assistance in checking the English of this article. Končelík, J., Večeřa, P., & Orság, P. (2010). Dějiny českých médií 20. století [A history of the Czech media of the 20th century]. Prague: Portál. Nordenstreng, K. (in press). The rise and fall of the International Organization of Journalists based in Czechoslovakia. Useful recollections part III. Prague: Karolinum. The authors declare no conflict of interests. The authors declare no conflict of interests. Nordenstreng, K., Björk, U. J., Beyersdorf, F., Høyer, S., & Lauk, E. (2016). A history of the International Move- ment of Journalists: Professionalism versus politics. Houndmills: Palgrave Macmillan. Media and Communication, 2017, Volume 5, Issue 3, Pages 95–102 About the Authors Markéta Ševčíková is an independent researcher in the field of media history from Prague, Czech Re- public. Her research focuses mainly on Czechoslovak journalists and the International Organization of Journalists. She studied at the Charles University, Faculty of Humanities and Faculty of Social Sci- ences. She has worked as a media analyst, freelance editor and a PR specialist in commercial and non-governmental organizations. Kaarle Nordenstreng is Professor Emeritus of Journalism and Mass Communication at the Univer- sity of Tampere, Finland. He worked as a freelance journalist starting in the mid-1950s and served as head of research in the Finnish Broadcasting Company in the late 1960s. His research has focused on theories of communication and global media. He has been Vice President of the International As- sociation for Mass Communication Research (IAMCR, 1972–1988) and President of the International Organization of Journalists (IOJ, 1976–1990). For more, see http://www.uta.fi/cmt/en/contact/staff/ kaarlenordenstreng/index.html 102 Media and Communication, 2017, Volume 5, Issue 3, Pages 95–102
https://openalex.org/W2168401290	https://zenodo.org/record/1281136/files/article.pdf	English	null	Frequency Tolerances and Spurious Emission Limitations	IRE transactions on communications systems	1,981	public-domain	6,931	ROBERT T. WATSON, MEMBER, IEEE. AND WILLIAM A. LUTHER ROBERT T. WATSON, MEMBER, IEEE. AND WILLIAM A. LUTHER inate any unnecessary cost increase of a transmitter attribut- able to the WARC-79 decisions. As different implementation dates apply to both Tables, even to the point of having subsets of dates in Table footnotes, those discussions will be given at more descriptive places in this article. Absrracr-Appendices 7 and 8 of the WARC-79 Final Acts contain, respectively; the Table of Transmitter Frequency Tolerances and the Table of Maximum Permitted Spurious Emission Power Levels. Decisions taken at that Conference on these two new Tables will eventually touch transmitter manufacturers and users everywhere. Frequency tolerances in most bands are tightened for new equipment effective January 2, 1985. TV broadcast for system M (NTSC) from 29.7 to 2450 MHz and broadcasting in the 535 to 1606.5-kHz bands remain the same. Earth and space‘services were introduced in the Table for the first time with impact from 4 MHz on up to 40 GHz after 1985. Relatively tight frequency tolerances are those for HF broad- casting, single sideband (SSB), and pulse modulation above 10 GHz. Peak envelope power rather than mean power is shown generally for SSB transmitters limits. The frequency tolerance limits, again, did not reach beyond 40 GHz. New spurious emission limits come into effect on that same date for flew transmitters operating between 235 MHz and 17.7 .GHz; although radiodetermination and emergency- type transmitters are excluded. There is also a special exception for spurious emissions of transmitters operating above 235 MHz where transmitters feed a common antenna or where an antenna farm exists [see Note 10 of Appendix 81, Spurious emissions have been sig- nificantly redefined. It is suggested those concerned with such limits be familiar with the new definition contained in RR 139 of Article 1 of the Final Acts. ’ p p From the sequence of events leading to final votes on Ap- pendices 7 and 8, the efforts of the International Radio Con- sultative Committee (CCIR from French translation) with re- spect to these tolerances and limits could not be overstated. Special discussion is needed, and is included below, on impor- tance of the CCIR to these technical discussions. CCIR Rec- ommendations on these Tables were essentially adopted by the WARC-79. JNTRODUCTION To understand how the frequency tolerances and spurious emission limits evolved ‘from the WARC, it is important to understand the CCIR process. In addition to a continuing review of these values over the intervening years between Administrative Conferences, both in the countries of the ITU Member Administrations and at various CCIR meetings, a very extensive effort took place in connection with the Special Preparatory Meeting (SPM) of the CCIR Study Groups. T WO SUBJECTS considered by the World Administrative Radio Conference (WARC-79), with broad impact on the world’s telecommunications community, are frequency tolerances and spurious emission limits. Values for both, as fixed by the Conference, eventually will apply to every trans- mitter used by the International Telecommunication Union (ITU) member nations ratifying their Final Acts, and will affect all transmitters manufactured in the world. T Pursuant to ITU Administrative Council Resolution 804 and commensurate CCIR Resolution 69, a special joint meet- ing of CCIR Study Groups-known as the Special Prepara- tory Meeting-was held in Geneva from October 23, to Novem- ber 17, 1978, about 10 months prior to commencement of WARC-79. The purpose of the meeting was to prepare tech- nical bases for the anticipated World Administrative Radio Conference. The terms of reference of the Special Preparatory Meeting, in accordance with the Resolution 804, were as follows: A!though the Table of Transmitter Frequency Tolerances, and the Table of Maximum Permitted Spurious Emission Power Levels are found in Appendices 7 and 8, of the Final Acts of WARC-79, their authority is actually drawn from Article 5 (Nos. 304 and 306). Article 5 deals with general technical characteristics of stations in a succinct manner that requires conformance to the two Tables. New and more strict values of these tolerances and limits become effective after 1985. They do not come into effect along with most provisions of the Final Acts. Delegates to the Conference recognized a potential economic impact with any new Table value, and consciously tried to minimize or elim- “To prepare a Report providing technical bases for the WARC-I9 and for the use of Administrations in prepar- ing their proposals to that Conference. ROBERT T. WATSON, MEMBER, IEEE. AND WILLIAM A. LUTHER There is some doubt that an Administrative Con- ference, such as the WARC, could have appropriately con- sidered, withm the scheduled time, all of the technical and economic aspects for the numerous individual standards rep- resented by each of the tolerance and limit numbers in these two Tables. W. ‘:,A. Luther is with the Engineering Division, Field Operation Bureau, Federal Communications Commission, Washington, DC 20554. Manuscript received April 8, 1981; revised April 29, 1981. The views expressed herein are those of the authors and do not necessarily reflect either the views of the National Telecommunications and Infor- mation Administration or of the Federal Communications Comission. R. T, Watson is with the U.S. Department of Commerce, National Telecommunications and Information Administration, Annapolis, MD 21401. W. ‘:,A. Luther is with the Engineering Division, Field Operation Bureau, Federal Communications Commission, Washington, DC 20554. IEEE TRANSACTIONS ON COMMUNICATIONS, VOL. COM-29, NO. 8, AUGUST 1981 1130 R. T, Watson is with the U.S. Department of Commerce, National Telecommunications and Information Administration, Annapolis, MD 21401. ‘ A i i Manuscript received April 8, 1981; revised April 29, 1981. The views expressed herein are those of the authors and do not necessarily reflect either the views of the National Telecommunications and Infor- mation Administration or of the Federal Communications Comission. N i l IEEE TRANSACTIONS ON COMMUNICATIONS, VOL. COM-29, NO. 8, AUGUST 1981 IEEE TRANSACTIONS ON COMMUNICATIONS, VOL. COM-29, NO. 8, AUGUST 1981 SPM DISCUSSIONS-FREQUENCY TOLERANCES Over a hundred delegates were involved in discussions on the relatively large number of frequency tolerance standards. Dr. Kaji of Japan was chosen as the Chairman of the overall Technical Characteristics Committee, including frequency tolerances, and Mr. George of the Federal Republic ofGermany was selected to chair specifically the Frequency Tolerances subcommittee. A total of 11 official documents from Adminis- trations were negotiated in the meeting, involving 87 Adminis- trations. g g) Safety-related functions of frequency tolerances. g g) Safety-related functions of frequency toleranc WATSON AND LUTHER: FREQUENCY TOLERANCES AND SPURIOUS EMISSION LIMITATIONS quebcy tolerances. In the case of some Administrations, not only %e& there multiple meetings prior to the SPM seeking tolerance contributions and agreements among the many interested agencies, but advice was solicited from the indus- trial community, as well as from the worldwide professional society, IEEE. The IEEE consideration also provided an op- portunity for the academic community to participate. Taken together, these inputs were utilized not only for the SPM and the WARC, but are also expected to be for post-WARC imple- mentation. Jished agenda of the WARC-79, but economics could not be ignored in some cases. As far as frequency tolerances and spur- ious emission limits were concerned, the full four meeting weeks of the SPM were required for all of the arguments. Subcommittees at SPM met practically every day on these sub jects, with the offdays devoted to evaluating results of pre- vious discussions and preparing for the next days’ sessions. By comparison, the WARC-79 deliberations on these two subjects took just a few days using the SPM Conclusions [l] as a pri- mary reference for making decisions. Of course, from aboi& the mid-point of the WARC-79, meetings were being held seven days a week, often taking up to 15 h each day, some- times longer. Time was precious at WARC-79, meaning that it was important to use every available expedient to maximum advantage. The SPM Conclusions were one expedient used to reach rapid decision. In the case of frequency tolerances, only two of the several hundred values approved were modi- fied from that determined by the SPM. No changes at all were made by the WARC-79 to Table values of spurious emis- sion limits determined by the SPM, although the covered spectrum range was extended from 960 MHz to 17.7 GHz, with limits applying only to certain services. Frequency Tolerances Approach consciously considered the following: In deliberating values of frequency tolerance, the SPM y g a) Appendix 3 of the existing Radio Regulations. pp g g b) CCIR Report 181-2 concerning overall frequency toler- ances. c) Specific CCIR Study Group 9 conclusions concerning problems associated with radio-relay frequency-controlling systems (using a chain of repeaters where final repeater fre- quency variation perceived is an aggregate from all the indi- vidual transmitters in a system). y ) d) Individual Administrations’ proposals associated with sometime special concerns, relating to national criteria for particular tolerances. pp y g y Most Administrations attending the SPM realized the im- portance attached to SPM Conclusions in the two highly tech- nical areas of frequency tolerance and spurious limits. Conse- quently, most prepared their proposals on these subjects as they would normally for an Administrative-type conference. This would explain why only very limited change occurred at the WARC-79. It must be noted, however, that the SPM con- sidered no implementation dates, leaving that matter strictly to the WARC. p e) Ultimate values needed for operational or technical reasons. f) Economic impact of any change in value from that existing. JNTRODUCTION This Report is to be based on the texts approved by the XIVth Plenary Assembly of the CCIR as well as on new contributions submitted by Administrations and other participants in the work of the CCIR; the Report should be presented in a form consistent with the various items of the Agenda of the WARC-19.” The activities of the SPM were, for the most part, confined to technical matters which would be relevant to the estab- U.S. Government work not protected by U.S. copyright 1131 WATSON AND LUTHER: FREQUENCY TOLERANCES AND SPURIOUS EMISSION LIMITATIONS Scope of Rationale and Quantities of Standards Scope of Rationale and Quantities of Standards In the approach taken to frequency tolerances, it was real- ized that a great many numbers would need to be considered. The services, power levels, and types of modulation proposed by one Administration alone comprised close to 2000 indi- vidual cases of frequency tolerance. Furthermore, in order to gain worldwide accaptance, each value needed an adequate technical foundation. To consider the difficulty usually ex- perienced reaching firm agreement on even one standard, corresponding to one tolerance number in this negotiation, suggests the enormity of the challenge. The SPM considered proposals on a spectrum basis, band by band. The frequency tolerance for each station function, power level, and operational state of readiness was taken up and discussed with a corresponding technical, sometimes other, rationale. Consideration was given to the spectral en- vironment of the particular standard under discussion. Statio receiving particular emphasis included Earth and space, radio- determination, land mobile, maritime mobile, broadcast, eme gency, and radionavigation. Within the station environment, functional characteristics were discussed for each appropriate standard including Doppler shift, filter slope factors, channel width requirements, multiplexed carriers, crystals, and oscilla- tors. Frequencydetermining elements of a transmitter received particular attention, particularly with respect to temperature variation, frequency/temperature inversion points and high slopes, sealed ovens, temperaturecompensated oscillators, aging, cumulative frequency errors in heterodyne systems, statistical considerations, total translational error, and unin- gg y g Special attention was given, not only to the primary tech- nical aspects of spectrum utilization, but also to consideration of the parallel CCIR concerns of operational questions and future consequences as they might affect SPM recommenda- tions to the WARC. Such consideration was to a large extent directed toward the establishment, development, and improve ment of telecommunications in new or developing countries. This approach was utilized not only to satisfy the primary stated purpose of the SPM, but also to facilitate ease of under- standing by those WARC delegates not fully familiar with the disciplines discussed. p For these and other considerations that were carefully pre- sented in the individual nation input papers to the SPM, a concentrated broad-scope effort backed up proposals for fre- telligibility due to frequency errors. All in all, technical corn- ponent , equipment, system characteristics, and technical feasibility were considered for each standard. Operational and economic variation of the technical characteristics then was discussed as appropriate. aids nor for the amateur services. Scope of Rationale and Quantities of Standards It was deemed that these matters could best be handled by national regulations, if appropriate. Recognizing the continuing development of equipment and systems, and their present tentative nature, no frequency toler- ances were established for services operating above 40 GHz. The final, agreed Frequency Tolerance Table may be found in the SPM Report, Chapter 8, entitled “Technical Character- istics of Equipment and Emissions.” This chapter also contains introductory comments for the benefit of the WARC dele- gates. The latter is presented here to better summarize the deliberations. No presently applicable values of frequency tolerance change until at least 1985, if at all. These present values are all contained in the first tolerance column of the Table contained in Appendix 7 of the WARC-79 Final Acts [2] . Tolerances that do change, or become effective on January 2,1985, are only for newly installed transmitters. Transmitters installed through January 1, 1985 will retain present tolerances until 1990 or later, of course, if no change is indicated. “In proposing frequency tolerance values for trans- mitters, the SPM has taken account of: (1) the need for efficient frequency spectrum utilization, (2) h i l i f h i (2) the operational requirements of the various com- munication systems, Band: 4 to 29.7 MHz Few other WARC agreements were reached in as short a time. The most influential WARC input document on fre- quency tolerances was, as indicated, the SPM Conclusions. Other WARC input documents on frequency tolerances in- cluded the very few additions or exceptions submitted by the participating Administrations. It was emphasized in WARC Committee that SPM Conclusions reflected future needs and, therefore, did not always correspond to present equipment specifications. Attention was drawn to the fact that in a few cases, unsupportably loose tolerance values proposed were such that a corresponding actual frequency instability would cause an emission to exceed the limits established for its channel. This was cited as leading to inefficient use of the radio frequency spectrum and a good reason to adopt the well- considered SPM values. New separate listings were made under Fixed Sta- tions for 1) single-sideband and independent-side- band emissions (recognizing the predominant modu- lation types), 2) class FIB emissions, and 3) other classes of emission. The Mobile Ship Station Class A1 emission was changed from 200 ppm to 50 ppm. Space and Earth station values are new to the band after 1985. Band: 29.7 to 100 MHz For Fixed Stations, while power levels remain the same at 200 W for the immediate future, these are reduced to 50-W levels in 1985. Space and Earth stations, after 1985, are new to the band. Band: 100 to 470 MHz Land Stations classified as Base Stations have been split into the subbands of 100-235 MHz, 235- 401 MHz, and 401 470 MHz after 1985. The same subbands are found after 1985 for Land-Mobile Stations. Also new in the band are Earth and space stations. Frequency subdivisions were introduced into the Table where necessary for some services, to clarify the presentation of tolerances. The value quoted for a particular category was normally for the most important service in that category. Other important types of spectrum usage in the same category required special consideration that was’contained in explana- tory footnotes. In order to distinguish clearly between in-force footnotes carried forward, and the newly proposed ones, the latter were given numerical designations by the SPM to enable a clearer understanding of change by WARC delegates. Band: 470 to 2450 MHz Space and Earth station tolerances after 1985 are the only changes. Band: 2450 MHz to 10 500 GHz Again, post 1985, Earth and space station tolerances take effect. WARC BAND-BY -BAND RESULTS-FREQUENCY TOLERANCES (3) the technical feasibility of achieving the standards laid down against a background of environmental and economic constraints, Analysis of significant decisions in the Frequency Tolerance Table follows. This is done on a band basis, as the Final Acts are arranged. (4) the basic differences between fixed and mobile sta- tions, and especially hand-held equipment, necessitating in some cases a relaxed value of tolerance for the mobile stations, Band: 535 to 1606.5 kHz (1605 kHz in Region 2) The upper limit had been 1605 for all Regions. Some Administrations wanted to raise the 10-Hz tolerance for broadcast stations to 20 Hz; it had been 10 Hz for most of the world with a 20-Hz Note for the North American Regional Broadcasting Agreement (NARBA) countries. The NARBA ex- ception was maintained. Band: 535 to 1606.5 kHz (1605 kHz in Region 2) The upper limit had been 1605 for all Regions. Some Administrations wanted to raise the 10-Hz tolerance for broadcast stations to 20 Hz; it had been 10 Hz for most of the world with a 20-Hz Note for the North American Regional Broadcasting Agreement (NARBA) countries. The NARBA ex- ception was maintained. (5) the accumulation of frequency errors of individual frequency sources in the case of some multi-hop radio relay systems necessitating some relaxation in the over- all tolerance limits. (5) the accumulation of frequency errors of individual frequency sources in the case of some multi-hop radio relay systems necessitating some relaxation in the over- all tolerance limits. Due consideration was given also to existing CCIR texts, where appropriate.” NEW TABLE FOOTNOTES Out of the many frequency tolerances decided, it isbelieve that the Table has no unnecessarily tight tolerance areas. Gen- erally, the tight tolerances were required by operational or interference considerations. It is recognized that some of these may take special attention to meet. Based upon SPM Conclusions, the WARC introduced 23 new footnotes to the Table of Frequency Tolerances. This was in addition to the 13 qualifying notations in the current Radio Regulations. The new ones are listed in Appendix 7 as footnotes 1, 4, 5, 7, 8 , 10, 12, 13, 14, 15, 16, 19, 20, 21, 22,23,24,25,26,28,29,32, and 36. Based upon SPM Conclusions, the WARC introduced 23 new footnotes to the Table of Frequency Tolerances. This was in addition to the 13 qualifying notations in the current Radio Regulations. The new ones are listed in Appendix 7 as footnotes 1, 4, 5, 7, 8 , 10, 12, 13, 14, 15, 16, 19, 20, 21, 22 23 24 25 26 28 29 32 and 36 y p Three of the areas having relatively tight tolerances are those of HF broadcasting, SSB emissions, and pulse modu- lation above 10 GHz (above 10 GHz being inherently more uncertain at this time). 22,23,24,25,26,28,29,32, and 36. . . WATSON AND LUTHER: FREQUENCY TOLERANCES AND SPURIOUS EMISSION LIMITATIONS and which receive their input from other television stations or which serve small isolated communities , For stations of 1 W (vision peak envelope power) or less this tolerance may be relaxed further to: 5 kHz in the band 100-470 MHz; 5 kHz in the band 100-470 MHz; ; 10 kHz in the band 470-960 MHz. ; 10 kHz in the band 470-960 MHz. (25) For trqnsmitters for system M (NTSC) the tolerance is 1000 Hz. However, for low-power transmitters using this system note (24) applies. (25) For trqnsmitters for system M (NTSC) the tolerance is 1000 Hz. However, for low-power transmitters using this system note (24) applies. (25) For trqnsmitters for system M (NTSC) the tolerance is 1000 H (25) For trqnsmitters for system M (NTSC) the tolerance is 1000 Hz. However, for low-power transmitters usin * All tolerances not indicated by hertz are in parts per million. * All tolerances not indicated by hertz are in parts per million. Band: 10.5 to 40 GHz Tolerances in the Table have been extended after 1985 not only to space and Earth stations, but also to broadcasting stations. the first time with impact from 4 MHz on up to 40 GHz after 1985. In the interim, power flux density limits tend to mini- mize any interference that might be caused by radiation of frequency spectra over an extra broad channel because of a loose tolerance. * All tolerances not indicated by hertz are in parts per million. Band: 4 to 29.7 MHz No tolerance values were established for meteorological 1133 WATSON AND LUTHER: FREQUENCY TOLERANCES AND SPURIOUS EMISSION LIMITATIONS WATSON AND LUTHER: FREQUENCY TOLERANCES AND SPURIOUS EMISSION LIMITATIONS TABLE I FREQUENCY TOLERANCE TABLE-BROADCASTING* Present Value As of 1985 for New (Until 1985 for Transmitters and New Band New:Transmitters) After 1990 for All , .. 9 to 535 kHz 10 Hz 535 to 1606.5 kHz (1605 kHz in Region 2) 10 Hz 10 Hz (6) i606.5 to 4000 HZ (1605 kHz in Region 2) 4 to 29.7 MHz 29.7 to 100 MHz 4 50 W (Other than TV) > 50 W 29.7 to 100 MHz 4 50 W (TV Sound and Vision) > 50 W 100 to 470 MHz (TV Sound and Vision) 4 100 W (TV Sound and Vision) > 100 W 470 to 2450 MHz (Other than TV) 470 to 960 MHz < 100 W (TV Sound and Vision >lo0 W in Band 470 to 960 MHz) 2450 to 10 500 MHz 10.5 to 40 GHz 20 15 50 20 100 1000 Hz 20 100 1000 Hz 100 100 1000 Hz none none agi 10 Hz (15) 10 Hz (15) (21) 2000 Hz (23) 2000 Hz (23) 500 Hz (24) (25) 500 Hz (24) (25) 2000 Hz (23) 500 Hz (24) (25) 500 Hz (24) (25) 100 500 Hz (24) (25) 500 Hz (24) (25) none 100 TABLE I FREQUENCY TOLERANCE TABLE-BROADCASTING (6) In countries covered by the North American Regional Broadcasting Agreement (NAREA) tolerance of 20 H (15) For A3E transmitters with carrier power of 10 kW or less the tolerance is 20 parts of 106 and 15 parts (23) For transmitters uf a mean power of 50 W or less operating at frequencies below 108 MHz, a tolerance of 3000 HZ applies. (24) In the faseof television stations of: q y , 50 W (vision peak envelope power) or less in the band 29.7-100 MHz; ( p p p ) ; 100 W (vision peak envelope power) or less in the band 100-960 MHz; ble to maintain this tolerance. For such stations, the tolerance is 2000 Hz. and which receive their input from other television stations or which serve small isolated communities, it may not, for operations reasons, be possi- ble to maintain this tolerance. For such stations, the tolerance is 2000 Hz. and which receive their input from other television stations or which serve small isolated communities, it may ble to maintain this tolerance. For such stations, the tolerance is 2000 Hz. HIGHLIGHTS-FREQUENCY TOLERANCES The limits of Appendix 17 and of Appendix Aer2 are for unwanted emissions, which of course, include both the outaf-band and the spurious. Taking a broad look at the 1979 WARC Frequency Toler- ance Table, it may be stated that the state of the art in almost every case comports with agreed tolerances, except perhaps above 10.5 GHz. State of the art here implies a worldwide operationally and economically useful technical specification. Regarding the perceived impact on world users, these new tolerances should provide a .significantly improved spectrum efficiency in terms of a harmful interference-free environmen Due to the economic, operational, and technical considera- tions given, along with the somewhat politically oriented WARC decisions, these tolerances will have wide acceptance and promote a broader use of telecommunications than could have otherwise been possible. While costs resulting from the tougher specifications may be somewhat higher to the more developed nations, overall costs will be significantly lower than would have been the case if only technical considerations had, prevailed. HIGHLIGHTS-FREQUENCY TOLERANCES In going from the current tolerances to those to be phased in after 1985, the range of values is from no change, tightening down to around 5 percent of the current values, depending upon requirements of the service. To better understand the tighter broadcasting tolerances, a listing of them is presented in Table I. g p Above 10.5 GHz, the tolerances are more tentative. This is due to such factors as 1) less developed art in this band, 2 ) for a given percentage of frequency tolerance to carrier frequency, p q Earth and space services were introduced in the Table for 1134 IEEE TRANSACTIONS ON COMMUNICATIONS, VOL. COM-29, NO. 8, AUGUST 1981 a higher actual frequency variation results, and 3) higher costs for the Same amount of power. In the 10.5 to 40CHz band, three new classes of stations need to eventually comply with tolerances while the existing two station types also have tightened values. Finally, it is noted that broadcasting comes under some tolerance control in 1985 in the highest band, generally applicable to the Broadcast Satellite service. . cult than spurious emissions to either control or measure because of presence of the energy associated with the funda- mental emission, energy that is usually about 99 percent of all radio frequency energy produced by the transmitter. Out- of-band emissions always exist when the fundamental fre- quency radiation is turned “on” with normal modulation. Their level is only dependent upon purity of the oscillation, linearity of the modulation process, and response character- istics of the final amplifier, subsequent tuned filter, and in minor part, the antenna, none of which are perfect. As they are always found to one extent or another, account is usu- ally taken of them in the frequencyassignmerit process so that no other special consideration by the system user is needed, unless adjacent channel interference is caused. In an effort to limit adjacent channel interference, Article 5 of the Final Acts addresses limits for outaf-band emissions’ as they are promulgated in the Radio Regulations for specific services (by general or specialized administrative conferences). At present, outaf-band emissions have specified maximum per- mitted power levels only for transmitters of the SSB radio- telephony Maritime-Mobile service operating in the MF and HF bands, as well as for transmitters of the aeronautical- mobile (route) service operating between 2.85 and 17.97 MHz. RR 305 of Article 5 refers to Appendices 17 and 27 Aer2 as well as to limits that may be specified in CCIR Recommendations. FUTURE CONSIDERATIONS-FREQUENCY TOLERANCES Spurious emissions, on the other hand, have much greater potential for causing interference to radiocommunications systems, and so have received the greater attention. Spurs may occur from a transmitter in literally any portion of the radio frequency spectrum, often far removed from the fundamental emission, and even sometimes with no predictable or calcul- able mathematical relationship to the fundamental frequency. As far as future frequency tolerance efforts are concerned, the WARC-79 issued Recommendation 69 which invited the CCIR to pursue study in the following four areas: 1) to continue its study of frequency tolerances with a view to the reduction of the frequency space required for a given channel; The latter situation has been found when components of a transmitter inadvertently form a resonant circuit with some coincidental feedback mechanism that derives fundamental transmitter energy and oscillates at whatever the incidental resonant frequency happens to be. This particular occurrence does not happen often, but has been experienced. Radiated levels of these and other spurious emissions are relatively simple to detect and measure with appropriate equipment such as spectrum analyzers, good attenuators, and calibrated field- strength meters. Given the propensity for some spurious emissions tb cause interference anywhere in the spectrum, and their relative importance, it is necessary for frequency mana- gers to be aware of their potential. Happily, these do not ofte exist to the point of creating interference. 2) to consider.whether or not in certain cases it is possible to predict ultimate values of tolerances, which it would not be necessary to make more stringent under currently known conditions of operation and to state what these tolerance values might be; g 3) to report upon the possibility of achieving such ulti- mate values of tolerances consistent with economic and design requirements and other practical consider- ations; 4) to indicate which, if any, of the tolerances specified in Appendix 7 have already attained these ultimate values. WARC-79 SPURIOUS LIMITS Only a short time was needed to adopt within the Final Acts, the Conclusions of the SPM on spurious limits,notwith- standing ten formal submissions from Administrations on the subject having a range of variations. It was, however, discus- sion on adding to the SPM frequency range for limits, that led to spirited interchange. Fortunately, the WARC was able to agree upon spurious emission limits that carry up to 17.7 GHz, although exclusion was provided for several significant uses. It was clear that insufficient data were available to find appropri- ate limits for the important space services, and systems usually found above 10 GHz using digital modulation. The Conference delegates readily agreed that these unresolved matters required immediate attention and that study should be undertaken of them, especially of space’services, on an urgent basis. Of all the types of spurious emissions encountered, by far the ones found most often, and which are cause for greatest concern, are harmonics and third-order intermodulation pro- ducts. In the experiences of the authors, these two types of undesirable emissions together account for nine out of ten cases of spurious emission interference to radiocommunica- tion services. SPURIOUS EMISSION LIMITATIONS To put spurious emission limitations into perspective, it must be ‘realized that these emissions are only half of the story. The general category of unwanted emissions is com- prised of two basic subparts, spurious emissions and’out-of- b a d emissions. Article 5 of the WARC-79 Final Acts states that every effort should be made to keep unwanted emissions at the lowest values ‘which the state of the technique (for doing so) and the nature of the (transmitter) service permit. Spurious emissions were redefined at the WARC-79 based upon previous work of the CCIR. They include patticularly harmonic emissions, parasitic emissions, intermodulation products, and frequencyconversion products, none of which occur, by definition, immediately outside the transmitted necessary bandwidth as a result of the modulation process. It would be possible, however, for a spurious emission to re- sult from a parasitic oscillation, or from a thirdarder inter- modulation product, and be sufficiently close to the nec- Outaf-band emissions are those resulting from the modu- lation process and which are found immediately outside the transmitted necessary bandwidth. They would include both transmitter noise and modulation splatter closely associated with the fundamental radiation. These are much more diffi- 1135 WATSON AND LUTHER: FREQUENCY TOLERANCES AND SPURIOUS EMISSION LIMITATIONS that all the significant spurious einission limits would be in- cluded in the measurement equipment bandwidth. essary bandwidth that it might mingle or be confused with otherwise ordinary out-of-band emissions. This situation does not occur often at all, so it is not a significant problemto worry about distinguishing between outofhand emissions and spurious emissions. q p The final, agreed Table of Maximum Permissible Levels of Spurious Emissions will be found in the SPM Report, Chapter 8, immediately following Frequency Tolerances. Other, related technical guidance was provided to the WARC, excluding as before, any mention of implementation dates, considered to be ex6lhsively the domain of the forthcoming Administrative Conference. Spurious emissions have specified, maximum permitted power levels in all radio Services (except radiodetermination) operating with fundamental radiation up to 960 MHz (after the implementation schedule of Appendix 8-only up to 235 MHz at present). Frequencies above 960 MHz, up to 17.7 GHz, are included in the future but exceptions are made in this band for systems using digital modulation, space services, again the radiodetermination service, and certain transmitters intended for use in emergency situations where the object is to attract aid, whatever the means. SPM DISCUSSIONS-SPURIOUS LIMITS A fewer number of SPM delegates were involved in spurious limit working groups than for frequency tolerances, and dis- cussions took substantially less time because there were fewer values to consider. It is significant, however, to realize that all of the SPM Conclusions in this matter were adopted at WARC- 79, without change to limits, much like occurred for the fre- quency tolerance values. W. Kilpatrick of the USA headed the Spurious Limits ’Working Group. A comparable number of input documents from Administrations were negotiated, ulti- mately involving the 87 countries, and including ITU recog- nized private operating agencies, international organizations, and scientific groups. p y p g In the case of spurious emission limits below 235 MHz, there was no tightening by WARC-79 of the basic values al- ready existing. In fact, effective in 1985, under Note 9 of the Spurious Emission Table (Appendix 8 of the Final Acts), Administrations may move from a 1- to a 10-mW level for spurious emissions of transmitters operating in the range 30-235 MHz with mean power over 25 W, unless harmful interference is caused by the lessened restriction. Furthermore, considering the types of power levels of typical frequency modulated maritime-mobile radiotelephone transmitters oper- ating above 30 MHz, the general absence of related serious interference cases, and the complicating special requirements of Note 5 to Appendix 8 (Spurious Limits) for such trans- mitters, the WARC-79 took the conscious decision to delete Note 5 at the end of 1984. It would be possible, of course, for the 1982 Mobile WARC to reconsider this action in Appendix 17, if believed necessary at that time. With this relaxation of the limits (deletion of Note 5), basically all communications transmitters operated up to 960 MHz will have the same re- quirements, regardless of the service in which they are used. Agreements on spurious emission limits were easily achieved, except above 960 MHz. Protracted discussions ensued and it became impossible for the SPM to reach any conclusion in the following two important areas: a) above 960 MHz in any service b) the bandwidth in which a spurious emission is measured, except that the measuring equipment should have a band- width “. . . sufficiently wide to accept all significant com- ponents . . Spectrum Allocations Above 40 GHz WEST E. KATZENSTEIN, ROBERT P. MOORE, AND HAROLD G. KIMBALL (WARC-79) revised the International Table of Frequency Al- locations to reflect increased interest and activity in this spectrum. WARC-79 was the first conference since 1959 which was competent to treat the region above 40 GHz. The objectives of the new Table above 40 GHz were to represent all potential users and to provide guidance and protection for development. Propagation phenomena played a major role in the definition and assignment of bands, and the desire to pro- vide flexibility (e.g., adding Fixed and Mobile) was also important. This latter aspect is reflected in the high degree of band-sharing which is embodied in the new Table. Increased usage above 40 GHz will provide unique challenges to the spec- trum manager and will likely reveal opportunities for more efficient use of the spectrum in the future. Absrrocr-The 1979 World Administrative Radio Conference (WARC-79) significantly revised the International Table of Frequency Allocations above 40 GHz to reflect a, high level of interest and activi in this portipn of the spectrum., The new Table of Allocations was created with the objectives of stimulating development of this spectrum resource by prqviding guidance,and protection to users and of providing each potential user bands in all parts of the Spectrum suitable to his charter. Thus propagation phenomena played a major role ini defining the new table-as, did the desire of some Admin- istrations to add services such as Fixed and Mobile. This paper discusses the approach used in creating.the new table, summarizes the allocations, discusses some bands of special interest, indicates’how future refinement of the table will likely occur, and addresses the challenge presented to the frequency manager by this part of the spectrum. FUTURE CONSIDERATIONS-SPURIOUS LIMITS The WARC-79 recognized the need to continue examining transmitter spurious emission levels in a number of areas, especially where agreements could not be reached. As the CCIR is the mechanism for conducting technical studies be- tween ITU Administrative Conferences,,Recommendation 66 of the Final Acts was specially addressed to that organization. Four specific areas of study were established by WARC-79 with the intent of eventually having appropriate recommenda- tions for consideration at future Administrative Conferences: The WARC-79 recognized the need to continue examining transmitter spurious emission levels in a number of areas, especially where agreements could not be reached. As the CCIR is the mechanism for conducting technical studies be- tween ITU Administrative Conferences,,Recommendation 66 of the Final Acts was specially addressed to that organization. IEEE TRANSACTIONS ON COMMUNICATIONS, VOL. COM-2‘ NO. 8, AUGUST 1981 IEEE TRANSACTIONS ON COMMUNICATIONS, VOL. COM-2‘ NO. 8, AUGUST 1981 a) Spurious emissions from space services transmissions. a) Spurious emissions from space services transmissions. a) Spurious emissions from space services transmissions. quency Tolerance Table. However, because providing greater spurious emission suppression involves much more than changing a component, as for example, a crystal oscillator in the case of frequency tolerances, the Conference determined that a decade and a half would be needed for any new spurious emission limits. A final, applicable date of January 1 , 1994 was determined, effectively applying to transmitters operating only between 235 MHz and 17.7 GHz. p p b) Spurious emission levels in all frequency bands in gen- eral, emphasizing those frequency bands, services, and modula- tion techniques not presently covered. q p y c) Measurement techniques for spurious emissions, includ- ing the determination of reference levels for wide-bid trans- missions as well as the applicability of reference measurement bandwidths. d) Categorizing of emissions (of any type) in terms of “mean power” to facilitate the interpretation and measure- ment of “mean power” as it applies to the various classes of emissions. REFERENCES [ I ] CCIR, “Technical bases for the World Administrative Radio Conference 1979,” Report of the Joint Meeting of CCIR Study Groups Special Preparatory Meeting for the WARC-79, Interna- tional Radio Consultative Committee, International Telecommu- nication Union, Geneva, Switzerland, 1978. p y g Four specific areas of study were established by WARC-79 with the intent of eventually having appropriate recommenda- tions for consideration at future Administrative Conferences: , , , [2] ITU, “Final Acts of the World Administrative Radio Conference, Geneva, 1979,” International Telecommunication Union, Geneva, Switzerland, 1980. Manuscript received December 1980. Ce te , C a a e,C 93555 H. G. Kimball is with the Communications and Data Systems Di- Washingtoh, DC 20546. vision, Office of Space Tracking and Data Systems, NASA Headquarters, a usc pt ece ved ece be 980 Center, China Lake, CA 93555. W. E. Katzenstein and R. P. Moore are with the Naval Weapons Manuscript received December 1980. Center, China Lake, CA 93555. W. E. Katzenstein and R. P. Moore are with the Naval Weapons H. G. Kimball is with the Communications and Data Systems Di- Washingtoh, DC 20546. vision, Office of Space Tracking and Data Systems, NASA Headquarters, SPM DISCUSSIONS-SPURIOUS LIMITS ..” a) above 960 MHz in any service b) the bandwidth in which a spurious emission is measured, except that the measuring equipment should have a band- width “. . . sufficiently wide to accept all significant com- ponents . . ..” Limits are newly applied, of course, for most transmitters operating between 235 and 960 MHz. These are effective for transmitters installed after January 1, 1985, and to all trans- mitters, regardless of installation date, after January 1, 1994. Exclusion is provided by Note 10 where more than one trans- mitter feeds a common antenna, or at an “antenna farm,’’ where it is difficult to realize the limits when intermodulation products result from induced RF voltages, because trans- mitters are so close to one another. In any event, it is clear from the WARC-79 Final Acts that spurious emission power should be kept as low as practicable, not only in this one case, but in all cases, and on all frequencies. Inability to establish limits above 960 MHz was a result of “last minute” data provided from FixedSatellite service interests that indicated existing satellite technology could not achieve a third-order intermodulation reduction of 30 dB, throwing into question the entire matter of spurious emission limits for space services in the microwave frequency range, i.e ., about above 1 GHz. Measurement bandwidths were discussed in terms of how to measure wide-band emissions. As it was generally conceded that widehand emissions do not especially occur below 960 MHz (the highest frequency where SPM agreement on limits was possible), the discussions on how to make measurements were concluded with the understanding The first conversion date of January 2, 1985 found by WARC-79 is exactly the same first new date under the Fre- 1136 11. SUMMARY OF SPECTRUM ALLOCATIONS ABOVE 40 GHz U SE OF THE REGION of the electromagnetic spectrum above 40 GHz represents both a large expansion in spec- trum resources and the development of new capabilities [I]. The 1979 World Administrative Radio Conference U Table I summarizes the allocations above 40 GHz. The total width of the spectrum allocated (235 GHz) indicates the ex- tent of this new spectium resource made accessible by ail- vances in the state of the art of telecommunications equip- ments. There are some striking differences between the ap- proach to allocation above and below 40 GHz [2]. For ex- ample, there are no bands allocated exclusively. This reflects U S . Government work not protected by US. copyright
https://openalex.org/W2272263698	https://bmcpublichealth.biomedcentral.com/counter/pdf/10.1186/s12889-016-2802-x	English	null	Predictors of residential stability among homeless young adults: a cohort study	BMC public health	2,015	cc-by	6,179	Abstract Background: Homelessness episodes have been shown to be associated with serious health outcomes among youth. This study was undertaken to estimate the probability of reaching residential stability over time and to identify predictors of residential stability among homeless young adults aged 18 to 25 years. Methods: A prospective cohort study was carried out in Montréal, Canada, between April 5th 2006 and January 21th 2009. Interviews conducted every three months included questions on life conditions and social and mental health factors that are known to influence residential trajectories. Residential status was determined, starting on the first day after recruitment; each follow-up day was classified as a homeless day or a housed day. A period of 90 days was used to define residential stability; therefore the main study outcome was the occurrence of the first consecutive 90 housed days during the follow-up period. Kaplan-Meier and Cox proportional-hazards regression analyses were conducted. Results: Of the 359 participants, 284 reached 90 days of residential stability over the study period, representing an annual probability of 80.5 %. In multivariate analysis, youth who had a high school degree, had a formal sector activity, and those who had sought psychological help were more likely to reach residential stability. Being a man, injecting substances, and having an informal sector activity were associated with a decreased probability to reach residential stability. Conclusion: Exposure to factors related to opportunities that promote social integration increases the chance of reaching residential stability. On the other hand, factors related to high level of street entrenchment seem to interfere with stabilization. Maximum efforts should be made to prevent chronic homelessness among youth, targeting not only individual impairments but also hinging on services adapted to foster social connections among the youth. Keywords: Homelessness, Youth, Residential stability, Housing, Cohort Studies have shown that street youth experience major residential transitions over relatively short time periods, alternating between lack of any housing, extremely pre- carious housing, and stable and autonomous housing [2–4]. Homelessness is a dynamic phenomenon; thus longitudinal designs are of paramount importance when studying the course of housing and its determinants [5]. Yet, only a few longitudinal studies have allowed quanti- tative examination of residential trajectories of homeless youth either adolescents or young adults [6–13]. Of these, five included adolescents [7–11] and two [7, 10] focused specifically on newly homeless youth. Although undoubtedly valuable, those studies do not specifically consider the situation of young adults. © 2016 Roy et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: Elise.Roy@usherbrooke.ca 1Addiction Research and Study Program, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 150, Place Charles-Le Moyne, room 200, Longueuil, QC J4K 0A8, Canada Full list of author information is available at the end of the article Abstract Indeed the course Roy et al. BMC Public Health (2016) 16:131 DOI 10.1186/s12889-016-2802-x Roy et al. BMC Public Health (2016) 16:131 DOI 10.1186/s12889-016-2802-x Background l Homelessness has deleterious effects on the develop- ment and health of youth. Precarious living conditions, hostile social environments, poor access to services, even just the struggle of day-to-day street survival are some of the main factors that significantly contribute to those effects. As a result, there is an increased risk of death: reported standardized mortality ratios show that mortal- ity rates for homeless youth are 2.7 to 37.3 times higher than for other young people [1]. * Correspondence: Elise.Roy@usherbrooke.ca 1Addiction Research and Study Program, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 150, Place Charles-Le Moyne, room 200, Longueuil, QC J4K 0A8, Canada Full list of author information is available at the end of the article Roy et al. BMC Public Health (2016) 16:131 Page 2 of 8 Page 2 of 8 on the “Life History Calendar” technique [16] and the residential follow-back calendar [17, 18] was used to document the main outcome of this study. Residential status was assessed day-by-day for the whole period since the previous interview (or during the 3 months prior to the intake interview). To help participants re- member their housing status by situating them in time, the interview started with questions about recent signifi- cant life events. Participants were asked if they had ex- perienced any of 33 positive or negative life events. This information was then noted on a calendar and used to guide the interview documenting the participant’s sleep- ing arrangements and locations on a daily basis for the whole time period. Based on this information, residential status was determined for each follow-up day, starting on the first day after recruitment (referred to as Day 1). Each follow-up day was classified as a homeless day or a housed day, the latter corresponding to the following sit- uations: the youth resided (1) in his or her own home; (2) in his or her partner’s home or with his or her parents or with relatives, friends, acquaintances, or families of friends without the reason being a need for temporary as- sistance; (3) in housing resources (excluding emergency or short-term shelters); (4) in a campground, hotel or motel (not as an emergency measure); or (5) in a place where a person works and lives (e.g., farm, fairground). Methods Between April 2006 and May 2007, study interviewers recruited participants through regular visits to all major street youth agencies in Montréal, Canada. As in our previous studies [1], youth were considered street- involved if they had used the services of Montréal street youth agencies at least three times in the previous year or had been without a place to sleep more than once during the same period. Only street youth who had ex- perienced at least one 24-h episode of homelessness in the previous 30 days were eligible for this study. Such epi- sode was defined as having spent at least one night in a place unfit for human habitation or having been housed temporarily in an emergency shelter or with friends or ac- quaintances. Other eligibility criteria included being 18 to 25 years of age, speaking French or English, being able to provide informed consent and to complete an interviewer- administered questionnaire, and planning to stay in the Montréal area for the following year. As for exposure variables, we focused on life condi- tions and social and mental health factors that are known to influence residential trajectories among home- less people [3–5, 20]. Each interview included questions about socio-demographic variables, previous homeless- ness experience, use of formal resources to get off the street, involvement in formal and informal sectors of ac- tivity, experience with the justice system, personal social network, psychological distress, suicidal thoughts and attempts, and markers of intensive drug use including experience of drug overdose and injection drug use. Substance use disorders and other mental health diagno- ses were measured only at baseline. g y The initial interview included signing a consent form and providing contact information. Six follow-up interviews took place every three months until January 2009. Detailed contact information was updated at each interview and thorough follow-up procedures were used. Participants received financial compensation (CAD $30) for each interview. This study was conducted with the approval of the Comité d’éthique de la recherche en santé chez l’humain du Centre Hospitalier Universitaire de Sherbrooke et de l’Université de Sherbrooke and conformed to the principles embodied in the Declaration of Helsinki. Formal sector activity was defined as having at least one source of income among the following during the last 3 months: full-time job, part-time job, occasional jobs, having a welfare cheque or unemployment cheque, and loans and grants from government sources. Methods Informal sector activities included sources of income from family or friends, prostitution, procuring (pimping), sale of Background l Youth spending a day in transitional facilities such as a police station, jail, prison, correctional halfway house, hospital, detoxification or rehabilitation center, or other similar re- sources were considered as housed on these days if these stays had been preceded by housed days; otherwise, these days were considered as homeless days. Further details about our research methodology and study instruments have been previously described [19]. of homelessness among young adults remains poorly documented despite the many characteristics distinguish- ing them from adolescents and from older street-involved populations. It is well acknowledged that the young adult or late adolescent stage is crucial to a normal outcome in adult functioning [14]. During this stage, many develop- mental tasks need to be completed to successfully transi- tion to adulthood. Social factors clearly influence the probability of a successful transition in terms of self‐suffi- ciency. One of these is certainly homelessness where young people living in unstable housing are more likely to experience living conditions that can hinder adult devel- opment [15]. Increased understanding of residential tra- jectories of homeless young adults will help formulate better public health interventions related to housing. We report below the results of a prospective cohort study which objectives were to estimate the probability of reach- ing residential stability over time and to identify predictors of residential stability among homeless young adults. Measurements All questionnaires were administered by interviewers trained specifically for this study. A questionnaire based Roy et al. BMC Public Health (2016) 16:131 Page 3 of 8 Page 3 of 8 Page 3 of 8 personal property (e.g., pawn shop), sale of drugs, artistic performances on the street or in the subway, begging, squeegeeing, and theft, fraud or concealment. was defined as the occurrence of the first consecutive 90 housed days during the follow-up period. The occur- rence of this event (i.e., 90 days of residential stability) was defined as happening on day i if the 90 days follow- ing day i represented 90 consecutive housed days. Using the Kaplan-Meier method, we estimated the cumulative probability of youth returning to a housed status for 90 consecutive days during follow-up. The probability of reaching a first episode of 90-day residential stability over one year was also computed using the following formula: 1-e-mean per-person incidence rate*length of observation [25]. Predictors of 90-day residential stability were assessed using Cox proportional-hazards regression. The measures of these predictors were obtained from ques- tionnaires on Day i or previously. Formal resources to get off the street included community-based or institutional services providing help finding a job, obtaining financial or legal assistance, and finding housing during the last 3 months. Experience with the justice system was defined as involvement in at least one of the following situations during the last 3 months: police arrest, awaiting trial or sentencing, having sentences with probation, being on conditional release, being in de- tention or owing money after getting a ticket. Having a so- cial network at risk was defined as currently having regular interactions with persons (i.e., boyfriend, girlfriend or anyone else excluding formal resource providers) who have problems with alcohol or drugs, having sex in ex- change for money or drugs, injecting drugs or being homeless. The presence (in the last three months) of sup- port from family, friends or boyfriend/girlfriend in the form of emotional support, instrumental support, advice or protection was also examined at every visit. Univariate analyses were conducted and factors with p- values less than 0.20 were retained for multivariate analyses. Following the purposeful selection procedure, significant variables at a level alpha of 0.05 and those with a confound- ing effect were retained in the final model. Measurements A variable was considered as a confounder if its removal from the model changed a significant coefficient by more than 20 %. SAS 9.3 software was used to perform the analyses. Psychological distress was assessed at each visit by the K10 scale developed and validated by Kessler and coll [21]. It consists of 10 questions on non-specific psycho- logical distress symptoms a person may have experi- enced in the most recent four-week period. The final score ranges between 10 and 50 and a score equal or greater than 30 was considered as severe psychological distress. Questions about diagnoses of major depression, bipolar disorders, anorexia/bulimia, and schizophrenia were taken from the World Mental Health Composite International Diagnostic Interview (CIDI) version 2.1. [22]. Anxiety and alcohol or drug-related disorders were assessed using the simplified version (CIDIS) developed by Kovess and colleagues [23]. The CIDI and the CIDIS are well-validated tools that can be administered by lay in- terviewers and produce psychiatric diagnoses according to the fourth version of the Diagnostic and Statistical Manual of Mental Health Disorders published by the American Psychiatric Association [24]. Having at least two mental health disorders was defined as demonstrating two or more of the following diagnoses: major depression, bipolar disorder, anorexia/bulimia, schizophrenia or anxiety. Results Of the 419 youth recruited, 359 (85.7 %) completed at least 90 days of follow-up, corresponding to 64,479 days of follow-up at risk (mean 180 days; range: 1–630, median 114 days). Two hundred and eighty-four participants (79.1 %) reached 90 days of residential stability over the study period, representing an annual probability of 80.5 %. The median survival time was 129 days and the corre- sponding interquartile range was 39 to 343 (Fig. 1). Most participants were men (79 %) and median age at study entry was 22 years (Table 1). At baseline, almost three-quarters of the participants reported having been homeless during their lives (not necessarily continuously) for more than a year (median: 2.00, IQR : 0.75–4.00). In all, 143 subjects presented at least one mental health problem in the last year (40 %). Results of univariate and multivariate Cox proportional- hazards regression analyses are shown in Table 2. Youth who had a high school degree, had a formal sector activity, and those who had sought psychological help were more likely to reach residential stability compared to those who did not. On the other hand, being a man, injecting substances, and having an informal sector activity were associated with a decreased probability to reach residential stability in multivariate analysis. Of note, participants excluded from the analyses (n = 60) were similar to those included with respect to all predictors identified except for injection drug use where 22 % of participants reported drug injection compared to 10 % of non-participants (p = 0.036). Analyses Frequency distributions were calculated to characterize the sample with respect to baseline characteristics. A period of 90 days was used to define residential stability, and therefore, only subjects who completed at least 90 days of follow-up were included in the analyses. A 90-day period was considered sufficiently long to repre- sent significant stability; it was also comparable to the minimum timeframe used in the most recent literature [11]. More specifically, 90 days of residential stability Roy et al. BMC Public Health (2016) 16:131 Page 4 of 8 Fig. 1 Cumulative probability to reach a housed status for 90 consecutive days during follow-up Fig. 1 Cumulative probability to reach a housed status for 90 consecutive days during follow-up began after quite a long period, that is, 129 days or 4.3 months after recruitment. In addition, a majority of participants already had a significant lifetime homeless- ness experience at study entry. The data suggest that in our sample, homelessness has not been a short term event but rather a relatively long-lasting issue. It is inter- esting to note that among factors leading to stability, a number of them concern access to resources that Slesc- nik et al. call the social system (employment, school and medical care) [9]. Youth who had sought psychological assistance, who had earned a high school diploma and who were working during follow-up were 40 to 50 % more likely to achieve stability than the other youth. These results are consistent with those of studies in the United States looking at samples that are comparable, in terms of age. In their longitudinal study of homeless youth in California, Tevendale et al. found that less involvement in informal sector activities predicted membership in the “consistently sheltered” versus “inconsistently sheltered” group during follow-up [11]. Moreover being able to go back home was more important than degree of individual impairment such as substance use and mental health problems. In their Ohio study, Slesnick et al. concluded that youth who had more social connections at the begin- ning of the study were more likely to see their number of homeless days decrease during follow-up [9]. In our study, while background characteristics such as lifetime duration Discussion Our study is the first to prospectively examine the prox- imal predictors of residential stability in a cohort of homeless young adults. It is also the first to define the outcome using number of consecutive days of housing in places fit for human habitation during the study period. Only a few prospective studies have looked at residential stability among homeless youth and none have determined housing status on a day-to-day basis [7–10, 13]. Various measures of housing and residential trajectories have been reported, all based on questions about current housing status at time of interview or re- call of various living situations since last interview. Given these significant methodological differences, it is difficult to compare our data to existing literature. Fur- thermore, the notion of stability considered in our study did not take into account qualitative aspects of housing such as satisfaction and security [26]. However, our study results, as those of similar studies, point to the same conclusion: stability defined on the basis of lasting episodes of housing is a reachable goal for many home- less youth, at least for a three-month period. An important finding is that the annual probability of reaching at least one episode of 90 consecutive housed days during follow-up was high (80.5 %). Although this is very good news, our figures show that for half of the study participants, the first 90-day period of stability Roy et al. Discussion Some comments are warranted concerning the fact of homelessness or diagnoses of substance use or mental health disorders were not associated with the outcome, exposure to factors related to opportunities that promote social integration during follow-up was key. This demon- strates the importance of providing services adapted to the needs of young people so they can build up confidence and develop skills to engage in society, which in turn en- courage them to get off the streets. g g Our results show that drug injection reduces by 30 % the chances of having at least one episode of residential stability during the follow-up period. The link between problematic substance use and homelessness is complex. The literature suggests there is a reciprocal relationship, where worsening of housing conditions can lead to in- creased consumption that can then jeopardize the capacity of achieving housing stability [19, 27]. Therefore, it is diffi- cult to determine the direction of the causal link, if any. More specific studies on drug injection among street youth have shown that homelessness episodes increase the risk of initiation into injection drug use in this population [28, 29]. The question as to whether drug injection perpetuates homelessness remains poorly documented. It is plausible, however, considering that injection is a very intense way of consuming drugs. Similarly, intense drug consumption was found to be negatively associated with transitioning out of homelessness among street-involved youth in Vancouver [13]. Indeed, intensive use is often associated with a life- style linked to high level of street entrenchment [30, 31] which, in turns, interferes with reintegration into main- stream society. Likewise, being involved in informal sector activities also decreased the risk of reaching residential sta- bility among our study participants. Finally, being female predicted a greater likelihood of attaining a first episode of stability, although the associ- ation was marginally significant. Similarly Tevendale et al.’s study showed that females were more likely than males to follow “consistently” and “short term inconsist- ently sheltered” trajectories as opposed to a “late term consistently sheltered” trajectory. However this associ- ation no longer applied in the multivariate analysis. The relatively small number of women typically ob- served in cohorts of homeless makes it difficult to es- timate the probability of reaching residential stability according to gender. Yet the literature suggests that the experience of women and men is different which could influence the probability of stabilizing. Discussion BMC Public Health (2016) 16:131 Page 6 of 8 Page 6 of 8 outcome was adapted from the Residential Follow-Back Calendar designed by the New Hampshire Dartmouth Research Center [18]. Tsemberis et al. [17] assessed the psychometric properties of the Calendar and demon- strated its high test-retest reliability, sensitivity to change and concurrent validity. In terms of limitations, our data collection method was based on self-reports, which may have introduced the possibility of both recall and social desirability biases. The impact of such biases was however limited by our use of the Life History Calendar technique, the short time spans between interviews (3 months), and the interviewers’ open and non-judgmental attitudes. Sec- ondly, our decision to focus on life conditions and social and mental health factors that could predict residential stability in the course of homelessness might have pre- vented us to identify important background factors such as childhood-related factors. Thirdly, our results may not be generalizable to all street youth. The majority of partic- ipants already had a relatively long history of homeless- ness at recruitment. Despite their young age, many of them had been involved in the street economy, arrested or convicted, and reported high risk networks, suggesting an already significant level of street-entrenchment at baseline. Finally, keeping up with homeless populations can be par- ticularly challenging. In our study, 87 % of subjects re- cruited at baseline were re-interviewed at three months, 72 % at 12 months, and 45 % at 18 months. These figures are comparable to that of Tevendale et al., who reported a two-year follow-up percentage of 42 %. Losses to follow- up may have affected our estimates of residential stability, especially after 12 months of follow-up. On one hand, it is plausible that subjects who stabilized for long periods dropped out of our study more frequently than less stable youth, leading to deflated housing stability estimates. On the other hand, it is conceivable that subjects who did not stabilize were more likely to be lost to follow- up due to chaotic lifestyles that jeopardized their con- tinued participation. It is less clear that the correlate analysis could be biased. To affect the magnitude or direction of risk ratio estimates, drop-out rates would have to differ according to presence of both a correlate and housing stability [33]. Discussion BMC Public Health (2016) 16:131 Page 5 of 8 Page 5 of 8 y p p Number of subjects at baseline (%) Baseline variables Male 284 (79.1) Age at first homelessness episode (≥16 years old) 222 (61.8) Age (≥22 years) 182 (50.7) Lifetime cumulative homelessness (≥1 year) 259 (72.1) Alcohol abuse or dependence (last 12 months) 119 (33.2) Drug abuse or dependence (last 12 months) 233 (64.9) Mental health problem (excluding abuse or dependence to alcohol or drugs; last 12 months) 143 (39.9) ≥2 mental health problems (last 12 months) 73 (20.3) Time-dependent variables High school or more 85 (23.7) Formal sector activity (last 3 months) 120 (33.4) Informal sector activity (last 3 months) 315 (87.7) Experience with the justice system (last 3 months) 277 (77.2) High psychological distress (last month) 87 (24.2) Social network at risk (currently) 308 (86.0) Formal resources to get off the street (last 3 months) 184 (51.3) Formal resource providing help to find housing (last 3 months) 72 (20.1) Seeking psychological help (last 3 months) 135 (37.6) Program or therapy for alcohol or drugs (last 3 months) 60 (16.7) Active support from family, friends or boyfriend/girlfriend (last 3 months) 315 (91.8) Active support from a formal individual (last 3 months) 118 (34.4) Suicidal ideation (last 3 months) 55 (15.3) Attempted suicide or intentional overdose (last 3 months) 20 (5.6) Accidental overdose (last 3 months) 26 (7.2) Injection (last 3 months) 78 (21.7) Number of subjects at baseline (%) Table 2 Crude and multivariate Cox proportional-hazards regression of predictors of 90-days residential stability (n = 359) Crude HR (95 % CI)b AHRa (95 % CI) Baseline variables Male 0.763 (0.579 - 1.004) 0.726 (0.545–0.969) Age at first homelessness episode (≥16 years old) 1.247 (0.979 - 1.587) - Lifetime cumulative homelessness (≥1 year) 0.810 (0.627 - 1.047) - Drug abuse or dependence (last 12 months) 0.761 (0.598 - 0.968) - ≥2 mental health problems (last 12 months) 1.319 (0.997 - 1.744) - Time-dependent variables High school or more 1.501 (1.149 - 1.962) 1.470 (1.122–1.926) Formal sector activity (last 3 months) 1.659 (1.308 - 2.104) 1.507 (1.181–1.924) Informal sector activity (last 3 months) 0.632 (0.461 - 0.866) 0.674 (0.488–0.931) Consultation for emotional or nervous problems (last 3 months) 1.377 (1.085 - 1.747) 1.359 (1.069–1.727) Injection (last 3 months) 0.665 (0.494 - 0.894) 0.692 (0.507–0.944) aAHR Adjusted Hazard Ratio bOnly the variables with p-value ≤0.20 were included in the multivariate analysis Roy et al. Discussion For ex- ample, while it is generally acknowledged that young street women are more victimized than males, they also make more use of their social networks than young men to cope with homelessness [32]. In the first case, this could com- promise the stabilization process while in the second case, this could be the reverse. Some comments are warranted concerning the fact that having received formal support to find housing was not a significant predictor of the study outcome. It is well acknowledged that securing housing for homeless people provides the stability often needed to access education, employment and all other assets necessary to develop an independent life. We do not believe these results should be interpreted as conflicting with the literature. In fact, at time of study, supportive youth-focused housing resources were very limited in Montréal. This could have affected our power to de- tect an association. Our study presents a number of strengths and limita- tions. The instrument we used to assess the study Roy et al. BMC Public Health (2016) 16:131 Page 7 of 8 Page 7 of 8 Page 7 of 8 Competing interest Competing interest p g The authors declare that they have no competing interest. 13. Cheng T, Wood E, Feng C, Mathias S, Montaner J, Kerr T, et al. Transitions into and out of homelessness among street-involved youth in a Canadian setting. Health & place. 2013;23:122–7. doi:10.1016/j.healthplace.2013.06.003. Epub 2013 Jun 22. healthplace.2013.06.003. Authors’ contributions d f h d ER conceived of the study, was responsible for its design and coordination and drafted the manuscript. MR, LF and JFB participated in the design of the study and the writing of the manuscript. DB carried out the statistical analyses. EL helped to draft the manuscript. All authors read and approved the final manuscript. 14. Ammerman SD, Ensign J, Kirzner R, Meininger ET, Tornabene M, Warf CW, et al. Homeless young adults ages 18–24: examining service delivery adaptations. Nashville: National Health Care for the Homeless Council, Inc; 2004. 15. Collins ME. Transition to adulthood for vulnerable youths: a review of research and implications for policy. Social Service Review. 2001;75:271–91. doi:10.1086/322209. Acknowledgements This study was supported by a grant (no. CBG-101825) from the Canadian Institutes of Health Research. We would like to acknowledge the excellent work of the research coordinator, Éric Vaillancourt. We extend a special thank you to the research staff and the participants of this study. 16. Caspi A, Moffitt TE, Thornton A, Freedman D, Amell JW, Harrington H, et al. The life history calendar: a research and clinical assessment method for collecting retrospective event-history data. International journal of methods in psychiatric research. 1996;6:101–14. Author details 1 17. Tsemberis S, McHugo G, Williams V, Hanrahan P, Stefancic A. Measuring homelessness and residential stability: the residential time-line follow- back inventory. Journal of community psychology. 2007;35:29–42. doi:10.1002/jcop.20132. 1Addiction Research and Study Program, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 150, Place Charles-Le Moyne, room 200, Longueuil, QC J4K 0A8, Canada. 2Département de Psychoéducation et de Psychologie, Université du Québec en Outaouais, 283, boul. Alexandre-Taché, bureau 3712, C.P. 1250, Succursale Hull, Gatineau, QC J8X 3X7, Canada. 3École de Santé Publique, Université Montréal, 7101 Avenue du Parc, 3ième étage, Montréal, QC H3N 1X9, Canada. 4Faculty of Medicine and Health Sciences, Université de Sherbrooke, 150, Place Charles-Le Moyne, room 200, Longueuil, QC J4K 0A8, Canada. 5Charles-LeMoyne Hospital Research Centre, 150, Place Charles-Le Moyne, room 200, C.P. 11, Longueuil, QC J4K 0A8, C d 6L d D i I tit t f M di l R h J i h G l H it l 18. New Hampshire Dartmouth Research Center. Residential follow-back calendar. Lebanon: Dartmouth Medical School; 1995. 18. New Hampshire Dartmouth Research Center. Residentia calendar. Lebanon: Dartmouth Medical School; 1995. 19. Roy E, Robert M, Vaillancourt E, Boivin JF, Vandermeerschen J, Martin I. Residential trajectory and HIV high-risk behaviors among Montréal street youth—a reciprocal relationship. Journal of urban health. 2011;88:767–78. doi:10.1007/s11524-011-9574-5. 20. Johnson G, Chamberlain C. From youth to adult homelessness. The Australian journal of social issues. 2008;43:563–82. Canada. 6Lady Davis Institute for Medical Research, Jewish General Hospital, 3755, Côte Ste-Catherine, Montréal, QC H3T 1E2, Canada. Canada. 6Lady Davis Institute for Medical Research, Jewish General Hospital, 3755, Côte Ste-Catherine, Montréal, QC H3T 1E2, Canada. 3755, Côte Ste-Catherine, Montréal, QC H3T 1E2, Canada. 21. Kessler RC, Andrews G, Colpe LJ, Hiripi E, Mroczek DK, Normand SL, et al. Short screening scales to monitor population prevalences and trends in non-specific psychological distress. Psychol Med. 2002;32:959–76. References 1. Roy E, Haley N, Leclerc P, Sochanski B, Boudreau JF, Boivin JF. Mortality in a cohort of street youth in Montréal. JAMA. 2004;292:569–74. 1. Roy E, Haley N, Leclerc P, Sochanski B, Boudreau JF, Boivin JF. Mortality in a cohort of street youth in Montréal. JAMA. 2004;292:569–74. in a cohort of street youth in Montréal. JAMA. 2004;292:569–74. 23. Kovess V, Fournier L, Lesage A, Amiel-Lebigre F, Caria A. Two validation studies of the CIDIS; a simplified version of the CIDI. Psychiatric Network. 2001;4:10–24. 2. Kelly K, Caputo T. Health and street/homeless youth. Journal of health psychology. 2007;12:726–36. 2. Kelly K, Caputo T. Health and street/homeless youth. Journal of health psychology. 2007;12:726–36. 3. Krüsi A, Fast D, Small W, Wood E, Kerr T. Social and structural barriers to housing among street-involved youth who use illicit drugs. Health & social care in the community. 2010;18:282–8. 3. Krüsi A, Fast D, Small W, Wood E, Kerr T. Social and structural barriers to housing among street-involved youth who use illicit drugs. Health & social care in the community. 2010;18:282–8. 3. Krüsi A, Fast D, Small W, Wood E, Kerr T. Social and structural barriers to housing among street-involved youth who use illicit drugs. Health & social care in the community. 2010;18:282–8. 4. Karabanow J. How young people get off the street: exploring paths and processes. In: Hulchanski J, Campsie P, Chay S, Hwang S, Paradis E, editors. Finding home: policy options for addressing homelessness in Canada (e-book). Toronto: Cities Centre, University of Toronto; 2009. 5. Toro PA, Lesperance TM, Braciszewski JM. The Heterogeneity of Homeless Youth in America: Examining typologies. National alliance to end homelessness. Research Institute, October 5, 2011. www.endhomelessness. org/library/entry/the-heterogeneity-of-homeless-youth-in-america- examining-typologies. Accessed 1 April 2015. 6. Barber CC, Fonagy P, Fultz J, Simulinas M, Yates M. Homeless near a thousand homes: outcomes of homeless youth in a crisis shelter. Am J Orthopsychiatry. 2005;75:347–55. 7. Rosenthal D, Rotheram-Borus MJ, Batterham P, Mallett S, Rice E, Milburn NG. Housing stability over two years and HIV risk among newly homeless youth. AIDS and behavior. 2007;11:831–41. 8. Slesnick N, Kang MJ, Bonomi AE, Prestopnik JL. Six- and twelve-month outcomes among homeless youth accessing therapy and case management services through an urban drop-in center. Health Serv Res. 2008;43:211–29. 24. American Psychiatric Association. DSM-IV, Manuel Diagnostique et Statistique des Troubles Mentaux. 4th ed. Paris: Masson; 2000 [French translation]. 4. Karabanow J. Received: 30 June 2015 Accepted: 1 February 2016 Received: 30 June 2015 Accepted: 1 February 2016 Received: 30 June 2015 Accepted: 1 February 2016 22. Kessler RC, Ustun TB. The World Mental Health (WMH) Survey Initiative version of the World Health Organization (WHO) Composite International Diagnostic Interview (CIDI). International journal of methods in psychiatric research. 2004;13:93–121. Conclusion 9. Slesnick N, Bartle-Haring S, Dashora P, Kang MJ, Aukward E. Predictors of homelessness among street living youth. Journal of youth and adolescence. 2008;37:465–74. The results of this study are encouraging in that most homeless youth achieve residential stability even after relatively long stays in the street. Our findings suggest that efforts to prevent chronic homelessness among youth should not only target individual impairments but also build on services that foster social connections among the youth. 10. Milburn NG, Rice E, Rotheram-Borus MJ, Mallett S, Rosenthal D, Batterham P, et al. Adolescents exiting homelessness over two years: the risk amplification and abatement model. Journal of research on adolescence. 2009;19:762–85. 11. Tevendale HD, Comulada WS, Lightfoot MA. Finding shelter: two-year housing trajectories among homeless youth. J Adolesc Health. 2011; 49:615–20. 12. Kidd SA, Karabanow J, Hughes J, Frederick T. Brief report: youth pathways out of homelessness - preliminary findings. J Adolesc. 2013;36:1035–7. doi:10.1016/j.adolescence.2013.08.009. 31. Karabanow J, Naylor T. Pathways Towards Stability: Young People’s Transitions Off of the Streets. Canadian Homelessness Research Network, The homeless Hub, 2013. http://homelesshub.ca/resource/3-pathways- towards-stability-young-people%E2%80%99s-transitions-streets. Accessed 5 Februay 2016. 32. Novac S, Serge L, Eberle M, Brown J. On Her Own: Young Women and Homelessness in Canada. Canadian Housing and Renewal Association, 2002. http://ywcacanada.ca/data/research_docs/00000271.pdf. Accessed 12 December 2015. 33. Moyses S, Nieto FJ. Epidemiology. Beyond the Basics. 2nd ed. Sudbury, Massachusetts: Jones and Bartlett Publishers; 2007. Roy et al. BMC Public Health (2016) 16:131 33. Moyses S, Nieto FJ. Epidemiology. Beyond the Basics. 2nd ed. Sudbury, Massachusetts: Jones and Bartlett Publishers; 2007 References How young people get off the street: exploring paths and processes. In: Hulchanski J, Campsie P, Chay S, Hwang S, Paradis E, editors. Finding home: policy options for addressing homelessness in Canada (e-book). Toronto: Cities Centre, University of Toronto; 2009. 25. Kelsey JL, Thompson WD, Evans AS. Methods in observational epidemiology. Monographs in Epidemiology and Biostatistics, Volume 10. New York: Oxford University Press; 1986. 26. Frederick TJ, Chwalek M, Hughes J, Karabanow J, Kidd S. How stable is stable? Defining and measuring housing stability. Journal of Community Psychology. 2014;42:964–79. doi:10.1002/jcop.21665. 5. Toro PA, Lesperance TM, Braciszewski JM. The Heterogeneity of Homeless Youth in America: Examining typologies. National alliance to end homelessness. Research Institute, October 5, 2011. www.endhomelessness. org/library/entry/the-heterogeneity-of-homeless-youth-in-america- examining-typologies. Accessed 1 April 2015. 27. Johnson G, Chamberlain C. Homelessness and substance abuse: which comes first? Australian Social Work. 2008;61:342–56. doi:10.1080/03124070802428191. 6. Barber CC, Fonagy P, Fultz J, Simulinas M, Yates M. Homeless near a thousand homes: outcomes of homeless youth in a crisis shelter. Am J Orthopsychiatry. 2005;75:347–55. 28. Roy É, Haley N, Leclerc P, Cédras L, Blais L, Boivin JF. Drug injection among street youths in Montréal: predictors of initiation. Journal of urban health. 2003;80:92–105. 7. Rosenthal D, Rotheram-Borus MJ, Batterham P, Mallett S, Rice E, Milburn NG. Housing stability over two years and HIV risk among newly homeless youth. AIDS and behavior. 2007;11:831–41. 29. Feng C, DeBeck K, Kerr T, Mathias S, Montaner J, Wood E. Homelessness independently predicts injection drug use initiation among street- involved youth in a Canadian setting. J Adolesc Health. 2003;52:499–501. doi:10.1016/j.jadohealth.2012.07.011. 8. Slesnick N, Kang MJ, Bonomi AE, Prestopnik JL. Six- and twelve-month outcomes among homeless youth accessing therapy and case management services through an urban drop-in center. Health Serv Res. 2008;43:211–29. 30. Stephens RC. The Street Addict Role: A Theory of Heroin Addiction. Albany: State University of New York Press; 1991. 31. Karabanow J, Naylor T. Pathways Towards Stability: Young People’s Transitions Off of the Streets. Canadian Homelessness Research Network, The homeless Hub, 2013. http://homelesshub.ca/resource/3-pathways- towards-stability-young-people%E2%80%99s-transitions-streets. Accessed 5 Februay 2016. 32. Novac S, Serge L, Eberle M, Brown J. On Her Own: Young Women and Homelessness in Canada. Canadian Housing and Renewal Association, 2002. http://ywcacanada.ca/data/research_docs/00000271.pdf. Accessed 12 December 2015. 33. Moyses S, Nieto FJ. Epidemiology. Beyond the Basics. 2nd ed. Sudbury, Massachusetts: Jones and Bartlett Publishers; 2007. Roy et al. BMC Public Health (2016) 16:131 Page 8 of 8 Page 8 of 8 • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step:
https://openalex.org/W2197057146	https://opencivilengineeringjournal.com/VOLUME/4/PAGE/38/PDF/	English	null	A Semi-Analytical Model for the Simulation of the Isolation of the Vibration Due to a Harmonic Load Using Pile Rows Embedded in a Saturated Half-Space	The Open civil engineering journal	2,010	cc-by	20,600	1. INTRODUCTION developed a theoretical model to study the vibration isolation effect of a pile row with the incident SV and Rayleigh waves as the sources. Kattis et al. [5, 6] used the 3-D frequency domain BEM to calculate the vibration isolation effect of a pile row under the same assumption. Recently, by means of the frequency domain BEM, the vibration isolation effect of four types of circular piles was studied by Tsai [7]. The vibration isolation methods can be classified into two categories: the active vibration isolation method and the passive isolation method. The active isolation method often is aimed to isolate the vibration source from its surrounding medium. Thus, the active vibration isolation facility is usu- ally installed around the vibration source or at a very close distance from the source. The passive isolation facility, on the other hand, usually is far away from the source and sur- rounds the protected structure. It should be noticed that many previous researches con- cerning vibration isolation treat the half-space as a single- phase elastic medium. Nevertheless, it is well-known that for the saturated soil, the pore fluid plays a very important role in the liquefaction and the shear failure of the soil. Thus, it is inappropriate to treat the saturated soil as a single-phase elas- tic medium. Although there have been many researches on the dynamic response of piles embedded in a poroelastic half-space, most of them are limited to the case of piles sub- jected to top harmonic loads. For example, based on Biot’s theory [8-10], Zeng and Rajapakse [11] analyzed the steady- state dynamic response of an axially loaded pile embedded in a poroelastic half-space. Wang et al. [12] extended the problem to the dynamic response of pile groups embedded in a poroelastic half-space. Jin et al. [13] studied the time- harmonic response of a pile under lateral loadings embedded in a poroelastic half-space. Besides, the frequency domain dynamic response of a pile embedded in a half-space porous medium and subjected to P and SV waves was investigated by Lu & Jeng [14]. However, up to now, very little attention has been paid on the vibration isolation using pile rows Roughly, there are two passive vibration isolation ap- proaches: the trench (open or in-filled) approach and the pile approach (pile rows or sheet piles). To date, many researches concerning vibration isolation by trenches or piles have been conducted. Address correspondence to this author at the Department of Civil Engineering, Jiangsu University, Zhenjiang, Jiangsu, 212013, P.R. China; Tel: +86(511)88791011; E-mail: ljfdoctor@yahoo.com 38 38 The Open Civil Engineering Journal, 2010, 4, 38-56 Open Access A Semi-Analytical Model for the Simulation of the Isolation of the Vibration Due to a Harmonic Load Using Pile Rows Embedded in a Saturated Half-Space Bin Xu1,2, Jian-Fei Lu3, and Jian-Hua Wang4 1Department of Civil Engineering, Shanghai JiaoTong University, Shanghai, 200030, P.R. China 2Department of Civil Engineering, Nanchang Institute of Technology, Nanchang, Jianxi, 330099, P.R. China 3Department of Civil Engineering, Jiangsu University, Zhenjiang, Jiangsu, 212013, P.R. China 4Department of Civil Engineering, Shanghai Jiao Tong University, Shanghai 200030, P.R. China Abstract: The isolation of the vibration due to a harmonic vertical load using pile rows embedded in a saturated poroelas- tic half-space is investigated in this study. Using the fundamental solution for a circular patch load and Muki’s method, the second kind of Fredholm integral equations describing the dynamical interaction between the pile rows and the satu- rated poroelastic half-space are obtained. Numerical solution of the integral equations yields the dynamic response of the pile-half-space system. The vibration isolation effect of the pile rows is investigated via the proposed semi-analytical model. Numerical results indicate that stiffer piles have better isolation vibration effect than flexible piles. Moreover, the pile length and the spacing between neighboring piles in one pile row have significant influence on the isolation vibration effect of pile rows, while the influence of the spacing between neighboring pile rows is relatively smaller. Poroelastic half-space, Biot’s theory, Fredholm integral equation, Pile rows, Passive vibration isolat Keywords: Poroelastic half-space, Biot’s theory, Fredholm integral equation, Pile rows, Passive v The Open Civil Engineering Journal, 2010, Volume 4 39 39 Based on Biot’s theory, the constitutive relation for a homogeneous porous medium has the following form [8- 10] embedded in poroelastic soils. Based on Biot’s theory, the effect of a pile row on the displacement amplitude reduction of soil subjected to an incident S wave was investigated in [15]. Also, using the Fourier-Bessel series expansion method with the aid of the translational addition theorem, the dy- namic response of the poroelastic medium behind a pile row when subjected to incident elastic waves was studied by Cai et al. [16]. Based on Biot’s theory and integral equation method, numerical simulations of the isolation of the moving loading induced vibration by pile rows embedded in a ho- mogeneous or layered poroelastic half-space were conducted in [17, 18], respectively. f ij ij ij ij p e μ + = 2 (3) M Me p f + = (4) (3) (4) where ij is the total stress of bulk material; ij denotes the strain tensor of the solid skeleton; fp is the excess pore fluid pressure and ij is the Kronecker delta; e and are the dilatation of the solid skeleton and the fluid volume increment for a unit volume porous medium. where ij is the total stress of bulk material; ij denotes the strain tensor of the solid skeleton; fp is the excess pore fluid pressure and ij is the Kronecker delta; e and are the dilatation of the solid skeleton and the fluid volume increment for a unit volume porous medium. In this paper, based on Muki’s method for piles [19, 20] and the fundamental solution for a circular patch load, a semi-analytical model for analyzing the isolation of the vi- bration due to a fixed vertical harmonic load using pile rows is developed. Compared with the researches in [17, 18], the most important contribution of this paper is that both the influence of the vertical and horizontal deformation of pile rows on the isolation effect is considered. As a result, three integral equations describing the vertical and horizontal pile- half-space interaction are needed for each pile. The remain- der of the paper is organized as follows. In the second Sec- tion, Biot’s theory is outlined. 2. BIOT’S THEORY where f , s and k ( k = 1, 2 , 3) are the scalar and vector potentials for the porous medium and ijk e is the Levi-Civita symbol. Moreover, in the Cartesian coordinate system, the vector potential k ( k = 1, 2, 3) satisfies the following condition In this study, the soil is considered as the porous me- dium, which is described by Biot’s theory [8-10]. Equations of motion for the bulk material and the pore fluid are ex- pressed in terms of the solid displacement ( iu ) and the infil- tration displacement ( iw ) as follows [8-10] , 0 i i = (8) 2 , , , ( ) i jj j ji j ji u M u Mw μ μ + + + + = b i f i u w + (1) , , j ji j ji Mu Mw + = f i i p i u mw b w + + (2) , 0 i i = (8) According to the analysis of Bonnet [22], although two displacement vectors are used in Biot’s theory, four inde- pendent variables exist in the porous medium. Consequently, the pore pressure has the following expression (2) , , j ji j ji f i i p i where and μ are Lame constants of the solid skeleton; b is the bulk density of the porous medium, which is de- fined by f s b + = ) 1( , where s is the density of the solid skeleton, is the porosity of the porous medium and f is the density of the pore fluid; / f a m = and a is the tortuosity of the porous medium; / p b k = , and k represent the viscosity of the pore fluid and the per- meability of the porous medium, respectively; the superim- posed dot on a variable denotes the time derivative. The Open Civil Engineering Journal, 2010, Volume 4 39 In the third Section, the ap- proach for establishing the fundamental solution for a verti- cal and a horizontal circular patch load applied in the poroe- lastic half-space is sketched. In the fourth Section, the Fred- holm integral equations describing the vertical and horizon- tal interaction between the piles and poroelastic half-space are established. In the fifth Section, the amplitude reduction ratio assessing the isolation vibration effect is defined. In the sixth Section, some numerical examples and corresponding analysis are presented. Finally, in the seventh Section, based on the research in this study, some conclusions are drawn. To derive the general solutions for Biot’s equations, the Fourier transform with respect to time and frequency is involved [21], which is defined as follows ( ) ( ) i t f f t e dt + = (5) 1 ( ) ( ) 2 i t f t f e d + = (6) (5) (6) where ( ) f t represents a function in the time domain, ( ) f is the Fourier transform of ( ) f t , t and denote time and frequency, respectively, and a bar over a variable denotes the Fourier transform. Based on the Helmholtz decomposition method, the solid displacement in the frequency domain has the following decomposition , , , i f i s i ijk k j u e = + + (7) (7) 1. INTRODUCTION For example, Emad and Manolis [1] utilized the boundary element method (BEM) with constant elements to research the vibration reduction effect of rectangular and circular open trenches. Beskos et al. [2] employed BEM with constant element to discuss the vibration reduction effects of the open and in-filled trench. Dasgupta et al. [3] applied the 3-D frequency domain BEM with the full space fundamental solution to investigate the isolation of the vibration due to a rigid surface foundation subjected to a harmonic loading via open and in-filled trenches. Avilles and Sanchez-Sesma [4] 1874-1495/10 2010 Bentham Open A Semi-Analytical Model for the Simulation The Open Civil Engineering Journal, 2010, Volume 4 39 3. THE FUNDAMENTAL SOLUTION FOR A POROELASTIC HALF-SPACE SUBJECTED TO A CIRCULAR PATCH LOAD IN THE FREQUENCY DOMAIN 2 2 0 ( , , , ) ( , , )sin( ) m m f r z f r z m = = (21) (21) where subscript m denotes the order of the term in the series, and 1f , 2f represent the corresponding potentials, dis- placements, the pore pressure and the stresses, respectively. As shown in the previous section, the governing equa- tions of Biot’s theory can be reduced to three Helmholtz equations for two scalar potentials and one vector potential. Herein, based on the potential method, the fundamental solu- tion for a poroelastic half-space subjected to a uniform patch load over a circular region with radius R (Fig. 2) will be es- tablished. The resultant of the patch load is assumed to be unity. Since the half-space considered here is axisymmetric with respect to the axis z (Fig. 2), it is more convenient to consider the current problem in a cylindrical coordinate system ( , , r z ). To derive the fundamental solutions for the poroelastic half-space in the cylindrical coordinate system ( , , ) r z , the Hankel integral transform is employed [24]. The m-th order Hankel transform is defined by ( ) 0 ˆ ( ) ( ) ( ) m m f rf r J r dr + = (22) ( ) 0 ˆ ( ) ( ) ( ) m m f r f J r d + = (23) (22) (23) In the cylindrical coordinate system, the vector potential for the solid skeleton and the pore fluid displacements can be represented by two scalar potentials, and [23], which satisfy the condition (8) automatically. Thus, in the cylindri- cal coordinate system, the Helmholtz equations (10)-(12) are reduced to where ( ) m J denotes the m-th order first kind of Bessel function and a caret above a variable denotes the Hankel transform, and denotes the horizontal wavenumber. Once the potentials for the porous medium ( f , s and ) are determined, the frequency domain displacement and pore pressure are given by (7), (9), while the stress of the porous medium is determined by (3). 1 1 0 ( , , , ) ( , , )cos( ) m m f r z f r z m = = (20) 1 1 0 ( , , , ) ( , , )cos( ) m m f r z f r z m = = (20) 2 2 0 ( , , , ) ( , , )sin( ) m m f r z f r z m = = (21) (20) 0 m= 2 2 0 ( , , , ) ( , , )sin( ) m m f r z f r z m = = (21) Xu et al. Xu et al. For the poroelastic half-space subjected to a harmonic load, potentials f , s , and displacements ru , zu , r w , z w and stresses rr , , zz , rz may be expanded into the series (20), while potentials and displacements u , w and stress z are expanded into the series (21) [24] Note that the expressions for the two constants f A , s A in (9) and the three complex wave numbers ( fk , sk and tk ) can be found in [23]. Note that the expressions for the two constants f A , s A in (9) and the three complex wave numbers ( fk , sk and tk ) can be found in [23]. tk ) can be found in [23]. Once the potentials for the porous medium ( f , s and ) are determined, the frequency domain displacement and pore pressure are given by (7), (9), while the stress of the porous medium is determined by (3). 3. THE FUNDAMENTAL SOLUTION FOR A POROELASTIC HALF-SPACE SUBJECTED TO A CIRCULAR PATCH LOAD IN THE FREQUENCY DOMAIN 2 2 2 2 2 2 2 2 1 1 ( ) 0 f f f k r r r r z + + + + = (13) 2 2 2 2 2 2 2 2 1 1 ( ) 0 s s s k r r r r z + + + + = (14) 2 2 2 2 2 2 2 2 1 1 ( ) 0 tk r r r r z + + + + = (15) 2 2 2 2 2 2 2 2 1 1 ( ) 0 tk r r r r z + + + + = (16) Substituting the m-th order component of the potentials f , s , , in equation (20) and (21) into (13)-(16) and performing m-th order Hankel transform with respect to the radial coordinate r, the general solutions of the potential ˆ fm , ˆ sm , ˆ m , ˆ m in the frequency-wavenumber domain are obtained as follows (13) (14) (15) ( ) ˆ ( , , ) ( , ) ( , ) f f z z m fm m m z A e B e = + (24) ( ) ˆ ( , , ) ( , ) ( , ) s s z z m sm m m z C e D e = + (25) ( ) ˆ ( , , ) ( , ) ( , ) t t z z m m m m z E e F e = + (26) ( ) ˆ ( , , ) ( , ) ( , ) t t z z m m m m z G e H e = + (27) (24) (16) The displacements for the solid frame can be expressed in terms of the above potentials in the cylindrical coordinate system ( , , ) r z as follows (26) (27) 2 1 f s ru r r r r z = + + + (17) 2 1 1 1 f s u r r r r z = + + (18) 2 2 2 1 1 ( ) f s zu r z z r r r r = + (19) where the superscript m denotes the m-th order Hankel trans- form, and ( , ) m A ~ ( , ) m H are arbitrary constants to be determined by the boundary condition and the continuity condition at the patch load plane (Fig. 2. BIOT’S THEORY , , f f f ii s s ii p A A = + (9) (9) The above equations for Biot’s theory can be reduced to the following Helmholtz equations [23] 2 f + k f 2 f = 0 (10) 2s + ks 2s = 0 (11) 2 + kt 2 = 0 (12) 2 f + k f 2 f = 0 2s + ks 2s = 0 2 + kt 2 = 0 (10) (11) (12) 40 The Open Civil Engineering Journal, 2010, Volume 4 40 The Open Civil Engineering Journal, 2010, Volume 4 3. THE FUNDAMENTAL SOLUTION FOR A POROELASTIC HALF-SPACE SUBJECTED TO A CIRCULAR PATCH LOAD IN THE FREQUENCY DOMAIN (28) Note that the real part of , , , f s t = in (28) should be always non-negative to guarantee the bounded condition at infinity. As mentioned above, when derive the integral equations for the pile rows, the fundamental solutions of the poroelas- tic half-space subjected to a vertical and a horizontal patch load over a circular domain with the radius equal to that of the piles are needed. These kinds of fundamental solutions can be derived by means of the general solution (24)-(27) and the displacement-potential relation, the pore pressure- potential relation and the stress-potential relation, the details of which are available in [14]. Following Muki and Sternberg [19, 20] and Pak & Jennings [25], the pile-soil is decomposed into two sub- problems: an extended poroelastic half-space and multiple fictitious piles. The response of the poroelastic half-space is governed by Biot’s theory, while the fictitious pile is de- scribed by a 1-D bar and beam vibration theory. For simplic- ity, the i-th pile is used to illustrate the establishment of the integral equations. For the i-th fictitious pile, its Young’s modulus and the density are expressed as [19, 20], 3. THE FUNDAMENTAL SOLUTION FOR A POROELASTIC HALF-SPACE SUBJECTED TO A CIRCULAR PATCH LOAD IN THE FREQUENCY DOMAIN 2). In (24)-(27), f , s and t are complex numbers associated with the vertical wavenumbers of the P1, the P2 and the S wave of the porous medium and 2 1 f s ru r r r r z = + + + (17) 2 1 1 1 f s u r r r r z = + + (18) 2 2 2 1 1 ( ) f s zu r z z r r r r = + (19) where the superscript m denotes the m-th order Hankel trans- form, and ( , ) m A ~ ( , ) m H are arbitrary constants to be determined by the boundary condition and the continuity condition at the patch load plane (Fig. 2). In (24)-(27), f , where the superscript m denotes the m-th order Hankel trans- form, and ( , ) m A ~ ( , ) m H are arbitrary constants to be determined by the boundary condition and the continuity condition at the patch load plane (Fig. 2). In (24)-(27), f , s and t are complex numbers associated with the vertical wavenumbers of the P1, the P2 and the S wave of the porous medium and (17) (18) (19) The Open Civil Engineering Journal, 2010, Volume 4 41 The Open Civil Engineering Journal, 2010, Volume 4 41 A Semi-Analytical Model for the Simulation 2 2 f fk = , 2 2 s sk = , 2 2 t tk = (28) d ( 2 d R = ), length L, modulus p E and the same density p . The spacing between two neighboring piles in one pile row is s and the separation between two adjacent pile rows is q. The distance between the first pile row and the center of the vibration source is s d (Fig. 1). 4. THE FREDHOLM INTEGRAL EQUATIONS FOR THE PILE- HALF-SPACE SYSTEM ( ) ( ) * i i p p s E E E = , ( ) ( ) * i i p p b = ( 1,2, , i m = ) (29) (29) 2 The Open Civil Engineering Journal, 2010, Volume 4 2 The Open Civil Engineering Journal, 2010, Volume 4 42 The Open Civil Engineering Journal, 2010, Volume 4 42 Xu et al. Xu et al. The extended poroelastic half-space are subjected to the following loads (Fig. 3): ( )( ) i kq z ( , , ) k x y z = which are uniformly distributed over the domain occupied by the i-th pile; ( ) * (0) i N , ( ) * (0) i k Q , ( ) * (0) i k M ( , ) k x y = and ( ) * ( ) i N L , ( ) * ( ) i k Q L , ( ) * ( ) i k M L ( , ) k x y = which are applied over the circular domain ( ) 0 i and ( )i L , respectively. As shown in Fig. (3), it is assumed that the axial force, the shear force and the bending moment of the i-th fictitious pile are denoted by ( ) * ( ) i N z , ( ) * ( ) i k Q z and ( ) * ( ) i k M z ( , ) k x y = , respectively. The i-th fictitious pile is subjected to the vertical and the horizontal distributed loads ( )( ) i kq z ( , , ) k x y z = along the length of the pile (Fig. 3). The top and the bottom of the i-th fictitious pile are subjected to ver- tical loads ( ) * (0) i N , ( ) * ( ) i i N L , horizontal loads ( ) * (0) i k Q , ( ) * ( ) i k Q L ( , ) k x y = and bending moments ( ) * (0) i k M , ( ) * ( ) i k M L ( , ) k x y = , respectively. a b Fig. (2). A uniform circular patch load applied in a saturated poroelastic half space: (a) a vertical uniform circular patch load; (b) a horizontal uniform circular patch load. 4.1. The Piles and the Half-Space System where Ep (i) and s E are Young’s modulus for the i-th pile and the poroelastic half-space with ) /( ) 2 3 ( μ μ μ + + = s E ; ( )i p , b are the densities for the i-th pile and the poroelas- tic half-space; ( ) * i p E and ( ) * i p are Young’s modulus and the density for the i-th fictitious pile. As shown in Fig. (1), the vibration source is a harmonic vertical patch load with frequency f ( / 2 f = ) and ap- plied to a circular surface domain with the diameter D . The passive vibration isolation system is multiple pile rows embedded in a poroelastic half-space. The total number of the piles is m . Each circular pile has the same diameter Fig. (1). Multiple pile rows used as vibration isolation system for a saturated poroelastic half space subjected to a harmonic vertical load. z L x pile a poroelastic half space o x y s q d ds q o i t F q e  D The vertical harmonic load o x y s q d ds q i t F q e  The vertical harmonic load The vertical harmonic The vertical harmonic load Fig. (1). Multiple pile rows used as vibration isolation system for a saturated poroelastic half space subjected to a harmonic vertical load. z L x pile a poroelastic half space y o i t F q e  D Fig. (1). Multiple pile rows used as vibration isolation system for a saturated poroelastic half space subjected to a harmonic vertical load. Fig. (1). Multiple pile rows used as vibration isolation system for a saturated poroelastic half space subjected 2 The Open Civil Engineering Journal, 2010, Volume 4 z 2R x o D1 D2 h a poroelastic half space y z 2R x o D1 D2 h a poroelastic half space y b z 2R x o D1 D2 h a poroelastic half space y a z 2R x o D1 D2 h a poroelastic half space y Fig. (2). A uniform circular patch load applied in a saturated poroelastic half space: (a) a vertical uniform circular patch load; (b) a horizontal uniform circular patch load. Fig. (3). The model for the i-th fictitious pile and the extended poroelastic half space. z y o an extended poroelastic half space x z L z ( ) 0 i   ( ) ( ) ( ) / i i zq z A ( )( ) i zq z ( )( ) i N L ( )i   ( )i L  fictitious pile i t F q e  ( )( ) i x Q L ( ) ( ) ( ) / i i xq z A ( ) ( ) (0) / i i x M A ( ) ( ) (0) / i i x Q A ( ) ( ) (0) / i i N A ( ) ( ) ( ) / i i N L A ( ) ( ) ( ) / i i x Q L A ( ) ( ) ( ) / i i x M L A ( )( ) i x M L ( )( ) i xq z ( )(0) i x M ( )(0) i N ( )(0) i x Q x z ( )( ) i zq z ( )( ) i N L itious pile ( )( ) i x Q L ( )( ) i x M L ( )( ) i xq z ( )(0) i x M ( )(0) i N ( )(0) i x Q ( )(0) i x Q x half space Fig. (3). The model for the i-th fictitious pile and the extended poroelastic half space. The Open Civil Engineering Journal, 2010, Volume 4 43 A Semi-Analytical Model for the Simulation The Open Civil Engineering Journal, 2010, Volume 4 4 43 When the pile-half-space system is subjected to external loads, generally, the piles will undergo both vertical and horizontal deformation. 2 The Open Civil Engineering Journal, 2010, Volume 4 As a result, the integral equations for the pile rows should account for the vertical and horizontal deformation of the piles. In this study, the compatibility con- dition that the axial strain of the fictitious piles and the verti- cal strain of the extended half-space along the piles’ axis are equal is used to construct the integral equation accounting for the vertical coupling of the pile-half-space system. On the other hand, the condition that the rotary angles of the axis of the fictitious piles and those of the extended half-space along the axis of the piles are equal is used to establish the Fredholm integral equations accounting for the horizontal interaction within the system. where ( )i kI ( , ) k x y = is the second moment of the pile cross section. 4.2. The Equations of Motion for the Piles The horizontal displacement ( ) ( ) p i ku z , the distributed horizontal load ( )( ) i kq z , the shear force ( ) * ( ) i k Q z and the bending moment ( ) * ( ) i k M z ( , ) k x y = of the i-th fictitious pile satisfy the following relations ( ) ( ) ( ) ( ) ( ) 2 * * * ( ) ( ) ( ) i i i i p i k k p k dQ z q z A u z dz = + , ( , ) k x y = (32) ( ) ( ) * * ( ) ( ) i i k k dM z Q z dz = , ( , ) k x y = (33) ( ) ( ) ( ) ( ) ( ) 2 * * * ( ) ( ) ( ) i i i i p i k k p k dQ z q z A u z dz = + , ( , ) k x y = (32) (32) 0 ( i = 1,2,......,m) (38) (38) where ( )( ) i zf z is the free field vertical strain due to the ver- tical harmonic source and ( )( , , ) G zz ijr z , ( )( , , ) G kz ijr z ( k = x, y ) represents the vertical strain at the center of z (i) due to a unit vertical and horizontal patch load at ( )j , respectively (Fig. 3), ijr is the horizontal distance between the axis of the i-th and j-th pile. Note that if i j = , rij will vanish. 4.3. The Fredholm Integral Equations for the Vertical Interaction Between the Piles and the Half-Space As mentioned above, the vertical compatibility condition between the i-th pile and the extended half-space is fulfilled by requiring the axial strain of the i-th fictitious pile and the vertical strain of the extended half-space along the axis of the i-th pile to be equal ( ) ( ) * ( ) ( ) , 0 p i s i z z z z z L = ( i = 1,2,......,m) (37) where ( ) * ( ) p i z z represents the axial strain of the i-th ficti- tious pile, ( )( ) s i z z is the vertical strain of the extended half-space along the axis of the i-th pile. ( ) ( ) * ( ) ( ) , 0 p i s i z z z z z L = ( i = 1,2,......,m) (37) (37) where ( ) * ( ) p i z z represents the axial strain of the i-th ficti- tious pile, ( )( ) s i z z is the vertical strain of the extended half-space along the axis of the i-th pile. 4.2. The Equations of Motion for the Piles For the i-th fictitious pile, the vertical displacement ( ) * ( ) p i zu z , the distributed vertical load ( )( ) z i q z and the axial force ( ) * ( ) i N z satisfy the following relations The vertical strain of the extended half-space along the axis of the piles is composed of two parts: the first part is due to the free wave field, while the second part is due to the loads applied to the half-space by all the fictitious piles. Thus, the vertical strain of the extended half-space along the axis of the i-th pile can be written as qz (i)(z) = dN* (i)(z) dz p* (i)A(i) 2uz* p(i)(z) (30) ( ) ( ) ( ) * * * ( ) ( ) * 0 1 ( ) (0) ( ) z p i p i i z z i i p u z u N d E A = + (31) (30) z s(i)(z) = zf (i)(z) + [N* ( j)(0)zz (G)(rij,z,0) j=1 m N* ( j)(L)zz (G)(rij,z, L) qz ( j)()zz (G)(rij,z,)d] 0 L ( ) ( ) ( ) * * , 1 { [ (0) ( , ,0) m j G j ij k kz k k x y j Q r z Q = = + + ( ) ( ) ( ) 0 ( ) ( , , ) ( ) ( , , ) ]} L G j G ij ij kz k kz L r z L q r z d + , ( i = 1,2,......,m) z s(i)(z) = zf (i)(z) + [N* ( j)(0)zz (G)(rij,z,0) j=1 m (31) where ( )i A denotes the cross-section area of the i-th pile. 4.2. The Equations of Motion for the Piles ( ) ( ) * * ( ) ( ) i i k k dM z Q z dz = , ( , ) k x y = (33) (33) Based on the beam theory, the bending moment ( ( ) * ( ) i k M z , ( , ) k x y = ), the rotary angle of the pile ( ( ) * ( ) p i k z , ( , ) k x y = ) and the horizontal displacement ( ( ) * ( ) p i ku z , ( , ) k x y = ) have the following form ( ) ( ) * * 0 ( ) ( ) z i i k k M z Q d = , ( , ) k x y = (34) ( ) ( ) ( ) * * * ( ) ( ) * 0 1 ( ) ( ) ( ) (0) z p i i p i k k k i i p k z z Q d E I = + , ( , ) k x y = (35) uk* p(i)(z) = 1 2Ep* (i)Ik (i) (z )2Qk* (i)()d 0 z +k* p(i)(0)z + uk* p(i)(0) , ( , ) k x y = (36) ( ) ( ) * * 0 ( ) ( ) z i i k k M z Q d = , ( , ) k x y = (34) (34) Using (30)-(36) and (37), (38), the Fredholm integral equation describing the vertical interaction between the i-th pile and the extended half-space has the form N* (i)(z) Ep* (i)A(i) + N* (i)(z)[zz (G)(rii,z, z+) zz (G)(rii,z,z)] + [ N* ( j) 0 L () j=1 m zz (G)(rij,z,) d N* (i)(z) Ep* (i)A(i) + N* (i)(z)[zz (G)(rii,z, z+) zz (G)(rii,z,z)] (35) + [ N* ( j) 0 L () j=1 m zz (G)(rij,z,) d (36) 44 The Open Civil Engineering Journal, 2010, Volume 4 Xu et al. 4.2. The Equations of Motion for the Piles Thus, using (30)-(36) and (41), the following supplementary equations for ( ) * (0) p i zu are derived ( ) ( ) 0 ( ) ( , , ) L G a ij ij kz r z d + ( ) ( ) ( ) ( ) ( ) * * (0) ( ) (0) ( )]} ( ) p j b p j c i k ij k ij zf z u z z + + = , ( i = 1,2,......,m) ( ) ( ) ( ) ( ) ( ) * * (0) ( ) (0) ( )]} ( ) p j b p j c i k ij k ij zf z u z z + + = , ( i 1 2 m) ( ) ( ) ( ) ( ) ( ) * * (0) ( ) (0) ( )]} ( ) p j b p j c i k ij k ij zf z u z z + + = , } k ij k ij zf ( i = 1,2,......,m) (39) (39) where ( )( , , ) G zz iir z z , ( )( , , ) G zz iir z z + denote the vertical strain of the extended half-space at the center of ( ) z i when the patch vertical load applied on ( )i approaches ( ) z i from up and down side, respectively, and ( ) ( ) ( ) ( ) * * 1 0 0 ( ,0, ) [ ( ) ( ) (0, ) ] G L L m zz ij j j c ij j U r N d N d = + ( ) ( ) * 1 (0)[ (0) ] m p j d z ij ij j u = + * ( ) ( ) ( ,0, ) { [ ( ( ) j k L G m kz ij j U r Q d z + ( ) ( ) ( ) ( ) * * 1 0 0 ( ,0, ) [ ( ) ( ) (0, ) ] G L L m zz ij j j c ij j U r N d N d = + ( ) ( ) * 1 (0)[ (0) ] m p j d z ij ij j u = + * ( ) ( ) , 1 0 ( ,0, ) { [ ( ( ) j k L G m kz ij j k x y j U r Q d z = = + ( ) ( ) ( ) * 0 ( ) ( ,0, ) L a G p j ij kz ij k U r d + + ( ) ( ) ( ) * (0) (0) (0) (0)]} d p j e ij k ij u + ( )(0) i zf u = , 1,2,......, i m = (42 ( ) ( ) ( ) ( ) * * 1 0 0 ( ,0, ) [ ( ) ( ) (0, ) ] G L L m zz ij j j c ij j U r N d N d = + ( ) ( ) * 1 (0)[ (0) ] m p j d z ij ij j u = + ( ) 2 * ( ) ( ) ( ) * ( , ) ( , , ) j L p a G ij zz ij j p z r z d E = (40a) ( ) ( ) ( ) 2 ( ) * 0 ( ) ( , , ) L b j j G ij p zz ij z A r z d = (40b) ( ) 2 * ( ) 2 ( ) * ( ) ( ) * 0 ( ) ( ) ( ) 2 j p a i ij k i i p x A Q d E I = (40c) ( ) ( ) ( ) 2 ( ) * 0 ( ) ( , , ) L b j j G ij p kz ij z A r z d = , ( k = x, y ) (40d) ( ) ( ) ( ) 2 ( ) * 0 ( ) ( , , ) L c j j G ij p kz ij z A r z d = , ( k = x, y ) (40e) (40a) (40b) (40c) * ( ) ( ) , 1 0 ( ,0, ) { [ ( ( ) j k L G m kz ij j k x y j U r Q d z = = + ( ) 2 * ( ) 2 ( ) * ( ) ( ) * 0 ( ) ( ) ( ) 2 j p a i ij k i i p x A Q d E I = (40c) , { m k x y j = = + (40c) ( )(0) i zf u = , 1,2,......, i m = (42) (42) Following the similar procedures, the surface vertical displacement ( , 0) zs u z = x for the poroelastic half space in the presence of pile rows can be calculated as follows where ij is the Kronecker delta and where ij is the Kronecker delta and ( ) 2 * ( ) ( ) ( ) * ( , ) ( , , ) j L p c G ij zz ij j p z U r z d E = (43a) ( ) ( ) ( ) 2 ( ) * 0 ( ) ( , , ) j L d j G ij p zz ij z A U r z d = (43b) ( ) ( ) ( ) 2 ( ) * 0 ( ) ( , , ) L d j j G ij p kz ij z A U r z d = , ( , k x y = ) (43c) ( ) ( ) ( ) 2 ( ) * 0 ( ) ( , , ) L e j j G ij p kz ij z A U r z d = , ( , k x y = ) (43d) ( ) ( ) * 1 0 ( ,0, ) ( ,0) ( ,0) [ ( ) G L m zz j j zs zf j U r u u N d = = + x x x * ( ) ( ) 2 ( ) ( ) * 0 ( ) ( ,0, ) ] p L j j p j G z zz j A u U r d + x ( ) ( ) * , 1 0 ( ,0, ) { [ ( ) j L G m kz j j k k x y j U r Q d z = = + x ( ) ( ) 2 ( ) ( ) * '* 0 ( ) ( ,0, ) ]} j L j j p j G p k kz j A u U r d + x (41 (43a) (43b) (41) 4.2. The Equations of Motion for the Piles ( ) ( ) ( ) ( ) * * 0 ( ) ( , ) (0) ( )] j L j a p j b ij z ij N z d u z ( ) ( ) * , 1 0 ( , , ) { [ ( ( ) G L m ij kz j k k x y j r z Q d z = = ( ) ( ) 0 ( ) ( , , ) L G a ij ij kz r z d + tance between the surface point x and the axis of the j-th pile. In equation (39), uz* p(i)(0) ( i = 1,2,......,m) are also un- known. Thus, additional equations for uz* p(i)(0) ( i = 1,2,......,m) are needed for the closure of the system. The supplementary equations for ( ) * (0) p i zu can be derived assuming that the vertical displacement of the i-th pile top and the vertical displacement of the extended half-space at the center of ( ) 0 i are equal, i.e., ( ) ( ) * (0) (0) p i s i z z u u = . Note that ( )(0) s i zu can be obtained via (41) by setting x coin- cide with the center of the i-th pile top. 4.4. The Fredholm Integral Equations Accounting for the Horizontal Interaction the Piles and the Half-Space (48) ( ) ( ) 2 ( ) ( ) * '* ' 0 ( ) ( ,0, ) ]} L j j p j G p k k k j A u U r d + x , ( , k x y = ) (48) where ( ,0) kf u x ( , ) k x y = represents the free field horizon- tal displacement, ( ) ' ( ,0, ) G k k ij U r , ( )( ,0, ) G zk ij U r ( , , ) k k x y = denotes the horizontal displacement at the sur- face point x ( x y = + x i j) due to a unit patch horizontal and vertical load applied at ( )j , respectively. For the close of the system, additional equations for ( ) * (0) p i ku (k = x, y; i = 1,2,......,m) are needed. Supposing that the horizontal displacement of the i-th fictitious pile top is equal to the horizontal displacement of the extended half- space at the i-th pile top, supplementary equations for ( ) * (0) p i ku (k = x, y; i = 1,2,......,m) in (46) can be obtained via (48) by setting x coincide with the center of the i-th pile top. 4.4. The Fredholm Integral Equations Accounting for the Horizontal Interaction the Piles and the Half-Space (46) where where Likewise, the rotary angle of the extended half-space along the axis the i-th pile consists of two parts: the first part is the contribution from the free wave field, while the second part is due to the loads applied to the half-space by the fictitious piles (Fig. 3). 4.4. The Fredholm Integral Equations Accounting for the Horizontal Interaction the Piles and the Half-Space where ( ,0) zf u x represents the free field vertical displace- ment, ( )( ,0, ) G zz j U r x , ( )( ,0, ) G kz j U r x , ( k = x, y ) denotes the vertical displacement at the surface point x ( x y = + x i j) due to a unit patch vertical and horizontal load applied at ( )j , respectively, j r x is the horizontal dis- where ( ,0) zf u x represents the free field vertical displace- ment, ( )( ,0, ) G zz j U r x , ( )( ,0, ) G kz j U r x , ( k = x, y ) denotes the vertical displacement at the surface point x ( x y = + x i j) due to a unit patch vertical and horizontal load applied at ( )j , respectively, j r x is the horizontal dis- The horizontal compatibility between the i-th pile and the extended half-space is realized by assuming the rotary angle of the i-th fictitious pile ( ( ) * ( ) p i k z ( , ) k x y = ) and the ro- tary angle of the extended half-space along the axis of the pile ( ( )( ) s i k z ( , ) k x y = ) to be equal The Open Civil Engineering Journal, 2010, Volume 4 45 A Semi-Analytical Model for the Simulation (44) ( ) ( ) ( ) ( ) * * 0 ( ) (z, ) (0) ( )] L j a p j b ij z ij N d u z ( )( ) f i k z = , ( , ; 1,2,......, ) k x y i m = = (46) where ( ) ( ) ( ) ( ) * * 0 ( ) (z, ) (0) ( )] L j a p j b ij z ij N d u z ( ) ( ) * ( ) ( ) s i p i k k z z = , 0 z L < < , i = 1,2,......,m, , k x y = (44) ( ) ( ) * ( ) ( ) s i p i k k z z = , 0 z L < < , i = 1,2,......,m, , k x y = (44) where ( )( ) s i k z and ( ) * ( ) p i k z ( , k x y = ) denote the rotary angle of the extended half-space and the i-th fictitious pile in the xoz and the yoz plane, respectively. 4.4. The Fredholm Integral Equations Accounting for the Horizontal Interaction the Piles and the Half-Space As a result, taking proper account of the discontinuity of the integrand, the rotary angle of the extended poroelastic half-space along the axis the i-th pile has the form ( ) ( ) ( ) ( ) 2 * ' 0 ( ) ( , , ) 1 G L a i i ij p k k ij z A r z d = , ( , , ) k k x y = (47a) ( ) ( ) ( ) ( ) 2 * ' 0 ( ) ( , , ) G L b i i ij p k k ij z A r z d = , ( , , ) k k x y = (47b) ( ) 2 * ( ) ( ) ( ) * ( , ) ( , , ) j L p a G ij zk ij j p z r z d E = , ( , ) k x y = (47c) ( ) ( ) ( ) 2 ( ) * 0 ( ) ( , , ) L b j j G ij p zk ij z A r z d = , ( , ) k x y = (47d) (47c) ( ) ( ) ( ) ( ) ( ) * ( ) ( ) ( )[ ( , , ) ( , , )] s i i i G G k kf k kk ii kk ii z z Q z r z z r z z + = + ( ) ' ( ) '* ' , 1 0 ( , , ) { [ ( ) G L m k k ij i k k x y j r z Q d = = + ( ) ( ) ( ) 2 ( ) * '* ' 0 ( ) ( , , ) ]} G L j j p j p k k k ij A u r z d + ( ) ( ) ( ) * 1 0 ( , , ) [ ( ) G L m j i zk ij j j r z N d = + ( ) ( ) ( ) 2 ( ) * * 0 ( ) ( , , ) ] G L j j p j p z zk ij A u r z d + , ( 1,2,......, , , ) i m k x y = = (45) (47d) For the extended half-space, the horizontal displacement at the surface point ( ,0) x along k ( , k x y = ) direction has the following expression ( ,0) ( ,0) ks kf u u = x x ( ) ( ) ( ) * 1 0 ( ,0, ) [ ( ) j L G m j i zk j j j U r N d = + x * ( ) ( ) 2 ( ) ( ) * 0 ( ) ( ,0, ) ] p L j j p j G z zk j A u U r d + x ( ) ' ( ) '* ' , 1 0 ( ,0, ) { [ ( ) G L m k k j j k k x y j U r Q d = = + x ( 1,2,......, , , ) i m k x y = = (45) (45) where ( )( ) i kf z ( , ) k x y = is the free field rotary angle along the axis of the i-th pile due to the harmonic vertical load, ( ) ' ( , , ) G k k ijr z , ( ) ( , , ) G zk ijr z ( , , ) k k x y = represents the rotary angle at the center of ( ) z i due to a unit patch horizon- tal and vertical load applied on ( )j , respectively; ( )( , , ) G kk iir z z , ( )( , , ) G kk iir z z + ( , ) k x y = denote the rotary angle at the center of ( ) z i when the patch load applied on ( )i located at z and z+ , respectively. 6. NUMERICAL RESULTS AND DISCUSSIONS As in this study, ) , ( z takes different values at = z and + = z , integral equation (53) is one kind of weakly singular integral equation. Due to the discontinuity of ( , ) / z at z, thus, it is appropriate to divide the integral in (53) into two parts at point z, which leads to (50b) (50c) (50c) 0 0 ( , ) ( , ) ( , ) ( ) ( ) ( ) L z L z z z z d d d + = + (54) 0 (k, k = x, y) (50d) (50d) To implement the quadrature approach, the interval 0 z L is divided into N segments evenly. And within each segment 1 + j j z z of the N segments, ( ) is approximated by 6. NUMERICAL RESULTS AND DISCUSSIONS The integral equations accounting for the vertical and horizontal interaction between pile rows and the half-space can be solved numerically. The methodology for solving integral equations (39), (46) was detailed in [25]. To show the main idea for solving the second kind of Fredholm inte- gral equations in this paper, the Fredholm integral equations in this paper is denoted by the following simple form ( 1,2,..., , , ) i m k x y = = (49) ( 1,2,..., , , ) i m k x y = = ( 1,2,..., , , ) i m k x y = = (49) 0 ( , ) ( ) ( ) ( ) ( ), 0 L z a z z d f z z L + = (53) where where (53) ( ) 2 * ( ) ( ) ( ) * ( , ) ( , , ) j L p c G ij zk ij j p z U r z d E = , ( , ) k x y = (50a) ( ) ( ) ( ) 2 ( ) * 0 ( ) ( , , ) L d j j G ij p zk ij z A U r z d = , ( , ) k x y = (50b) ( ) ( ) ( ) 2 ( ) * ' 0 ( ) ( , , ) L c j j G ij p k k ij z A U r z d = , (k, k = x, y) (50c) ( ) ( ) ( ) 2 ( ) * ' 0 ( ) ( , , ) L b j j G ij p k k ij z A U r z d = , (k, k = x, y) (50d) (50a) where ( ) a z ( ) f z are known functions and should be con- tinuous within the interval 0 z L , ( ) is the unknown function to be determined, ( , ) / z is the kernel func- tion for the integral equation. 4.4. The Fredholm Integral Equations Accounting for the Horizontal Interaction the Piles and the Half-Space 46 ( ) ' ( ) '* ' , 1 0 ( ,0, ) { [ ( ) G L m k k ij j k k x y j U r Q d = = + ( ) ( ) ( ) ( ) ' '* 0 ( ) ( ,0, ) (0) ( )]} L G p a j c ij ij ij k z k U r d z + + ( ) ( ) ( ) ( ) * '* 1 1 (0)[ ( ) ] (0) ( ) m m p j d p j d k ij ij k ij j j u z u z = = + + ( ) (0) i kf u = , where A is a rectangle with its width and length determined by the wavelength of the Rayleigh wave and the width of pile rows. 5. DEFINITION OF THE AMPLITUDE REDUCTION RATIO To assess the isolation vibration effect of pile rows, the amplitude reduction ratio rA , which is the ratio between the amplitude of the vertical displacement of the half-space in the presence of the pile rows and that of the free field solu- tion, is defined as follows 1 1 ( ) ( ( ) ( )) / 2 , j j j j z z z z + + = + (55) (55) Substitution of (55) into the integration over the interval 1 + j j z z , one has 1 + j j , 1 ( , ) ( ) (1/ 2)[ ( ) j j z j z z d z + = 1 1 ( , ) ( )] j j z j z z z d + + + ( ) ( , 0) ( , 0) z zs r f u z A u z = = = x x (51) (51) where x y = + x i j, ( , 0) zs u z = x , ( )( , 0) z f u z = x where x y = + x i j, ( , 0) zs u z = x , ( )( , 0) z f u z = x is the amplitude of the vertical displacement for the half-space with the pile rows and the amplitude of the vertical dis- placement for the free field solution. The displacement ( )( , 0) z f u z = x can be obtained by performing the inverse Hankel transform on the frequency-wavenumber domain solution of the vertical displacement in Section 3. 4.4. The Fredholm Integral Equations Accounting for the Horizontal Interaction the Piles and the Half-Space Thus, using (30)-(36) and (48), the following sup- plementary equations for ( ) * (0) p i ku are derived Using equations (30)-(36) and (44), (45), the following second kind of Fredholm integral equations in the xoz and the yoz plane are obtained as follows ( ) ( ) ( ) * ( )[ ( , , ) ( , , )] i G G k kk ii kk ii Q z r z z r z z + ( ) ' ( ) '* ' , 1 0 ( , , ) { [ ( ) G L m k k ij i k k x y j r z Q d = = + ( ) ( ) ( ) '* ' ( ) ( ) * 0 0 1 ( ) ( ) ( ) ( , , ) G z L i a k ij k k ij j j p k z Q d r z d E I + ( ) ( ) ( ) ( )( ) '* '* (0) ( ) (0) ( )]} p j a p j b j k ij k ij z u z + + ( ) ( ) ( ) * 1 0 ( , , ) [ ( ) G L m j i zk ij j j r z N d = + ( ) ( ) ( ) * 1 0 ( ,0, ) [ ( ) G L m j i zk ij j j U r N d = ( ) ( ) ( ) * * 0 ( ) (0, ) (0) L j c p j ij z i N d u + + ( ) ( ) ( ) * 1 0 ( ,0, ) [ ( ) G L m j i zk ij j j U r N d = L ( ) ( ) ( ) ( ) * * ( ) (0, ) (0) (0)] L j c p j d ij z ij N d u + + ( ) ( ) ( ) ( ) * * ( ) (0, ) (0) (0)] L j c p j d ij z ij N d u + + 46 The Open Civil Engineering Journal, 2010, Volume 4 Xu et al. 5. DEFINITION OF THE AMPLITUDE REDUCTION RATIO 1 1 (1/ 2)[ ( ) ( )][ ( , ) ( , )] j j j j z z z z z z + + = + , 1 j j z z + (56) 1 1 (1/ 2)[ ( ) ( )][ ( , ) ( , )] j j j j z z z z z z + + = + , 1 1 (1/ 2)[ ( ) ( )][ ( , ) ( , )] j j j j z z z z z z + + = + , (56) 1 j j z z + (56) 1 j j z z + Using (56) to evaluate the integrations over the N sub- intervals in (53), then, the integral equation (53) is reduced to the following discrete form Woods [26] proposed an average amplitude reduction ratio rA for assessing the isolation vibration effect. The av- erage amplitude reduction ratio rA is defined as follows: 1 ( ) ( ) (1/ 2)[ ( ) N i i j j a z z z = + 1 1 ( )][ ( , ) ( , )] ( ) j j i j i i z z z z z f z + + + = , 1,2,3... 1 i N = + (57) 1 ( ) ( ) (1/ 2)[ ( ) N i i j j a z z z = + 1 1 ( )][ ( , ) ( , )] ( ) j j i j i i z z z z z f z + + + = , 1 r r A A A dA A = (52) 1 r r A A A dA A = (52) 1,2,3... 5. DEFINITION OF THE AMPLITUDE REDUCTION RATIO 1 i N = + (57) (57) The Open Civil Engineering Journal, 2010, Volume 4 4 A Semi-Analytical Model for the Simulation 47 It is worth stressing that when 1 j + in the above equation equal to i , then, 1 ( , ) j i z z + will assume the values ) , ( i i z z , as the interval 1 j j z z + infinitely ap- proaches the point iz form the above; on the other hand, when j i = , then, ( , ) j i z z should take the value ) , ( i i z z + as the interval 1 j j z z + is infinitely close to the point iz from the below. observed that the amplitude reduction ratio r A right behind the pile rows is much smaller than those at other areas. For further comparison, the average amplitude reduction ratio rA for the quasi elastic half-space is calculated, which is 0.718, while the result of Kattis [6] for the elastic half-space is 0.712. The difference between the present solution and that of Kattis’ [6] is only 0.842%. Fig. (4). The contour of the amplitude reduction ratio rA for a single pile row with eight piles used as the passive system. 0.59 0.63 0.68 0.72 0.76 0.80 0.84 0.76 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.0 0.5 0.0 -0.5 -1.0 a single pile row with 8 piles embedded in an elastic half space / R y  / R x  a single pile row with 8 piles embedded in an elastic half space 0.59 0.63 0.68 0.72 0.76 0.80 0.84 0.76 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.0 0.5 0.0 -0.5 -1.0 a single pile row with 8 piles embedded in an elastic half space / R y  / R x  Discrete formula (57) can be used to reduce the integral equations (39), (46) to linear algebraic equations. After the numerical solution of the linear algebraic equations, the vertical displacement of the half-space can be obtained via (41) and the amplitude reduction ratio can be evaluated by (51) and (52). To verify the proposed method, a special case of our model will be compared with an existing result in Sec. 6.1. Comparison of our Result with an Existing Result The parameters for each pile assume the following values: 1.0 m d = , 5.0 m L = , 10 2 4.526 10 N/m p E = , 3 3 2.35 10 kg/m p = . The net spacing between two neighboring piles is 0.5 m s = . The vibration source is a harmonic vertical uniform patch load with magnitude 2 200 kN/m F Q = and applied over a circu- lar area with the diameter 0.8 m D = . Its frequency is 50 Hz f = . The distance between the pile row and the cen- ter of the vibration source is 7.5 m s d = (see Fig. 1). To reduce the poroelastic half-space to a quasi-elastic half- space, the parameters M , m , , p b , f are set to be 0.0001 Fig. (4). The contour of the amplitude reduction ratio rA for a single pile row with eight piles used as the passive system. 5. DEFINITION OF THE AMPLITUDE REDUCTION RATIO 6.1. Also, some numerical examples and corresponding analysis are given in Sec. 6.2 and Sec. 6.3. 6.2. Numerical Simulation of the Vibration Isolation using a Single Pile Row In this section, the passive isolation vibration system is a single pile row with nine piles embedded in a poroelastic half-space. The vibration source is a harmonic vertical uni- form patch load with the magnitude F Q and applied over a circular area with the diameter D and its frequency is f . The reference Rayleigh wavelength for the half-space is 5.0 m R = . The parameters for the poroelastic half-space, pile rows and the vibration source are given in Table 1. In the following, the influences of Biot’s parameter (M), Young’s modulus of the pile ( p E ), the pile length ( L ) and as in [27]. It should be noted that for an elastic medium there are singularities in the path of the integration when evaluat- ing the fundamental solution, which makes the numerical evaluation of the integral a formidable task. However, some researchers tend to evaluate this kind of integrals by use of a special viscoelastic model. In the viscoelastic model, the material damping is taken into account by using complex Lame constants i.e. 0(1 i ) s μ μ = + and 0(1 i ) s = + , where s denotes the damping ratio. In this paper, we use a damping ratio of 0.05 s = for the half-space and 8 2 0 1.32 10 N/m μ = , 8 2 0 1.32 10 N/m = , 3 3 1.75 10 kg/m s = . According to [6], the reference Rayleigh wavelength for the quasi elastic half-space is 5.0 m R = . the net spacing between two neighboring piles in a pile row (s) on the vibration isolation effect will be discussed. Note that when the influence of one parameter is examined, all the other parameters will remain fixed and take the typical val- ues as given in Table 1. 6.1. Comparison of our Result with an Existing Result As the first example, a special case of our solution is compared with an existing result. In our solution, a single pile row with eight piles embedded in the poroelastic half- space is used as the passive vibration isolation system again a harmonic vertical patch load. The parameters for each pile assume the following values: 1.0 m d = , 5.0 m L = , 10 2 4.526 10 N/m p E = , 3 3 2.35 10 kg/m p = . The net spacing between two neighboring piles is 0.5 m s = . The vibration source is a harmonic vertical uniform patch load with magnitude 2 200 kN/m F Q = and applied over a circu- lar area with the diameter 0.8 m D = . Its frequency is 50 Hz f = . The distance between the pile row and the cen- ter of the vibration source is 7.5 m s d = (see Fig. 1). To reduce the poroelastic half-space to a quasi-elastic half- space, the parameters M , m , , p b , f are set to be 0.0001 as in [27]. It should be noted that for an elastic medium there are singularities in the path of the integration when evaluat- ing the fundamental solution, which makes the numerical evaluation of the integral a formidable task. However, some researchers tend to evaluate this kind of integrals by use of a special viscoelastic model. In the viscoelastic model, the material damping is taken into account by using complex Lame constants i.e. 0(1 i ) s μ μ = + and 0(1 i ) s = + , where s denotes the damping ratio. In this paper, we use a damping ratio of 0.05 s = for the half-space and 8 2 0 1.32 10 N/m μ = , 8 2 0 1.32 10 N/m = , 3 3 1.75 10 kg/m s = . According to [6], the reference Rayleigh wavelength for the quasi elastic half-space is 5.0 m R = . As the first example, a special case of our solution is compared with an existing result. In our solution, a single pile row with eight piles embedded in the poroelastic half- space is used as the passive vibration isolation system again a harmonic vertical patch load. 6.2.1. The Effect of Biot’s Parameter (M) To study the influence of Biot’s parameter (M) on the vibration isolation effect of the pile row, Biot’s parameter (M) takes the following three different values: 8 2.0 10 Pa M = , 10 2.0 10 Pa and 12 2.0 10 Pa , respec- tively. The other parameters for the piles, the vibration source and the poroelastic half-space assume the values as given in Table 1. Fig. (4) shows the contour of the amplitude reduction ratio r A for the single pile row embedded in the quasi elas- tic half-space according to the present model. It can be Fig. 5(a)-(c) shows the amplitude reduction ratio r A on the surface for the poroelastic half-space for 48 The Open Civil Engineering Journal, 2010, Volume 4 Xu et al. Table 1. The Parameters for the Poroelastic Half-Space, Pile Rows and the Vibration Source in Sec. 6.2 Parameters Values (Pa) 2.6 X 107 (Pa) 2.6 X 107 s (kg/m3) 2.6 X 103 f (kg/m3) 1.0 X 103 0.3 a 2.0 0.97 M (N/m2) 2.8 X 108 bp (kg/(m3 .s)) 1.0 X 1010 D (the diameter of the circular area subjected to the vibration source) (m) 0.8 f (the frequency of the vibration source) (Hz) 20.0 QF (the magnitude of the harmonic vertical uniform load) (kN) 100 ds (the distance between the center of the first pile row and the vibration source ) (m) 7.5 d (the diameter of the piles) (m) 0.8 L (the pile length) (m) 10.0 Ep/Es 50 p (the density of the piles) ( kg/m3) 3.9 X 103 s (m) 0.8 q (the spacing between the adjacent pile rows) (m) 0.4 . The Parameters for the Poroelastic Half-Space, Pile Rows and the Vibration Source in Sec. 6.2 q (the spacing between the adjacent pile rows) (m) / p s E E =100, 200 and 500, respectively. Note that the result for the case of / p s E E =50 has already been presented in Sec.6.2.1 (see Fig. 5(a)). Fig. (8) illustrates the amplitude reduction ratio r A within the range / 0 ~ 2 R x = and 0 y = for the poroelastic half-space when / p s E E takes 50, 100, 200 and 500, respectively. 6.2.1. The Effect of Biot’s Parameter (M) The average amplitude re- duction ratio r A for the four cases of modulus ratios are: for / 50 p s E E = , 0.6586 r A = ; for / 100 p s E E = , 0.5926 r A = ; for / 200 p s E E = , 0.5644 r A = ; for / 500 p s E E = , 0.5489 r A = . One can be see clearly that the average amplitude reduction ratio rA decreases with the increasing / p s E E . Moreover, when / 200 p s E E , rA only decreases slightly with the increasing / p s E E . As a result, it can be concluded that a stiffer pile row will produce a better isolation vibration effect. 8 2.0 10 Pa M = , 10 2.0 10 Pa and 12 2.0 10 Pa , respec- tively. Fig. (6) illustrates the amplitude reduction ratio r A within the range / 0 ~ 2 R x = and 0 y = when Biot’s pa- rameter takes the above three values. The average amplitude reduction ratio r A for the three cases of Biot’s parameter (M) are as follows: for 8 2.0 10 Pa M = , 0.6586 r A = ; for 10 2.0 10 Pa M = , 0.6176 r A = ; for 12 2.0 10 Pa M = , 0.6166 r A = . One can see that the average amplitude re- duction ratio rA decreases slightly with the increasing M, which shows that Biot’s parameter M has a small effect on the vibration isolation effect of the pile row. 6.2.2. The Effect of Young’s Modulus of the Piles ( p E ) Young’s modulus ( p E ) of the pile is an important pa- rameter for the design of the pile row isolation system. Herein, the effect of Young’s modulus of the piles on the vibration isolation effect will be investigated. In this exam- ple, Young’s modulus of the piles assumes the following four values: / p s E E =50, 100, 200 and 500, respectively, with s E fixed and given in Table 1. 6.2.3. The Effect of the Pile Length The contour of the amplitude reduction ratio rA for a si different values of Biot’s parameter M: (a) 8 2.0 10 Pa M = ; (b) M 0.48 0.54 0.64 0.70 0.70 0.64 0.54 0.70 0.75 0.75 0.81 0.81 0.64 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.0 0.5 0.0 -0.5 -1.0 8 2.0 10 Pa M   / R y  / R x  0.58 0.61 0.64 0.67 0.61 0.64 0.58 0.70 0.70 0.67 0.58 0.58 0.70 0.73 0.73 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.0 0.5 0.0 -0.5 -1.0 12 2.0 10 Pa M   / R y  / R x  0.58 0.61 0.64 0.67 0.61 0.64 0.70 0.70 0.58 0.67 0.70 0.73 0.73 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.0 0.5 0.0 -0.5 -1.0 10 2.0 10 Pa M   / R y  / R x  8 2.0 10 Pa M   0.48 0.54 0.64 0.70 0.70 0.64 0.54 0.70 0.75 0.75 0.81 0.81 0.64 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.0 0.5 0.0 -0.5 -1.0 8 2.0 10 Pa M   / R y  / R x  10 2.0 10 Pa M   / R y  / R x  a b c Fig. (5). The contour of the amplitude reduction ratio rA for a single pile row with nine piles embedded in the poroelastic half space for different values of Biot’s parameter M: (a) 8 2.0 10 Pa M = ; (b) 10 2.0 10 Pa M = ; (c) 12 2.0 10 Pa M = . 6.2.3. The Effect of the Pile Length The influence of the pile length on the isolation vibration effect is examined in this section. The pile length takes three different values: L = 5.0 m, 10.0 m and 15.0 m, respec- tively. Fig. 7(a)-(c) shows the variation of the amplitude reduc- tion ratio r A on the surface of the poroelastic half-space for The Open Civil Engineering Journal, 2010, Volume 4 49 A Semi-Analytical Model for the Simulation 0.58 0.61 0.64 0.67 0.61 0.64 0.70 0.70 0.58 0.67 0.70 0.73 0.73 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.0 0.5 0.0 -0.5 -1.0 10 2.0 10 Pa M   / R y  / R x  a b c Fig. (5). The contour of the amplitude reduction ratio rA for a single pile row with nine piles embedded in the poroelastic half space for different values of Biot’s parameter M: (a) 8 2.0 10 Pa M = ; (b) 10 2.0 10 Pa M = ; (c) 12 2.0 10 Pa M = . 0.48 0.54 0.64 0.70 0.70 0.64 0.54 0.70 0.75 0.75 0.81 0.81 0.64 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.0 0.5 0.0 -0.5 -1.0 8 2.0 10 Pa M   / R y  / R x  0.58 0.61 0.64 0.67 0.61 0.64 0.70 0.70 0.58 0.67 0.70 0.73 0.73 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.0 0.5 0.0 -0.5 -1.0 10 2.0 10 Pa M   / R y  / R x  0.58 0.61 0.64 0.67 0.61 0.64 0.58 0.70 0.70 0.67 0.58 0.58 0.70 0.73 0.73 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.0 0.5 0.0 -0.5 -1.0 12 2.0 10 Pa M   / R y  / R x  a c Fig. (5). 6.2.3. The Effect of the Pile Length 0.58 0.61 0.64 0.67 0.61 0.64 0.58 0.70 0.70 0.67 0.58 0.58 0.70 0.73 0.73 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.0 0.5 0.0 -0.5 -1.0 12 2.0 10 Pa M   / R y  / R x  a c 0.58 0.61 0.64 0.67 0.61 0.64 0.58 0.70 0.70 0.67 0.58 0.58 0.70 0.73 0.73 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.0 0.5 0.0 -0.5 -1.0 12 2.0 10 Pa M   / R y  / R x  b b a 12 2.0 10 Pa M   12 2.0 10 Pa M   c Fig. (5). The contour of the amplitude reduction ratio rA for a single pile row with nine piles embedded in the poroelastic half space for different values of Biot’s parameter M: (a) 8 2.0 10 Pa M = ; (b) 10 2.0 10 Pa M = ; (c) 12 2.0 10 Pa M = . 0.0 0.4 0.8 1.2 1.6 2.0 0.60 0.65 0.70 0.75 0.80 Ar 12 2.0 10 Pa M   8 2.0 10 Pa M   10 2.0 10 Pa M   / R x  Fig. (6). Variation of the amplitude reduction ratio r A for a single pile row with nine piles embedded in the poroelastic half space within the range 0 / 2 R x and 0 y = when Biot’s parameter (M) takes the three different values. 0.0 0.4 0.8 1.2 1.6 2.0 0.60 0.65 0.70 0.75 0.80 Ar 12 2.0 10 Pa M   8 2.0 10 Pa M   10 2.0 10 Pa M   / R x  Fig. (6). Variation of the amplitude reduction ratio r A for a single pile row with nine piles embedded in the poroelastic half space within the range 0 / 2 R x and 0 y = when Biot’s parameter (M) takes the three different values. 50 The Open Civil Engineering Journal, 2010, Volume 4 Xu et al. a b c Fig. (7). 6.2.3. The Effect of the Pile Length The contour of the amplitude reduction ratio Ar for a single pile row with nine piles embedded in the poroelastic half space for differen values of the ratio of Young’s modulus between the piles and the poroelastic half space: (a) Ep / Es 100; (b) Ep / Es = 200; (c) Ep / Es 500. 6.2.3. The Effect of the Pile Length 0.46 0.51 0.55 0.60 0.65 0.60 0.55 0.51 0.70 0.70 0.60 0.74 0.74 0.79 0.79 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.0 0.5 0.0 -0.5 -1.0 / 100 p s E E  / R y  / R x  0.52 0.57 0.62 0.57 0.52 0.47 0.47 0.67 0.67 0.57 0.73 0.73 0.78 0.78 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.0 0.5 0.0 -0.5 -1.0 / 200 p s E E  / R y  / R x  0.56 0.61 0.56 0.51 0.45 0.45 0.51 0.66 0.66 0.56 0.72 0.72 0.77 0.77 0.51 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.0 0.5 0.0 -0.5 -1.0 / 500 p s E E  / R y  / R x  Ar / 50 E E 0.46 0.51 0.55 0.60 0.65 0.60 0.55 0.51 0.70 0.70 0.60 0.74 0.74 0.79 0.79 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.0 0.5 0.0 -0.5 -1.0 / 100 p s E E  / R y  / R x  0.52 0.57 0.62 0.57 0.52 0.47 0.47 0.67 0.67 0.57 0.73 0.73 0.78 0.78 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.0 0.5 0.0 -0.5 -1.0 / 200 p s E E  / R y  / R x  0.52 0.57 0.62 0.57 0.52 0.47 0.47 0.67 0.67 0.57 0.73 0.73 0.78 0.78 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.0 0.5 0.0 -0.5 -1.0 / 200 p s E E  / R y  / R x  / 100 p s E E  0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 / R x  a b c Fig (7) The contour of the amplitude reduction ratio A for a single pile row with nine piles embedded in the poroelastic half space for differen 0.56 0.61 0.56 0.51 0.45 0.45 0.51 0.66 0.66 0.56 0.72 0.72 0.77 0.77 0.51 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.0 0.5 0.0 -0.5 -1.0 / 500 p s E E  / R y  / R x  a 0.56 0.61 0.56 0.51 0.45 0.45 0.51 0.66 0.66 0.56 0.72 0.72 0.77 0.77 0.51 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.0 0.5 0.0 -0.5 -1.0 / 500 p s E E  / R y  / R x  b b a b c Fig. 6.2.4. The Effect of the Pile-Pile Net Spacing The net spacing between two neighboring piles in a pile row is an important parameter for the design of pile rows for vibration isolation. In this section, the value of s takes the following three values: 0.8 m, 1.2 m and 1.6 m, respec- tively. The other parameters for the pile, the poroelastic half- space and the vibration source take the values as given in Table 1. L =5.0 m and 15.0 m, respectively. The result for the case of L =10.0 m is illustrated in Fig. 5(a). Fig. (10) illustrates that the amplitude reduction ratio r A within / 0 ~ 2 R x = and 0 y = for the poroelastic half-space when the pile length takes L = 5.0 m, 10.0 m and 15.0 m, respectively. The aver- age amplitude reduction ratios r A for L = 5.0 m, 10.0 m and 15.0 m equal to 0.8653, 0.6586, 0.6884, respectively. It can be seen that the length of piles has a significant influence on the average amplitude reduction ratios r A when 10.0 m L . However, when L is larger than 10.0 m, with increase of the pile length, r A only change slightly, which agrees with the result in [7]. Fig. 11(a), (b) shows the variation of the amplitude re- duction ratio r A on the surface of the poroelastic half-space for s =1.2 m and 1.6 m, respectively. The result for the case of s =0.8 m is given in Fig. 5(a). Fig. 5(a) and Fig. (11) clearly indicate the decay of the isolation vibration effect behind the pile row with the in- a b Fig. (9). The contour of the amplitude reduction ratio Ar for the single pile row with nine piles for different values of the pile length: (a) L = 5.0 m; (b) L = 15.0 m. 6.2.3. The Effect of the Pile Length (7). The contour of the amplitude reduction ratio Ar for a single pile row with nine piles embedded in the poroelastic half space for different values of the ratio of Young’s modulus between the piles and the poroelastic half space: (a) Ep / Es 100; (b) Ep / Es = 200; (c) Ep / Es 500. 0.56 0.61 0.56 0.51 0.45 0.45 0.51 0.66 0.66 0.56 0.72 0.72 0.77 0.77 0.51 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.0 0.5 0.0 -0.5 -1.0 / 500 p s E E  / R y  / R x  a / 500 p s E E  Fig. (7). The contour of the amplitude reduction ratio Ar for a single pile row with nine piles embedded in the poroelastic half space for different values of the ratio of Young’s modulus between the piles and the poroelastic half space: (a) Ep / Es 100; (b) Ep / Es = 200; (c) Ep / Es 500. Fig. (8). The amplitude reduction ratio Ar within the range 0 x / R 2 and y = 0 when the ratio of Young’s modulus between the piles and the poroelastic half takes the values 50, 100, 200 and 500, respectively. 0.0 0.4 0.8 1.2 1.6 2.0 0.5 0.6 0.7 Ar / 500 p s E E  / 200 p s E E  / 100 p s E E  / 50 p s E E  / R x  0.0 0.4 0.8 1.2 1.6 2.0 0.5 0.6 0.7 Ar / 500 p s E E  / 200 p s E E  / 100 p s E E  / 50 p s E E  / R x  Fig. (8). The amplitude reduction ratio Ar within the range 0 x / R 2 and y = 0 when the ratio of Young’s modulus between the piles and the poroelastic half takes the values 50, 100, 200 and 500, respectively. A Semi-Analytical Model for the Simulation The Open Civil Engineering Journal, 2010, Volume 4 51 Figs. 9(a), (b) show the variation of the amplitude reduc- tion ratio r A on the surface of the poroelastic half-space for 6.3. Numerical Simulation of the Vibration Isolation using Multiple Pile Rows In the following, the influences of the number of the pile rows, the net spacing between two neighboring piles and the spacing between neighboring pile rows on the vibration iso- lation effect are discussed. It is noted that when the influence of one parameter is examined, all the other parameters will remain unchanged and take the typical values as given above and in Table 1. In this section, the vibration isolation effect of multiple pile rows is investigated. The vibration source is a harmonic vertical uniform patch load with the magnitude F Q and the frequency f . The patch load is applied over a circular do- main with the diameter D . A single pile row with seven 0.65 0.69 0.74 0.74 0.78 0.69 0.82 0.78 0.82 0.87 0.74 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.5 1.0 0.5 0.0 -0.5 -1.0 -1.5 s =1.6 m / R y  / R x  a b Fig. (11). The contour of the amplitude reduction ratio Ar for the single pile row with nine piles for different values of the net spacing (s) between two neighboring piles: (a) s = 1.2 m; (b) s = 1.6 m. 0.61 0.65 0.69 0.73 0.77 0.77 0.65 0.81 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.5 1.0 0.5 0.0 -0.5 -1.0 -1.5 s =1.2 m / R y  / R x  0.65 0.69 0.74 0.74 0.78 0.69 0.82 0.78 0.82 0.87 0.74 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.5 1.0 0.5 0.0 -0.5 -1.0 -1.5 s =1.6 m / R y  / R x  0.61 0.65 0.69 0.73 0.77 0.77 0.65 0.81 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.5 1.0 0.5 0.0 -0.5 -1.0 -1.5 s =1.2 m / R y  / R x  b a Fig. (11). The contour of the amplitude reduction ratio Ar for the single pile row with nine piles for different values of the net spacing (s) between two neighboring piles: (a) s = 1.2 m; (b) s = 1.6 m. Fig. (12). 6.2.4. The Effect of the Pile-Pile Net Spacing 0.75 0.75 0.77 0.80 0.83 0.85 0.77 0.88 0.80 0.90 0.90 0.90 0.93 0.72 0.72 0.72 0.72 0.83 0.83 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.0 0.5 0.0 -0.5 -1.0 L =5.0 m / R y  / R x  0.65 0.68 0.68 0.65 0.65 0.65 0.72 0.75 0.79 0.79 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.0 0.5 0.0 -0.5 -1.0 L =15.0 m / R y  / R x  0.75 0.75 0.77 0.80 0.83 0.85 0.77 0.88 0.80 0.90 0.90 0.90 0.93 0.72 0.72 0.72 0.72 0.83 0.83 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.0 0.5 0.0 -0.5 -1.0 L =5.0 m / R y  / R x  0.65 0.68 0.68 0.65 0.65 0.65 0.72 0.75 0.79 0.79 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 1.0 0.5 0.0 -0.5 -1.0 L =15.0 m / R y  / R x  b Fig. (9). The contour of the amplitude reduction ratio Ar for the single pile row with nine piles for different values of the pile length: (a) L = 5.0 m; (b) L = 15.0 m. A Fig. (10). The amplitude reduction ratio Ar within the range 0 x / R 2 and y = 0 when the pile length takes the values L = 5.0 m, 10.0 m and 15.0 m, respectively. 0.0 0.4 0.8 1.2 1.6 2.0 0.6 0.7 0.8 0.9 Ar L=5.0 m L=10.0 m L=15.0 m / R x  0.0 0.4 0.8 1.2 1.6 2.0 0.6 0.7 0.8 0.9 Ar L=5.0 m L=10.0 m L=15.0 m / R x  Fig. (10). The amplitude reduction ratio Ar within the range 0 x / R 2 and y = 0 when the pile length takes the values L = 5.0 m, 10.0 m and 15.0 m, respectively. The Open Civil Engineering Journal, 2010, Volume 4 Xu et al. Xu et al. 52 creasing pile net spacing s. The average amplitude reduction ratios for the three cases of net spacing are: for s = 0.8 m, r A =0.6586; for s = 1.2 m , 0.7133; for s = 1.6 m, 0.7486, which shows that the average amplitude reduction ratio r A has a considerable increase with the increasing net spacing s. 6.2.4. The Effect of the Pile-Pile Net Spacing Thus, it can be concluded that a better isolation vibration effect can be realized by smaller net pile spacing. piles, two pile rows with seven and six piles and three pile rows with seven, six and seven piles, respectively are con- sidered in this example (Fig. 12), respectively. The net spac- ing between two neighboring piles in a pile row is 0.2 m s = . The other parameters for the pile, the poroelastic half-space and the vibration source are given in Table 1 ex- cept that 0.4 m d = and 9 2 1.735 10 N/m p E = . The refer- ence Rayleigh wavelength is assumed to be 5.0 m R = . 6.3. Numerical Simulation of the Vibration Isolation using Multiple Pile Rows Three kinds of pile rows used as vibration isolation system for the poroelastic half space subjected to a harmonic vertical load: (a) a single pile row with seven piles; (b) two pile rows with seven and six piles, respectively; (c) three piles rows with seven, six and seven piles, respectively. s x o (a) (b) d o s s q q o d y x x y (c) q y s s s x q o d y (c) q s s s s y Fig. (12). Three kinds of pile rows used as vibration isolation system for the poroelastic half space subjected to a harmonic vertical load: (a) a single pile row with seven piles; (b) two pile rows with seven and six piles, respectively; (c) three piles rows with seven, six and seven piles, respectively. The Open Civil Engineering Journal, 2010, Volume 4 53 A Semi-Analytical Model for the Simulation 53 6.3.1. The Effect of the Number of the Pile Rows ratios for the single pile row, the two piles rows, the three pile rows are r A =0.7118, 0.5408, 0.4466, respectively. In this example, we consider the following three cases of pile rows embedded in the poroelastic half-space: a single pile row with seven piles; two pile rows with seven and 6 piles, respectively; three pile rows with seven, six and seven piles, respectively (Fig. 12). All the parameters for the pile row, the poroelastic half-space and the circular patch load assume the values as given above and in Table 1. 6.3.2. The Effect of the Net Spacing Between Two Neigh- boring Piles In this section, we consider two pile rows embedded in the poroelastic half-space. The first and second pile row has seven and six piles, respectively (Fig. 12(b)). The net spac- ing between two neighboring piles in a pile row takes the following three different values: s = 0.2 m, 0.4 m, 0.8 m, respectively, while the parameters for the poroelastic half- space, the piles and the vibration source assume the typical values as given above and in Table 1. Fig. 13(a)-(c) plot the variation of the amplitude reduc- tion ratio r A on the surface of the poroelastic half-space for the three different kinds of the pile rows. The variation of the amplitude reduction ratios r A within / 0 ~ 2 R x = and 0 y = for three cases of pile rows are also shown in Fig. (14). Fig. 15 (a), (b) plot the amplitude reduction ratio r A on the surface of the poroelastic half-space for s = 0.4 m and 0.8 m, respectively. Note that the result of r A for the two pile rows with s = 0.2 m is given in Fig. 13(b). For the two pile rows with s = 0.2 m, 0.4 m, 0.8 m, respectively, the As expected, it follows from Fig. (13) that the increase of the number of pile rows will enhance the isolation vibration effect of the pile rows. The average amplitude reduction a b c Fig. (13). The variation of the amplitude reduction ratio Ar on the surface for the poroelastic half space for three different piles rows: (a) the single pile row with seven piles; (b) two pile rows with seven and six piles, respectively; (c) three piles rows with seven, six and seven piles, respectively. 6.3.1. The Effect of the Number of the Pile Rows 0.72 0.72 0.69 0.66 0.63 0.60 0.57 0.3 0.2 0.1 0.0 -0.1 -0.2 -0.3 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 a single pile row with seven piles / R y  / R x  0.93 0.83 0.73 0.64 0.54 0.44 0.34 0.3 0.2 0.1 0.0 -0.1 -0.2 -0.3 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 two pile rows with seven, six piles / R y  / R x  0.93 0.84 0.75 0.67 0.58 0.49 0.40 0.32 0.32 0.32 0.23 0.3 0.2 0.1 0.0 -0.1 -0.2 -0.3 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 three pile row with seven, six, seven piles / R y  / R x  0.93 0.83 0.73 0.64 0.54 0.44 0.34 0.3 0.2 0.1 0.0 -0.1 -0.2 -0.3 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 two pile rows with seven, six piles / R y  / R x  0.93 0.83 0.73 0.64 0.54 0.44 0.34 0.3 0.2 0.1 0.0 -0.1 -0.2 -0.3 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 two pile rows with seven, six piles / R y  / R x  0.72 0.72 0.69 0.66 0.63 0.60 0.57 0.3 0.2 0.1 0.0 -0.1 -0.2 -0.3 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 a single pile row with seven piles / R y  / R x  a single pile row with seven piles / R x  b a 0.93 0.84 0.75 0.67 0.58 0.49 0.40 0.32 0.32 0.32 0.23 0.3 0.2 0.1 0.0 -0.1 -0.2 -0.3 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 three pile row with seven, six, seven piles / R y  / R x  three pile row with seven, six, seven piles three pile row with seven, six, seven piles Fig. (13). The variation of the amplitude reduction ratio Ar on the surface for the poroelastic half space for three different piles rows: (a) the single pile row with seven piles; (b) two pile rows with seven and six piles, respectively; (c) three piles rows with seven, six and seven piles, respectively. Fig. (13). 7. CONCLUSIONS The numerical simulation of the isolation of the vibration due to a harmonic vertical load applied on the surface of a poroelastic half-space by pile rows has been carried out in this study. The second kind of Fredholm integral equations describing the dynamic interaction between the piles and the half space are obtained using Muki’s method. The vibration isolation effect of pile rows is analyzed via the proposed semi-analytical model. As the semi-analytical model pro- posed in this paper avoids the discretization of the whole calculation domain, thus, it can save CPU time substantially for the current full 3-D problem. To investigate the vibration isolation effect of pile rows, the influence of Biot’s parame- ter (M), Young’s modulus of the pile ( p E ), the pile length (L), the spacing between two neighboring piles (s) in a pile row are investigated via the semi-analytical model. Also, the influence of the number of the pile rows (n) and the spacing (q) between two adjacent pile rows are examined. Fig. (14). The variation of the amplitude reduction ratio Ar within the range 0 x / R 2 and y = 0 for three kinds of pile rows. 6.3.3. The Effect of the Spacing Between Two Adjacent Pile Rows To examine the effect of the spacing between neighbor- ing pile rows, only two pile rows with the number of piles seven, six in the first and the second row, respectively are considered in this section (see Fig. 12(b)). The value of q takes 0.4 m, 0.8 m and 1.2 m, respectively, while the parameters for the poroelastic half-space, the piles and the From the numerical results of this study, the following conclusions can be drawn: 1. The larger M corresponds to a slightly better vibration isolation effect than the smaller M. 1. The larger M corresponds to a slightly better vibration isolation effect than the smaller M. a b Fig. (15). The variation of the amplitude reduction ratio r A on the surface for the poroelastic half space for two pile rows with seven and six piles, respectively, for different values of the net spacing (s) between two neighboring piles: (a) s = 0.4 m; (b) s = 0.8 m. Xu et al. 54 vibration source are given as above and in Table 1. vibration source are given as above and in Table 1. average amplitude reduction ratios are r A =0.5408, 0.6299, 0.7583, respectively. One can see that the average amplitude reduction ratio r A for multiple pile rows increase with the increasing net spacing s. Thus, it can be concluded that the pile rows with a smaller separation between neighboring piles is generally more efficient in vibration isolation. Fig. 16(a), (b) depicts the amplitude reduction ratio r A on the surface of the half-space for q = 0.8 m and 1.2 m respectively. The result of r A for the case of q = 0.4 m is given in Fig. 13(b). For the two pile rows with q = 0.4 m, 0.8 m and 1.2 m, the average amplitude reduction ratios are r A = 0.5408, 0.5482, 0.5568, respectively. From the result for the average amplitude reduction ratios and Fig. (16), one can observe that compared with other parameters, the space between two adjacent pile rows has a relatively smaller ef- fect on the vibration isolation of the pile rows. Fig. (14). The variation of the amplitude reduction ratio Ar within the range 0 x / R 2 and y = 0 for three kinds of pile rows. 6.3.3. The Effect of the Spacing Between Two Adjacent Pile Rows 0.0 0.4 0.8 1.2 1.6 2.0 0.0 0.2 0.4 0.6 0.8 1.0 Ar one pile row with seven piles two piles rows with seven, six piles three piles rows with seven, six, seven piles / R x  0.0 0.4 0.8 1.2 1.6 2.0 0.0 0.2 0.4 0.6 0.8 1.0 Ar one pile row with seven piles two piles rows with seven, six piles three piles rows with seven, six, seven piles / R x  one pile row with seven piles two piles rows with seven, six piles three piles rows with seven, six, seven pile 6.3.1. The Effect of the Number of the Pile Rows The variation of the amplitude reduction ratio Ar on the surface for the poroelastic half space for three different piles rows: (a) the single pile row with seven piles; (b) two pile rows with seven and six piles, respectively; (c) three piles rows with seven, six and seven piles, respectively. Fig. (13). The variation of the amplitude reduction ratio Ar on the surface for the poroelastic half space for three different piles rows: (a) the single pile row with seven piles; (b) two pile rows with seven and six piles, respectively; (c) three piles rows with seven, six and seven piles, respectively. The Open Civil Engineering Journal, 2010, Volume 4 Xu et al. 7. CONCLUSIONS Biot, “Theory of propagation of elastic waves in a fluid- saturated porous solid, II: higher frequency range”, J. Acoust. Soc. Am., Vol. 28, pp. 179-191, 1956. [10] M. A. Biot, “Mechanics of deformation and acoustic propagation in porous media”, J. Appl. Phys., Vol. 33, pp. 1482-1498, 1962. [11] X. Zeng, and R. K. N. D. Rajapakse, “Dynamic axial load transfer from elastic pile to poroelastic medium”, J. Eng. Mech., ASCE, 1999, 125: 1048-1055. [12] J. H. Wang, X. L. Zhou, and J. F. Lu, “Dynamic response of pile groups embedded in a poroelastic medium”, Soil Dyn. Earthquake Eng., Vol. 23, pp. 235-242, 2003. [13] B. Jin, and Z. Zhong, “Lateral dynamic compliance of pile embed- ded in poroelastic half-space”, Soil Dyn. Earthquake Eng, Vol. 21, pp. 519-525, 2001. [14] J. F. Lu, and D. S. Jeng “Poroelastic model for pile-soil interaction in a half-space porous medium due to seismic waves”, Int. J. Numer. Anal. Meth. Geomech., Vol. 32, pp. 1-41, 2008. [15] Y. Q. Cai, G. Y. Ding, and C. J. Xu, “Screening of plane S waves by an array of rigid piles in poroelastic soil”, J. Zhejiang Univ. Sci. A, Vol. 9, pp. 589-599, 2008. [16] Y. Q. Cai, G. Y. Ding, and C. J. Xu, “Amplitude reduction of elas- tic waves by a row of piles in poroelastic soil”, Comput. Geotech, Vol. 36, pp. 463-473, 2009. [17] J.F. Lu, B. Xu, and J. H. Wang, “Numerical analysis of isolation of the vibration due to moving loads using pile rows”, J. Sound Vi- brat., Vol. 319, pp. 940-962, 2009. [18] J.F. Lu, B. Xu, and J. H. Wang, “A numerical model for the isola- tion of moving-load induced vibrations by pile rows embedded in layered porous media” Int J Solids Struct Vol 46 pp 3771- [3] B. Dasgupta, D. E. Beskos, and I. G. Vordouclakis, “Vibration isolation using open or filled trenches. Part 2: 3-D homogeneous soil”, Comput. Mech., Vol. 6, pp. 129-142, 1990. 2. The length of the piles is a most important factor for the vibration isolation effect. Usually, longer piles will pro- duce a better vibration isolation effect. The recom- mended optimal length of the piles for vibration isolation purpose ranges between 1.5 ~ 2.0 R , in which R is the wavelength of the Rayleigh wave of the porous half- space. , p , , pp , [4] J. Avilles, and F.J. 7. CONCLUSIONS Sanchez-Sesma, “Foundation isolation from vibration using piles as barriers”, J. Eng. Mech., ASCE, Vol. 114, pp. 1854-1870, 1988. [5] S. E. Kattis, D. Polyzos, and D. E. Beskos, “Modelling of pile wave barriers by effective trenches and their screening effectiveness”, Soil Dyn. Earthquake Eng., Vol. 18, pp. 1-10, 1999. y q g pp [6] S. E. Kattis, D. Polyzos, and D. E. Beskos, “Vibration isolation by a row of piles using a 3-D frequency domain BEM”, Int. J. Numer. Methods Eng., Vol. 46, pp. 713-728, 1999. 3. Young’s modulus ( p E ) of the piles also has significant influence on the effect of vibration isolation. Generally, the vibration isolation effect can be improved by increas- ing Young’s modulus of the piles ( p E ). [7] P. H. Tsai, Y. F. Zheng, and T. L. Jen, “Three-dimensional analysis of the screening effectiveness of hollow pile barriers for founda- tion-induced vertical vibration”, Comput. Geotech., Vol. 38, pp. 489-499, 2007. [8] M. A. Biot, “Theory of propagation of elastic waves in a fluid- saturated porous solid, I, low frequency range”, J. Acoust. Soc. Am., Vol. 28, pp. 168-178, 1956. 4. The net spacing between two neighboring piles is also a key factor for the vibration isolation effect. Usually, smaller net pile spacing will result in a better vibration isolation effect. [9] M. A. Biot, “Theory of propagation of elastic waves in a fluid- saturated porous solid, II: higher frequency range”, J. Acoust. Soc. Am., Vol. 28, pp. 179-191, 1956. [10] M. A. Biot, “Mechanics of deformation and acoustic propagation in porous media”, J. Appl. Phys., Vol. 33, pp. 1482-1498, 1962. 5. Multiple pile rows tend to produce a better vibration iso- lation effect than a single pile row. Moreover, the spacing between the pile rows has a relatively smaller impact on the vibration isolation effect. [11] X. Zeng, and R. K. N. D. Rajapakse, “Dynamic axial load transfer from elastic pile to poroelastic medium”, J. Eng. Mech., ASCE, 1999, 125: 1048-1055. [12] J. H. Wang, X. L. Zhou, and J. F. Lu, “Dynamic response of pile groups embedded in a poroelastic medium”, Soil Dyn. Earthquake Eng., Vol. 23, pp. 235-242, 2003. 7. CONCLUSIONS 0.81 0.74 0.67 0.60 0.88 0.95 0.60 0.60 0.53 0.53 0.4 0.2 0.0 -0.2 -0.4 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 s =0.4 m / R y  / R x  0.80 0.77 0.74 0.74 0.77 0.77 0.74 0.74 0.74 0.84 0.87 0.90 0.77 0.70 0.80 0.6 0.4 0.2 0.0 -0.2 -0.4 -0.6 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 s =0.8 m / R y  / R x  b 0.80 0.77 0.74 0.74 0.77 0.77 0.74 0.74 0.74 0.84 0.87 0.90 0.77 0.70 0.80 0.6 0.4 0.2 0.0 -0.2 -0.4 -0.6 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 s =0.8 m / R y  / R x  0.81 0.74 0.67 0.60 0.88 0.95 0.60 0.60 0.53 0.53 0.4 0.2 0.0 -0.2 -0.4 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 s =0.4 m / R y  / R x  b Fig. (15). The variation of the amplitude reduction ratio r A on the surface for the poroelastic half space for two pile rows with seven and six piles, respectively, for different values of the net spacing (s) between two neighboring piles: (a) s = 0.4 m; (b) s = 0.8 m. The Open Civil Engineering Journal, 2010, Volume 4 55 A Semi-Analytical Model for the Simulation a b Fig. (16). The contour of the amplitude reduction ratio rA on the surface for the poroelastic half space for two pile rows with seven and six piles, respectively, for different spacing between two neighboring pile rows: (a) q = 0.8 m; (b) q = 1.2 m. ACKNOWLEDGMENTS [13] B. Jin, and Z. Zhong, “Lateral dynamic compliance of pile embed- ded in poroelastic half-space”, Soil Dyn. Earthquake Eng, Vol. 21, pp. 519-525, 2001. The project is supported by the National Natural Science Foundation of China with grant number No. 50578071, 50969007. Also, the research is supported by the returned overseas scholar funding from Chinese Education Ministry. Besides, this research is also supported financially by State Key Laboratory for GeoMechanics and Deep Underground Engineering (No. SKLGDUE08012X). [14] J. F. Lu, and D. S. Jeng “Poroelastic model for pile-soil interaction in a half-space porous medium due to seismic waves”, Int. J. Numer. Anal. Meth. Geomech., Vol. 32, pp. 1-41, 2008. [15] Y. Q. Cai, G. Y. Ding, and C. J. Xu, “Screening of plane S waves by an array of rigid piles in poroelastic soil”, J. Zhejiang Univ. Sci. A, Vol. 9, pp. 589-599, 2008. [16] Y. Q. Cai, G. Y. Ding, and C. J. Xu, “Amplitude reduction of elas- tic waves by a row of piles in poroelastic soil”, Comput. Geotech, Vol. 36, pp. 463-473, 2009. [2] D. E Beskos, G. Dasgupta, and I. G. Vardoulakis, “Vibration isola- tion using open or filled trench. Part 1: 2-D homogeneous”, Comput. Mech., Vol. 1, pp. 43-63, 1986. [1] K. Emad, and G. D. Manolis, “Shallow trenches and propagation of surface waves”, J. Eng. Mech., ASCE, Vol. 111, pp. 279-282, 1985. 7. CONCLUSIONS 0.86 0.80 0.73 0.66 0.59 0.52 0.52 0.45 0.38 0.38 0.3 0.2 0.1 0.0 -0.1 -0.2 -0.3 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 q =0.8m / R y  / R x  0.81 0.75 0.69 0.62 0.56 0.50 0.50 0.81 0.44 0.37 0.3 0.2 0.1 0.0 -0.1 -0.2 -0.3 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 q =1.2 m / R y  / R x  b 0.81 0.75 0.69 0.62 0.56 0.50 0.50 0.81 0.44 0.37 0.3 0.2 0.1 0.0 -0.1 -0.2 -0.3 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 q =1.2 m / R y  / R x  0.86 0.80 0.73 0.66 0.59 0.52 0.52 0.45 0.38 0.38 0.3 0.2 0.1 0.0 -0.1 -0.2 -0.3 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 q =0.8m / R y  / R x  b Fig. (16). The contour of the amplitude reduction ratio rA on the surface for the poroelastic half space for two pile rows with seven and six piles, respectively, for different spacing between two neighboring pile rows: (a) q = 0.8 m; (b) q = 1.2 m. [3] B. Dasgupta, D. E. Beskos, and I. G. Vordouclakis, “Vibration isolation using open or filled trenches. Part 2: 3-D homogeneous soil”, Comput. Mech., Vol. 6, pp. 129-142, 1990. [4] J. Avilles, and F.J. Sanchez-Sesma, “Foundation isolation from vibration using piles as barriers”, J. Eng. Mech., ASCE, Vol. 114, pp. 1854-1870, 1988. [5] S. E. Kattis, D. Polyzos, and D. E. Beskos, “Modelling of pile wave barriers by effective trenches and their screening effectiveness”, Soil Dyn. Earthquake Eng., Vol. 18, pp. 1-10, 1999. [6] S. E. Kattis, D. Polyzos, and D. E. Beskos, “Vibration isolation by a row of piles using a 3-D frequency domain BEM”, Int. J. Numer. Methods Eng., Vol. 46, pp. 713-728, 1999. [7] P. H. Tsai, Y. F. Zheng, and T. L. Jen, “Three-dimensional analysis of the screening effectiveness of hollow pile barriers for founda- tion-induced vertical vibration”, Comput. Geotech., Vol. 38, pp. 489-499, 2007. [8] M. A. Biot, “Theory of propagation of elastic waves in a fluid- saturated porous solid, I, low frequency range”, J. Acoust. Soc. Am., Vol. 28, pp. 168-178, 1956. [9] M. A. p (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. © Xu et al.; Licensee Bentham Open. REFERENCES [17] J.F. Lu, B. Xu, and J. H. Wang, “Numerical analysis of isolation of the vibration due to moving loads using pile rows”, J. Sound Vi- brat., Vol. 319, pp. 940-962, 2009. [1] K. Emad, and G. D. Manolis, “Shallow trenches and propagation of surface waves”, J. Eng. Mech., ASCE, Vol. 111, pp. 279-282, 1985. [1] K. Emad, and G. D. Manolis, “Shallow trenches and propagation of surface waves”, J. Eng. Mech., ASCE, Vol. 111, pp. 279-282, 1985. pp [18] J.F. Lu, B. Xu, and J. H. Wang, “A numerical model for the isola- tion of moving-load induced vibrations by pile rows embedded in layered porous media”, Int. J. Solids Struct., Vol. 46, pp. 3771- 3781, 2009. Received: June 25, 2009 56 The Open Civil Engineering Journal, 2010, Volume 4 Xu et al. [24] R. Muki, Ed. Asymmetric problem of the theory of elasticity for a semi-infinite solid and a thick plate. In: Progress in Solid Mechanics 1, Interscience, vol. 1, New York, Amaterdam North Holland, 1960. [19] R. Muki, and E. Sternberg, “On the diffusion of an axial load from an infinite cylindrical pile embedded in an elastic medium”, Int. J. Solids Struct., Vol. 5, pp. 587-606, 1969. [20] R. Muki, and E. Sternberg, “Elastostatic load transfer to a half- space from a partially embedded axially loaded rod”, Int. J. Solids Struc., Vol. 6, pp. 69-90, 1970. [25] R.Y. Pak, and P. C. Jennings, “Elasticdynamic response of pile under transverse excitations”, J. Eng. Mech., ASCE, Vol. 113, pp. 1101-1116, 1987. pp [21] I. N. Sneddon, Ed. Fourier transforms, New York: McGraw-Hill, 1951. [26] R.D. Woods, “Screening of surface waves in soil”, J. Soil Mech. Found. Eng., ASCE, Vol. 94(SM4), pp. 951-79, 1968. [22] G. Bonnet, “Basic singular solutions for poroelastic medium in the dynamic range”, J. Acoust. Soc. Am., Vol. 82, pp. 1758-1762, 1987. [27] B. Jin, “Dynamic displacements of an infinite beam on a poroelas- tic half-space due to a moving oscillating load”, Arch. Appl. Mech., Vol. 74, pp. 277-287, 2004. [23] J.F. Lu, D. S. Jeng, and W. D. Nie, “Dynamic response of a pile embedded in a porous medium and subjected to plane SH waves”, Comput. Geotech., Vol. 33, pp. 404-418, 2006. Accepted: December 17, 2009 Accepted: December 17, 2009 Revised: December 09, 2009 Received: June 25, 2009 © Xu et al.; Licensee Bentham Open. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.
https://openalex.org/W2081853051	https://www.scielo.br/j/rsbmt/a/FfgnX3CsDxCZVR9Vx3mRwRt/?lang=en&format=pdf	English	null	Performance of a rapid diagnostic test for the detection of visceral leishmaniasis in a large urban setting	Revista da Sociedade Brasileira de Medicina Tropical	2,013	cc-by	4,080	INTRODUCTION Visceral leishmaniasis (VL) has a wide global distribution, with 200,000 to 400,000 estimated cases per year1, and is the second largest cause of death among parasitic diseases worldwide2. Approximately 90% of cases occur in six countries, including India, Bangladesh, Sudan, South Sudan, Brazil and Ethiopia, with 20,000 to 40,000 estimated deaths per year1. In Brazil, VL is caused by Leishmania (Leishmania) infantum and has been geographically expanding in the past decade3. The mean annual incidence for the period 2001-2010 was 1.8/100,000 people, with a case fatality rate of 6.8%4. Belo Horizonte, a city of 2.4 million inhabitants located in the southeastern part of the country, reported a mean incidence of 5.2 cases/100,000 people during the same time period, with a case fatality rate of 13.4%5. The accuracy of immunochromatographic rapid tests based on the rK39 protein of Leishmania infantum has been assessed in studies from different regions2,9. The sensitivity of the rK39 RDT has been reported to vary between 37 and 100%, and the speciﬁ city was estimated between 59 and 100% among immunocompetent patients in endemic regions9-16. In addition, a meta-analysis of 13 studies reported an overall sensitivity and speciﬁ city of 93.9% and 90.6%, respectively15. In Brazil, estimates of the sensitivity and speciﬁ city of RDTs vary between 85.7 and 100% and 82.0 and 100%, respectively17-21, but few studies have been conducted in real-world scenarios. For example, among immunocompromised patients, the sensitivity of RDTs seems to be lower, with one study reporting an estimation of 77.3%16. Moreover, in a previous Brazilian study, the performance of the rK39 RDT was inferior to the use of a parasitological method, real-time PCR in peripheral blood and a prototype kit for a direct agglutination test (DAT) among human immunodeﬁ ciency virus (HIV)-positive patients22. Early diagnosis and treatment of VL have been recommended by the Brazilian National VL Surveillance and Control Program to reduce mortality6. According to the Brazilian Ministry of Health, a diagnosis of VL requires the identiﬁ cation of the parasite in a smear or culture and/or positive serological testing in patients presenting with fever and spleen enlargement7. The In Belo Horizonte, the rK39 RDT was implemented at emergency care units in 2010 to foster the early treatment of suspected VL cases with a positive test, according to World Health Organization (WHO) recommendations23. Case R Major Revista da Sociedade Brasileira de Medicina Tropical 46(5):589-593, Sep-Oct, 2013 http://dx.doi.org/10.1590/0037-8682-0145-2013 Case Report Major Article Case Report Major Article Performance of a rapid diagnostic test for the detection of visceral leishmaniasis in a large urban setting Alexandre Sampaio Moura[1],[2], Helen Maria Ramos de Oliveira Lopes[1], Maria Vitória Assumpção Mourão[1] and Maria Helena Franco Morais[1] 1] Secretaria Municipal de Saúde, Prefeitura Municipal, Belo Horizonte, MG. [2]. Curso de Medicina, Universidade Federal de Alfenas, Belo Horizonte, MG. Secretaria Municipal de Saúde, Prefeitura Municipal, Belo Horizonte, MG. [2]. Curso de Medicina, Universidade Federal d Address to: Dr. Alexandre Sampaio Moura. Curso de Medicina/UNIFENAS- BH. Rua Libano 66, Bairro Itapoã, 31710-030 Belo Horizonte, MG, Brasil. Phone: 55 31 3497-4302 e-mail: alexandre.moura@unifenas.br Received 18 July 2013 Accepted 2 October 2013 ABSTRACT Introduction: Rapid diagnostic tests (RDTs) may improve the early detection of visceral leishmaniasis (VL), but their real-world performance requires additional study. Therefore, we evaluated the performance of an rK39-based RDT (Kalazar DetectTM) for the detection of VL in an endemic, large urban area. Methods: Data were collected from a registry of rK39 RDT performed at 11 emergency care units in Belo Horizonte, Brazil, and from a national database of reportable communicable diseases of the Sistema de Informação de Agravos de Notiﬁ cação (SINAN). Results: The rapid rK39 test was performed in 476 patients, with 114 (23.9%) positive results. The analysis of rK39 RDT performance was based on 381 (80%) cases reported to the SINAN database, of which 145 (38.1%) were conﬁ rmed cases. Estimates for sensitivity and speciﬁ city were 72.4% (95% CI: 64.6- 79%) and 99.6% (95%CI: 97.6-99.9%), respectively. Positive and negative predictive values were estimated at 99.1% (95%CI: 94.9-99.8%) and 85.5% (95%CI: 80.8-89.1%), respectively. In addition, close agreement between the rK39 RDT and indirect immunoﬂ uorescence was observed. Conclusions: In summary, the rK39 RDT showed a high speciﬁ city but only moderate sensitivity. In endemic areas for VL, treatment may be considered in cases with clinical manifestations and a positive rK39 RDT, but those with a negative test should be subjected to further investigation. Keywords: Visceral leishmaniasis. Serologic test. Sensitivity and speciﬁ city. most widely available serological test for VL diagnosis in Brazil is indirect immunoﬂ uorescence (IFI)6; this diagnostic method is typically performed in referral laboratories around the country, and the results become available to the attending physician within a couple of days. As a delay in VL diagnosis may be associated with an increase in case fatality rates8, the use of rapid diagnostic tests (RDTs) can help reduce mortality. METHODS An RDT for VL was implemented in Belo Horizonte in May 2010 at 11 public emergency care units. This RDT provided by the Ministry of Health was marketed as Kalazar DetectTM (InBios International), an immunochromatographic test that provides the qualitative detection of antibodies against the recombinant K39 (rK39) protein of Leishmania. The use of the rK39 RDT was recommended in Belo Horizonte for the following situations: patients of any age with I) fever of any duration with spleen enlargement or II) fever lasting 14 or more days associated with any cytopenia (anemia, leukopenia or low platelet counts). Serum samples were obtained immediately after consultation, and a trained health professional performed the RDT at the point-of-care. A sample of serum was sent to Fundação Ezequiel Dias (FUNED), a national referral laboratory, which blindly performed indirect immunoﬂ uorescence (IFI) assays for VL; this method is considered the standard serological test in Brazil. According to the recommendations from the Ministry of Health, the reporting of all suspected VL cases to a public health authority is mandatory, and data are recorded at a national database of reportable communicable diseases (SINAN). This study included all patients who received the rK39 RDT at one of the 11 public emergency care units of Belo Horizonte from May 2010 to July 2011 and had SINAN data available. Clinical and demographic information was obtained from the SINAN database. The RDT results were obtained from spreadsheets that were regularly ﬁ lled out by the laboratory manager of each of the healthcare units, and the IFI results were obtained directly from the FUNED lab. Indirect immunoﬂ uorescence results were considered as reactive when the titers were equal or greater than 1:80. To assess the performance of the RDT among HIV patients, information on HIV serology was obtained from the SINAN database. Statistical analyses were conducted using OpenEpi (Open Source Epidemiologic S tatistics for Public Health, Version 2.3.1). INTRODUCTION The objective of the present study was to assess rK39 RDT performance in widespread use in a large urban area with a high incidence of VL. 589 Moura AS et al - Rapid diagnostic test for visceral leishmaniasis The following Ministry of Health deﬁ nition of a conﬁ rmed VL case was used to assess RDT performance: individuals from an area of occurrence of VL with fever and spleen enlargement I) in whom the parasite is identiﬁ ed in direct examination or culture and/or II) demonstrate a positive IFI reaction (titer equal or greater than 1:80) or, although not showing laboratory conﬁ rmation, III) demonstrate a favorable treatment response. Notably, all conﬁ rmed cases were followed by the city health department’s epidemiological bureau to assess patient's outcomes, particularly their response to treatment. Ethical considerations The study was approved by the ethics committee at the Belo Horizonte Health Department and was performed in accordance with the ethical standards of the Declaration of Helsinki. RESULTS The rapid diagnostic test for VL was performed in 476 patients throughout the study period. Among these patients, 381 (80%) had SINAN data available and were included in the study. There was a predominance of male patients (68.8%), with a mean age of 28 years (7-88 years) (Table 1). According to the Ministry of Health case deﬁ nition, 145 (38.1%) patients had conﬁ rmed VL, with 28 (19.3%) cases meeting the parasitological criteria, 112 (77.2%) meeting the serological (IFI) criteria TABLE 1 - Demographic and clinical characteristics of 381 patients that performed rK39 rapid diagnostic test for diagnosis of visceral leishmaniasis. Characteristics Number Percentage Male (%) 262 68.8 Age group (years) < 1 32 8.4 1-9 100 26.25 10-19 33 8.66 20-49 133 34.91 50-79 74 19.42 ≥ 80 9 2.36 Criteria for conﬁ rmation of visceral leishmaiasis* (n=145) parasitological 28 19.3 serological (IFI) 112 77.0 response to therapy 5 3.4 Conﬁ rmation criteria was ranked in the following order: parasitological, serological and response to therapy. IFI: indirect immunoﬂ uorescence for detection of Leishmania antibodies; rK39: recombinant K39. Criteria for conﬁ rmation of visceral leishmaiasis (n=145) Conﬁ rmation criteria was ranked in the following order: parasitological, serological and response to therapy. IFI: indirect immunoﬂ uorescence for detection of Leishmania antibodies; rK39: recombinant K39. Conﬁ rmation criteria was ranked in the following order: parasitological, serological and response to therapy. IFI: indirect immunoﬂ uorescence for detection of Leishmania antibodies; rK39: recombinant K39. 590 Rev Soc Bras Med Trop 46(5):589-593, Sep-Oct, 2013 and 5 (3.4%) demonstrating a favorable treatment response without positive results in parasitological or serological tests. TABLE 3 - Analysis of agreement between rK39 rapid diagnostic test and indirect immunoﬂ uorescence for the diagnosis of visceral leishmaniasis. Among the 145 conﬁ rmed VL cases, 105 had a positive RDT, and among the 236 non-conﬁ rmed cases, 235 had had a negative RDT. The sensitivity and speciﬁ city of rK39 RDT were estimated at 72.4% (95%CI: 64.6-79%) and 99.6% (95%CI: 97.6-99.9%), respectively (Table 2). Estimates for the positive (PPV) and negative (NPV) predictive values were 99.1% (95%CI: 94.8-99.8%) and 85.5% (95%CI: 80.8-89.1%), respectively. Because information on the ﬁ nal diagnosis was not available for 95 of the 476 patients who received the RDT, we conducted a sensitivity analysis including all patients for whom we had RDT results available. DISCUSSION Rapid diagnostic tests (RDTs) based on rK39 showed good accuracy for VL diagnosis when widely implemented in a large urban area. Moreover, the high PPV estimates support the World Health Organization23 and the Brazilian Ministry of Health7 recommendations for the use of RDTs to conﬁ rm a VL diagnosis in suspected cases. However, due to the moderate estimates for NPV, our ﬁ ndings suggest that patients with negative RDT results should undergo further investigation with additional serological tests and examination of bone marrow aspirates. Polymerase chain reaction for the detection of Leishmania deoxyribonucleic acid (DNA) in bone marrow or peripheral blood specimens might also be useful to conﬁ rm or rule out VL diagnosis in challenging situations23-26. Among the 28 patients with amastigotes detected in bone marrow aspirates, the rK39 RDT was positive in 17 (60.7%) cases. Of note, 14 (50%) of these 28 patients were HIV-positive. Information on HIV co-infection was available for 224 of the 381 included patients, and 93 of these patients were found to be HIV-positive. Among HIV-infected patients, the sensitivity and speciﬁ city of the RDT were estimated at 60% (95%CI: 40.7-76.6%) and 100% (95%CI: 94.7-100%), respectively. The positive and negative predictive values for this subset of patients were estimated at 100% (95% CI: 79.6-100%) and 87.2% (95% CI: 77.9-92.9%), respectively. An evaluation of rK39 RDT performance revealed better results for studies conducted in the Indian subcontinent as compared to those from East Africa or Brazil9. Differences in study design, the species of Leishmania responsible for VL cases and antibody concentrations might explain this variation in RDT performance. An initial evaluation of the performance of the rK39 RDT in India estimated its sensitivity and speciﬁ city at 100% and 98%, respectively11. Of note, this previous study considered conﬁ rmed VL cases to be only those with parasitological conﬁ rmation. A controlled study subsequently conducted in India by the same research group, which sought to validate another type of rK39 RDT (One Step InsureTM), reported a sensitivity of 100% and a speciﬁ city of 93% using the same case criteria as their previous study27. Information on the results of indirect immunoﬂ uorescence was available for 296 of the 381 included patients. RESULTS For this analysis, we considered two hypothetical extreme situations to address the missing data for a ﬁ nal diagnosis: I) all 95 patients with missing data had visceral leishmaniasis or II) all of these patients did not have VL. Considering the ﬁ rst scenario, the estimates for sensitivity, speciﬁ city, PPV and NPV were 47.1% (95%CI: 40.9-50.4%), 99.6% (95%CI: 97.6-99.9%), 99.1% (95%CI: 95.2-99.8%) and 64.9% (95%CI: 59.9-69.6%), respectively. For the latter situation, the estimates for the same parameters were 72.4% (95%CI: 64.3-79%), 97.3% (95%CI: 94.9-98.6%), 92.1% (95%CI: 85.7-95.8%) and 88.9% (95%CI: 85.3-91.8%), respectively. Indirect immunoﬂ uorescence RDT ≥ 1:80 < 1:80 total Positive 63 10 73 Negative 23 200 223 Total 86 210 296 RDT: rapid diagnostic test. Kappa 0.71 (CI95% 0.60-0.83). rK39: recombinant K39; CI: conﬁ dence interval. REFERENCES 1. Alvar J, Vélez ID, Bern C, Herrero M, Desjeux P, Cano J, et al. Leishmaniasis worldwide and global estimates of its incidence. PLoS ONE 2012; 7:e35671. Our study also found that the sensitivity of the RDT seemed to be lower among HIV-infected patients compared to the general population. However, we had no information regarding the cluster of differentiation 4 (CD4)-T cell counts or HIV staging of these patients with which to establish the impact of immunosuppression on test performance. In addition, we were unable to ﬁ nd any studies in the literature designed speciﬁ cally to address rK39 RDT accuracy among HIV-infected patients. A study in Ethiopia, a country with a high HIV prevalence, reported an RDT sensitivity of 77% for HIV-positive patients, which was lower than that observed for HIV-negative patients (87%), but this difference was not statistically signiﬁ cant15. In addition, a recent meta-analysis of diagnostics tests for VL in HIV-infected patients did not include the rK39 RDT due to the lack of previously published studies29. 2. Chappuis F, Su ndar S, Hailu A, Ghalib H, Rijal S, Peeling RW, et al. Visceral leishmaniasis: what are the needs for diagnosis, treatment and control? Nat Rev Microbiol 2007; 5:873-882. 3. Gontijo CMF, Melo MN. Leishmaniose visceral no Brasil: quadro atual, desaﬁ os e perspectivas. Rev Bras Epidemiol 2004; 7:338-349. 3. Gontijo CMF, Melo MN. Leishmaniose visceral no Brasil: quadro atual, desaﬁ os e perspectivas. Rev Bras Epidemiol 2004; 7:338-349. 4. Ministério da Saúde. Secretaria de Vigilância em Saúde. Letalidade de leishmaniose visceral. Brasil, Grandes Regiões e Unidades Federadas, 2000-2010 [Internet]. Brasília: Ministério da Saúde [cited 2013 Jul 13]. Available at: portal.saude.gov.br/ portal/arquivos/pdf/lv_letalidade_05_09_11.pdf 5. Prefeitura de Belo Horizonte. Vigilância Epidemiológica da Leishmaniose Visceral em BH [Internet]. Belo Horizonte: Prefeitura de Belo Horizonte [cited 2013 Jul 13]. Available at: http://portalpbh.pbh.gov.br/pbh/ecp/ comunidade.do?evento=portlet&pIdPlc=ecpTaxonomiaMenuPortal&app =s.aude&tax=22301&lang=pt_BR&pg=5571&taxp=0&/. 6. Ministério da Saúde. Secretaria de Vigilância em Saúde. Manual de Vigilância e Controle da Leishmaniose Visceral. Brasília; 2006. 120 p. 7. Ministério da Saúde. Secretaria de Vigilância em Saúde. Leishmaniose Visceral - Recomendações para Redução da Letalidade. Brasília; 2011. Finally, we found a close agreement between the RDT and the frequently used IFI test. A similar ﬁ nding was observed in a study comparing an RDT with the direct agglutination test (DAT)30. Nonetheless, a Brazilian study showed that IT- LEISHTM, but not Kalazar DetectTM, performed signiﬁ cantly better than IFI with greater speciﬁ city and PPV21. DISCUSSION A reactive IFI result was observed in 63 (86.3%) of the 73 samples that had previously been shown to be positive in the rK39 RDT, and 200 (89.6%) of the 223 samples from patients with a negative result on the RDT were also negative for IFI (Table 3). In Uganda, Kalazar DetectTM was used for the diagnosis of VL among 250 inpatients admitted with a clinical presentation compatible with VL28, and this RDT demonstrated good accuracy, with a sensitivity and speciﬁ city estimated at 82% and 99%, respectively. It is interesting to note that this Uganda study used conﬁ rmation criteria that included the detection of the parasite in a spleen aspirate smear, a positive direct agglutination test (i.e., titers greater than 1:12,800) or a good treatment response. TABLE 2 - Evaluation of rK39 rapid diagnostic test performance for diagnosis of visceral leishmaniasis. Cases RDT conﬁ rmeda not conﬁ rmed Total Positive 105 1 106 Negative 40 235 275 Total 145 236 381 RDT: rapid diagnostic test; aconﬁ rmed according to Brazilian Ministry of Health criteria; rK39: recombinant K39. TABLE 2 - Evaluation of rK39 rapid diagnostic test performance for diagnosis of visceral leishmaniasis. In a study conducted in Sudan using a different rK39 RDT (IT-LEISHTM) but also including a serological test (direct agglutination test) among the conﬁ rmation criteria for VL cases, RDT: rapid diagnostic test; aconﬁ rmed according to Brazilian Ministry of Health criteria; rK39: recombinant K39. 591 Moura AS et al - Rapid diagnostic test for visceral leishmaniasis similar results were found for sensitivity, speciﬁ city, PPV and NPV (81%, 97%, 98% and 71%, respectively)10. similar results were found for sensitivity, speciﬁ city, PPV and NPV (81%, 97%, 98% and 71%, respectively)10. similar results were found for sensitivity, speciﬁ city, PPV and NPV (81%, 97%, 98% and 71%, respectively)10. In summary, the rK39 RDT showed good accuracy for VL diagnosis and close agreement with IFI and may therefore be useful for the timely identiﬁ cation of cases in point-of-care treatment. However, this test is not sufﬁ cient to rule out VL, and further testing is warranted in suspected cases with negative RDT results. In Brazil, validation studies of the IT-LEISHTM have reported a sensitivity varying from 93% to 100% and a speciﬁ city between 94% and 96%18,20, considering conﬁ rmed cases only as those with a parasitological diagnosis. REFERENCES This ﬁ nding suggests that RDTs may be an effective substitute for IFI in most clinical situations, considering their rapid results and ease of use. 8. Oliveira JM, Fernandes AC, Dorval MC, Alves TP, Fernandes TD, Oshiro ET, Oliveira AL. Mortalidade por leishmaniose visceral: aspectos clínicos e laboratoriais. Rev Soc Bras Med Trop 2010; 43:188-193. 9. Cunningham J, Hasker E, Das P, El Saﬁ S, Goto H, Mondal D, et al. A global comparative evaluation of commercial immunochromatographic rapid diagnostic tests for visceral leishmaniasis. Clin Infect Dis 2012; 55:1312-1319 10. Ritmeijer K, Melaku Y, Mueller M, Kipngetich S, O´Keeffe C, Davidson RN. Evaluation of a new recombinant k39 rapid diagnostic test for Sudanese visceral leishmaniasis. Am J Trop Med Hyg 2006; 74:76-80. One limitation of our study involves the criteria used to conﬁ rm VL cases. We chose to use the Brazilian Ministry of Health criteria and therefore considered conﬁ rmed cases as not only those with parasitological diagnosis but also those with a reactive IFI and/or good treatment response. In our view, the use of the Ministry of Health criteria better reﬂ ects the real-world scenario of clinical practice. In addition, because we did not have information on the ﬁ nal diagnosis of the non-conﬁ rmed cases, interpretations of the estimates regarding speciﬁ city should consider this limitation. Finally, the clinical and demographic data were obtained from the SINAN database, and although reporting is mandatory, the attending physicians do not always report suspected cases. As a result, we were unable to obtain these data for approximately 20% of the cases. To address this gap, we performed a sensitivity analysis considering two extreme situations, and these results indicated that this lack of data did not change our main ﬁ nding of the low sensitivity and good speciﬁ city of the rK39 RDT. 11. Sundar S, Reed SG, Singh VP, Kumar PC, Murray HW. Rapid accurate ﬁ eld diagnosis of Indian visceral leishmaniasis. Lancet 1998; 351:563-565. 12. Zijlstra EE, Nur Y, Desjeux P, Khalil EAG, El-Hassan AM, Groen J. Diagnosing visceral leisimaniasis with the recombinant K39 strip test: experience from Sudan. Trop Med Int Health 2001; 6:108-113. 13. Veeken H, Ritmeijer K, Seaman J, Davidson R. Comparison of an rK39 dipstick rapid test with direct agglutination test and splenic aspiration for the diagnosis of kala-azar in Sudan. Trop Med Int Health 2003; 8:164-167. 14. DISCUSSION In addition, the PPV and NPV of 98% and 89%, respectively, were similar to those found in our study. Evaluations of the performance of Kalazar DetectTM have shown sensitivity values between 85.7% and 90% and a speciﬁ city between 82% and 100%18,19. The authors declare that there is no conﬂ ict of interest. In a multicentric study, four commercially available RDTs were compared, including Kalazar Detect™. Both at Brazilian and African centers, Kalazar Detect™ presented a lower sensitivity when compared to IT-LEISHTM, which must be taken into account when using these tests in clinical practice9. REFERENCES Terán-Ángel G, Rodríguez V, Silva R, Zerpa O, Schallig H, Ulrich M, Cabrera M. Herramientas no invasivas en Venezuela: comparación entre las pruebas inmunoserológicas DAT, rK26 y rK39 en el diagnóstico de leishmaniasis visceral. Biomedica 2010; 30:39-45. 15. Chappuis F, Rijal S, Soto A, Menten J, Boelaert M. A meta-analysis of the diagnostic performance of the direct agglutination test and rK39 dipstick for visceral leishmaniasis. BMJ 2006; 333:723. 16. Horst R, Tefera T, Assefa G, Ebrahim AZ, Davidson RN, Ritmeijer K. Field evaluation of rK39 test and direct agglutination test for diagnosis 592 Rev Soc Bras Med Trop 46(5):589-593, Sep-Oct, 2013 of visceral leishmaniasis in a population with high prevalence of human immunodeﬁ ciency virus in Ethiopia. Am J Trop Med Hyg 2009; 80: 929-934. 24. Cascio A, Calattini S, Colomba C, Scalamogna C, Galazzi M, Pizzuto M, et al. Polymerase chain reaction in the diagnosis and prognosis of Mediterranean visceral leishmaniasis in immunocompetent children. Pediatrics 2002; 109:e27. 17. Assis TSM, Braga ASC, Pedras MJ, Oliveira E, Barral A, Siqueira IC, et al. Multi-centric prospective evaluation of rk39 rapid test and direct agglutination test for the diagnosis of visceral leishmaniasis in Brazil. Trans R Soc Trop Med Hyg 2011; 105:81-85. 25. Antinori S, Calattini S, Longui E, Bestetti G, Piolini R, Magni C, et al. Clinical use of polymerase chain reaction performed on peripheral blood and bone marrow samples for the diagnosis and monitoring of visceral leishmaniasis in HIV-infected and HIV-uninfected patients: a single- center, 8-year experience in Italy and review of the literature. Clin Infect Dis 2007; 44:1602-1610. 18. Schallig HDFH, Canto-Cavalheiro M, Silva ES. Evaluation of the direct agglutination test and the rK39 dipstick test for the sero-diagnosis of visceral leishmaniasis. Mem Inst Oswaldo Cruz 2002; 97:1015-1018. 26. Fraga TL, Brustoloni YM, Lima RB, Dorval EC, Oshiro ET, Oliveira J, et al. Polymerase chain reaction of peripheral blood as a tool for the diagnosis of visceral leishmaniasis in children. Mem Inst Oswaldo Cruz 2010; 105:310-313. 19. Carvalho SFG, Lemos EM, Corey R, Dietze R. Performance of recombinant K39 antigen in the diagnosis of Brazilian visceral leishmaniasis. Am J Trop Med Hyg 2003; 68:321-324. 20. Amato Neto V, Amato VS, Tuon FF, Gakiya E, Marchi CR, Souza RM, et al. False-positive results of a rapid K39-based strip test and Chagas disease. Int J Infet Dis 2009; 13:182-185. 27. Sundar S, Pai K, Sahu M, Kumar V, Murray HW. REFERENCES Immunochromatographic strip-test detection of anti-K39 antibody in Indian visceral leishmaniasis. Ann Trop Med Parasitol 2002; 96:19-23. 21. Peruhype-Magalhães V, Machado-de-Assis TS, Rabello A. Use of the Kala-Azar Detect® and IT-LEISH® rapid tests for the diagnosis of visceral leishmaniasis in Brazil. Mem Inst Oswaldo Cruz 2012; 107: 951-952. 28. Chappuis F, Mueller Y, Nguimfack A, Rwakimari JB, Coufﬁ gnal S, Boelaert M, et al. Diagnostic accuracy of two rK39 antigen-based dipsticks and the formol gel test for rapid diagnosis of visceral leishmaniasis in northeastern Uganda. J Clin Microbiol 2005; 43:5973-5977. 22. Cota GF, Sousa MR, Freitas Nogueira BM, Gomes LI, Oliveira E, Assis TS, et al. Comparison of parasitological, serological, and molecular tests for visceral leishmaniasis in HIV-infected patients: a cross-sectional delayed-type study. Am J Trop Med Hyg 2013; 89:570-577. 29. Cota GF, Sousa MR, Demarqui FN, Rabello A. The diagnostic accuracy of serologic and molecular methods for detecting visceral leishmaniasis in HIV infected patients: meta-analysis. PLoS Negl Trop Dis 2012; 6:e1665. 23. World Health Organization. Visceral Leishmaniasis Rapid Diagnostic Test Performance [Internet]. Geneva: World Health Organization. Diagnostics Evaluation Series Nº 4. [cited 2013 Jul 13]; Available at http://www.who. int/tdr/publications/tdr-research-publications/vl-rdt-evaluation/en/ 30. Bern C, Jha SN, Joshi AB, Thakur GD, Bista MB. Use of the recombinant K39 dipstick test and the direct agglutination test in a setting endemic for visceral leishmaniasis in Nepal. Am J Trop Med Hyg 2000; 63:153-157. 593
https://openalex.org/W3017669273	https://www.nature.com/articles/s41598-020-70181-0.pdf	English	null	A fully open-source framework for deep learning protein real-valued distances	bioRxiv (Cold Spring Harbor Laboratory)	2,020	cc-by	9,655	OPEN Badri Adhikari As deep learning algorithms drive the progress in protein structure prediction, a lot remains to be studied at this merging superhighway of deep learning and protein structure prediction. Recent findings show that inter-residue distance prediction, a more granular version of the well-known contact prediction problem, is a key to predicting accurate models. However, deep learning methods that predict these distances are still in the early stages of their development. To advance these methods and develop other novel methods, a need exists for a small and representative dataset packaged for faster development and testing. In this work, we introduce protein distance net (PDNET), a framework that consists of one such representative dataset along with the scripts for training and testing deep learning methods. The framework also includes all the scripts that were used to curate the dataset, and generate the input features and distance maps. Deep learning models can also be trained and tested in a web browser using free platforms such as Google Colab. We discuss how PDNET can be used to predict contacts, distance intervals, and real-valued distances. Deep learning and covariance signals obtained from sequence alignments are accelerating the progress in the field of protein structure prediction1. It is exciting that information culled from sequences whose structures are not solved can serve as the primary input feature to predict contacts and distances. The most successful methods, presented in the recent CASP13 experiment (http://predictioncenter.org/), exploit such sequence databases and unanimously demonstrate that the key to enhancing the current progress is accurate contact and distance map prediction2–4. The distance prediction methods, in particular, are a major advancement in the area of ab initio or free modeling. Much remains to be investigated at this merging superhighway of deep learning and protein structure prediction. For example, we do not know if current deep learning methods are an ideal fit for solving the distance prediction problem. In addition, many types and combinations of features are used as inputs. These include secondary structure predictions, coevolution-based signals5, and raw features such as the pair frequen- cies matrix, the covariance matrix6, the compressed covariance matrix7, and the precision matrix8. How to best engineer these features for deep learning algorithms also remains an open question. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Department of Computer Science, University of Missouri-St. Louis, St. Louis MO, 63132, USA. email: adhikarib@ umsl.edu OPEN How useful the solitary deep learning algorithms are compared to the classical methods based on amino acid frequencies, such as the Chou-Fasman method9 and the Garnier-Osguthorpe-Robson (GOR) method10, also remains an open question. The irony behind current methods for structure prediction is that they rely on coevolution and conservation signals in multiple sequence alignments, which a protein sequence folding in a cell does not have access to. Hence, it should be possible to build accurate models without such input features. How accurately this can be done remains to be seen. An urgent need exists for a small and representative dataset packaged for fast development and investigation; thus, we created PDNET to meet this need and fill the information gap.h gi g p The protein inter-residue distance prediction problem is to predict a pair-wise distance matrix (2D) from a protein sequence (one-dimensional sequence of amino acids). It may be compared with the monocular or ste- reo depth estimation problem in computer vision as shown in Fig. 1. Image depth prediction methods such as the fully convolutional residual networks (FCRN)11 method take an image matrix as input and predict a depth matrix as output where each pixel has a predicted depth (distance from the camera to the object in the scene). The FCRN method is trained using various indoor and outdoor scene images. Similar to the depth prediction problem, in distance prediction, the input is a three-dimensional volume (height × width × channels) and the output is the same dimensions as the input (height × width) though with a single channel. The many character- istics of distance prediction, however, make it a unique deep learning problem. Unlike problems in computer \| https://doi.org/10.1038/s41598-020-70181-0 Scientific Reports \| (2020) 10:13374 www.nature.com/scientificreports/ Figure 1. Comparison of the protein inter-residue distance prediction problem with the ‘depth prediction from a single image problem’ in computer vision. In both problems, the input to the deep learning model is a volume and the output is a 2D matrix. The depth predictions for this specific image (top right corner) were obtained with a pre-trained fully convolutional residual network (FCRN)11. Figure 1. Comparison of the protein inter-residue distance prediction problem with the ‘depth prediction from a single image problem’ in computer vision. In both problems, the input to the deep learning model is a volume and the output is a 2D matrix. OPEN vision, which usually have one to three input channels, the distance prediction problem may have a few to a few hundred input channels depending on what and how many input features are used. Also, a distance map is symmetrical about the diagonal line, and each pixel on the map represents a distance between a pair of residues in the sequence. A single input feature, such as a precision matrix, can have more than 400 channels. Some of these input channels are tiled rows or columns obtained from one-dimensional predictions such as secondary structures and solvent accessibility. The comparison of this problem with a computer vision problem naturally raises the question of what the convolutional filters in each layer learn. Visualization of what the deep learning layers learn and how the input features translate over the layers is difficult; thus, little research has been done in this area12. Because we do not know what to expect in our visualizations, exploiting the techniques of explainable artificial intelligence used in computer vision is not directly useful for the distance prediction problem. Another unique feature of the distance prediction problem, compared to computer vision problems, is that a protein struc- ture and the corresponding distance maps cannot be augmented in the same ways that images can. For instance, distance maps cannot be scaled, rotated, or flipped. This is because an object in the real world (for example, a chair) may be tiny or large but in the case of protein structures, all proteins, small or large, are comprised of fixed size structural units such as an alpha helix. In a distance map, distance pixels away from the diagonal line are more useful for reconstructing the original structure. These distances, known as long-range distances, are also harder to predict as shown in Fig. 2. In a way, this is similar to saying that in the case of depth prediction, it is more important to predict the depth of objects closer (to the camera) in the scene than the objects that are far away. An ideal distance prediction algorithm should predict exact physical distances on the entire distance map accurately. This is extremely difficult and as such, a binary version of the problem known as contact-prediction has been more widely studied. OPEN The depth predictions for this specific image (top right corner) were obtained with a pre-trained fully convolutional residual network (FCRN)11. vision, which usually have one to three input channels, the distance prediction problem may have a few to a few hundred input channels depending on what and how many input features are used. Also, a distance map is symmetrical about the diagonal line, and each pixel on the map represents a distance between a pair of residues in the sequence. A single input feature, such as a precision matrix, can have more than 400 channels. Some of these input channels are tiled rows or columns obtained from one-dimensional predictions such as secondary structures and solvent accessibility. The comparison of this problem with a computer vision problem naturally raises the question of what the convolutional filters in each layer learn. Visualization of what the deep learning layers learn and how the input features translate over the layers is difficult; thus, little research has been done in this area12. Because we do not know what to expect in our visualizations, exploiting the techniques of explainable artificial intelligence used in computer vision is not directly useful for the distance prediction problem. Another unique feature of the distance prediction problem, compared to computer vision problems, is that a protein struc- ture and the corresponding distance maps cannot be augmented in the same ways that images can. For instance, distance maps cannot be scaled, rotated, or flipped. This is because an object in the real world (for example, a chair) may be tiny or large but in the case of protein structures, all proteins, small or large, are comprised of fixed size structural units such as an alpha helix. In a distance map, distance pixels away from the diagonal line are more useful for reconstructing the original structure. These distances, known as long-range distances, are also harder to predict as shown in Fig. 2. In a way, this is similar to saying that in the case of depth prediction, it is more important to predict the depth of objects closer (to the camera) in the scene than the objects that are far away. An ideal distance prediction algorithm should predict exact physical distances on the entire distance map accurately. This is extremely difficult and as such, a binary version of the problem known as contact-prediction has been more widely studied. OPEN y Although almost all currently successful methods2, 3, 13 formulate the distance prediction problem as a multi- class classification problem, undeniably, the ultimate goal is to accurately predict real valued distances14. Recently, some researchers have formulated the problem as a regression problem, which has demonstrated promising results15. Methods Dataset preparation. Our dataset is derived from the 3456 representative protein sequences and 150 test protein chains used to train, validate, and test the widely used DeepCov method16 for protein contact predic- tion. The 3,456 protein chains used for training and validating the DeepCov method are a subset of a set of 6,729 protein chains that have no domains homologous with the protein chains in the test set. The sequence lengths of the protein chains range from 50 to 500 residues and 50 to 266 residues in the training set and test set, respec- tively. The original set of 6,729 protein chains have less than 25% pairwise sequence similarity, which makes this the original set representative of the full Protein Data Bank (PDB) set. We used this list of 3,456 protein chains, the list of 150 protein chains, and the corresponding alignments to prepare our dataset. For each FASTA file in the DeepCov set, we downloaded the corresponding protein chain from http://www.pdb.org and cleaned it by removing rows containing non-standard amino acids and then renumbered the residues to match the FASTA https://doi.org/10.1038/s41598-020-70181-0 Scientific Reports \| (2020) 10:13374 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Example of long-range, medium-range, short-range, and local distances in a protein distance map. The distances between residue pairs 42–47, 42–52, 42–62, and 42–67 are examples of local, short, medium, and long-range distances, respectively (left). In the heatmap plot (right), the sequence separation domain for long, medium, and short-range distances, and local distances are [24+], [12, 23], [6, 11], and [0, 5], respectively. The labels in the x and y axis refer to the residue index in the corresponding protein sequence. The diagonal line shows that the residue pair i-i have a zero distance. Figure 2. Example of long-range, medium-range, short-range, and local distances in a protein distance map. The distances between residue pairs 42–47, 42–52, 42–62, and 42–67 are examples of local, short, medium, and long-range distances, respectively (left). In the heatmap plot (right), the sequence separation domain for long, medium, and short-range distances, and local distances are [24+], [12, 23], [6, 11], and [0, 5], respectively. The labels in the x and y axis refer to the residue index in the corresponding protein sequence. The diagonal line shows that the residue pair i-i have a zero distance. sequence. Pairwise Cβ distances between residues were calculated from the true structure (3D) to obtain a distance map (2D). Methods The protein sequence obtained from the true structure was used as input to predict various sequence features (1D) and pairwise features (2D), which are all translated to an input volume (2D input with various channels). This input volume is the input to a deep learning model, and the output labels are the true distances. To maintain consistency with the DeepCov method, in all our experiments we selected the first 130 chains when sorted alphabetically by PDB IDs (a random subset) in the entire DeepCov set as the validation set and the remaining 3,326 chains as the training set. The alphabetical ordering ensured a random selection because PDB IDs are automatically assigned and do not have meaning17. Since the 150 proteins in the test set are easier (i.e. they have much richer alignments), we further tested our methods on two hard datasets—131 CAMEO-HARD (https://www.cameo3d.org/) targets released between December 8, 2018 and June 1, 2019, and the CASP13 free-modeling targets, released in 2018. These sets are also used as test datasets by other successful methods such as trRosetta18 to evaluate the performance of their methods. Both of these datasets serve as a test set since they were released after the original DeepCov set (development set) was curated. yt g p p Notably, while our dataset may not be a fully balanced representation of all the protein structural folds as summarized in databases such as CATH19, it is a representative set of the protein sequence space. Designing and developing state-of-the-art models may require a much larger dataset. However, we assert that a framework such as PDNET can answer many fundamental questions about the applicability and limitations of deep learning when solving the distance prediction problem. Input features. Successful methods for contact and distance prediction use a variety of features predicted and derived from the input sequence. These include predicted secondary structures, coevolution features, solvent accessibility, position-specific scoring matrix derived features, Atchley factors, many pre-computed statistical potentials, alignment statistics such as the number of effective sequences, Shannon entropy sum, mean contact potential, normalized mutual information, covariance matrices, precision matrices, etc. Contact and distance prediction methods use a variety of combinations of these features; moreover, it is not fully understood, in gen- eral, which of them contain complementary information and which are redundant. Additionally, generating all of these features for an input protein sequence is computationally expensive, both in terms of time and resources. Methods L stands for the length of the protein sequence in the native (true) structure, and NC is the number of true contacts in the structure. The potential matrix captures the frequencies of the co-varying pair positions weighted by the value of each sequence in the input sequence alignment, and the Shannon entropy sum calculates the variability at each resi- due position. For our last input feature, we generated sequence profiles from the multiple sequence alignments. i Features derived directly from the multiple sequence alignments (MSAs) such as covariance or precision matrix are significantly important and have achieved expected improvements7. However, in this work, we inten- tionally skipped investigating such features because working with these features requires huge storage capacities, solid state disks, and many high-end GPUs. These resources may not be available to all those wishing to use our framework. In a separate work, we will elaborately discuss the importance of all of these features (a work in progress). Evaluation of predicted distance maps. A primary goal of evaluating the distances in a predicted dis- tance map is to assess their usefulness towards building full three-dimensional models. We used two sets of metrics for evaluating predicted distances: (a) the mean absolute error (MAE) of predicted distances, and (b) the precision of the contacts derived from the predicted distances. To calculate MAE, we first kept all the true distances below a certain distance threshold from the native structure, and calculated the mean absolute dif- ference between these true distances and corresponding predicted distances. Analogous to the definition of various types of contacts, we defined local, short-range, medium-range, and long-range distances as the dis- tances between residue pairs with sequence separation of [0, 5], [6, 11], [11, 23], and [23+] respectively. Previous studies have shown that long-range contacts, i.e., pairs separated by at least 23 residues in the sequence, are the most informative pairs for accurate reconstruction26, 27. Hence, we designed our evaluation metrics focusing on long-range distances (see Fig. 2). Here, we evaluate the mean absolute error of medium and long-range, as well as long-range only distances at distance thresholds of 8 Å and 12 Å ( MAE8 and MAE12 ). We are certain that other variations of MAE, such as the root mean squared deviation (RMSD) can also be relevant. When evaluating the predictions for the validation set, we observed a Pearson’s correlation coefficient of 0.9 between MAE8 and PNC. Methods Å fi Ideally, to obtain contacts from predicted distances, one would simply consider distances less than 8 Å as contacts. Such a technique, however, did not favor the evaluation of predicted distances. Hence, we resorted to a technique similar to the one that is currently used for evaluating predicted contacts. We assigned contact scores such that shorter predicted distances have higher scores than longer ones. A score of 0.5 is assigned for a predicted distance of 8 Å. Precisely, if Dij is the predicted distance between two residues, i and j, then the cor- responding score Pij is given by: Pij = 4.0 Dij if Dij >= 4.0 1.0 otherwise. Following the practice of evaluating predicted contacts by calculating the precision of top L/x contacts, we evalu- ated top L, and top NC long-range contacts ( PL and PNC ). Here L and NC stand for the length of the protein sequence and the total number of contacts in the native structure. We calculated precision as the ratio of the number of matches and the total number of contacts considered. For a protein sequence, if the corresponding true contact map does not have any long-range contacts, we exclude the target from evaluation. Our motivation for evaluating the precision of top NC contacts is driven by two insights. In a recent work28, we reported that selecting or evaluating the top L/x contacts does not work well for all proteins. Secondly, in the most recent CASP competition, the accessors of the contact prediction category have discussed many reasons for considering evaluation of the top NC contacts instead of fewer contacts27. Although the PNC metric is not discussed in most contact prediction method papers, we determined that as more and more accurate contact prediction methods are developed, PNC will emerge as a more informative, reliable, and widely adopted metric. Table 1 summarizes our evaluation metrics. Following the practice of evaluating predicted contacts by calculating the precision of top L/x contacts, we evalu- ated top L, and top NC long-range contacts ( PL and PNC ). Here L and NC stand for the length of the protein sequence and the total number of contacts in the native structure. We calculated precision as the ratio of the number of matches and the total number of contacts considered. Methods Based on recent research advancements18, 20, 21, we identified the top seven features that are complementary and most informative. They are: (1) sequence profiles, (2) secondary structure predictions and (3) solvent accessibil- ity predictions (with both 2 and 3 using PSIPRED22), (4) coevolutionary signals predicted using CCMpred5, (5) FreeContact23, (6) contact potentials calculated from multiple sequence alignments, and (7) Shannon entropy of the alignment column. The secondary structure predictions by PSIPRED represent the probabilities of each residue in the input sequence being a helix, strand, or coil, i.e. predicting whether each amino acid will be a part of a helix, beta-strand or a coil in the final model. Similarly, the solvent accessibility predictions by PSIPRED are binary predictions of hydrophobicity for each residue, i.e. predicting whether each amino acid will be ‘exposed’ to water or not. Looking into a predicted multiple sequence alignment, the coevolutionary predictions by CCM- pred and FreeContact capture the strength of covariance between all pairs of residue positions. These predictions themselves can be considered as contact predictions. However, deep learning algorithms can improve these pre- dictions by learning to detect noise, correct mistakes, and identify high-confidence and missing predictions24. With the predicted multiple sequence alignment as input, we can also calculate the contact potential matrix and the Shannon entropy sum at each residue position using the ‘alnstats’ C program in the MetaPSICOV package25. Scientific Reports \| (2020) 10:13374 \| https://doi.org/10.1038/s41598-020-70181-0 www.nature.com/scientificreports/ Prediction Metric Description Distances MAE8 MAE of the predicted long-range distances with corresponding true distances shorter than 8 Å MAE12 MAE of the predicted long-range distances with corresponding true distances shorter than 12 Å Contacts PL Precision of top L long-range predicted contacts PNC Precision of top NC long-range predicted contacts Prediction Metric Description Distances MAE8 MAE of the predicted long-range distances with corresponding true distances shorter than 8 Å MAE12 MAE of the predicted long-range distances with corresponding true distances shorter than 12 Å Contacts PL Precision of top L long-range predicted contacts PNC Precision of top NC long-range predicted contacts Table 1. Evaluation metrics used for evaluation of predicted distances and/or contacts. L stands for the length of the protein sequence in the native (true) structure, and NC is the number of true contacts in the structure. Table 1. Evaluation metrics used for evaluation of predicted distances and/or contacts. Methods layer’s activation was a ‘softmax’ layer and loss was calculated using categorical cross-entropy; meanwhile, for the PDNET-Distance, the last layer’s activation was left as ReLU. We trained a model for up to 64 epochs. During each epoch of training, we randomly cropped the input feature volumes to a 128 × 128 sub-volume. Before crop- ping, we also padded zeros of width 5 to all sides of the input volume similar to the AlphaFold approach3. Dur- ing prediction, however, we built a model as wide as the input sequence, i.e., we did not crop during prediction. layer’s activation was a ‘softmax’ layer and loss was calculated using categorical cross-entropy; meanwhile, for the PDNET-Distance, the last layer’s activation was left as ReLU. We trained a model for up to 64 epochs. During each epoch of training, we randomly cropped the input feature volumes to a 128 × 128 sub-volume. Before crop- ping, we also padded zeros of width 5 to all sides of the input volume similar to the AlphaFold approach3. Dur- ing prediction, however, we built a model as wide as the input sequence, i.e., we did not crop during prediction. Binned distance prediction. Since short (euclidean) distances between pairs with long-range separation (in sequence index) are more critical for structure reconstruction and other uses, we binned distances so that bins were narrower for shorter distances and wider for larger distances. Specifically, we used a fixed bin width of 0.2 Å for bins below 8 Å and an increasing bin width for larger distances (by a factor consisting of adding 0.2 Å for every next bin). These thresholds for the distance bins were 4, 4.2, 4.4, . . . , 8, 8.4, 9.0, 9.8, 10.8, 12, . . . , 21, 23.4, 26, and 26+ Å. Our technique differed slightly from the fixed-width binning technique used in methods such as RaptorX13 and AlphaFold3 and the two-width binning used in the DMPfold method29. We used the standard categorical cross-entropy loss to optimize our model with a softmax output layer as the last layer. t To translate the predictions into contact prediction probabilities, we summed all the probabilities in the bins below the 8 Å threshold. Similarly, to translate the predictions into distance maps, for each residue pair, we selected the distance bin with the highest probability and calculated the mean of the distance range as the predicted distance. Methods For a protein sequence, if the corresponding true contact map does not have any long-range contacts, we exclude the target from evaluation. Our motivation for evaluating the precision of top NC contacts is driven by two insights. In a recent work28, we reported that selecting or evaluating the top L/x contacts does not work well for all proteins. Secondly, in the most recent CASP competition, the accessors of the contact prediction category have discussed many reasons for considering evaluation of the top NC contacts instead of fewer contacts27. Although the PNC metric is not discussed in most contact prediction method papers, we determined that as more and more accurate contact prediction methods are developed, PNC will emerge as a more informative, reliable, and widely adopted metric. Table 1 summarizes our evaluation metrics. Residual network architecture. All successful methods for contact and distance prediction use residual networks and their variants2–4, 13, 18. We developed deep learning methods to predict contacts (PDNET-Contact), distance intervals (PDNET-Binned), and real-valued distances (PDNET-Distance), i.e., three separate models. Our deep learning architecture for contact prediction is a standard 128 block residual neural network with dropouts added in between the convolutional layers as described in our DEEPCON method21. Each residual block consists of the following layers: batch normalization, ReLU activation, 2D convolution using 3 × 3 filters, the dropout layer with a dropout rate of 0.3, another ReLU activation, and another similar 2D convolution layer. Each network has around 9.5 million parameters. For our PDNET-Contact method, we set the last layer’s activation as ‘sigmoid’ and loss was calculated using binary cross-entropy. Similarly, for PDNET-Binned, the last https://doi.org/10.1038/s41598-020-70181-0 Scientific Reports \| (2020) 10:13374 \| www.nature.com/scientificreports/ Figure 3. Reciprocating the distance matrix so that larger numbers represent shorter distances in the input distance matrix. The input distance matrix is shown in left and the reciprocated distance matrix is on the right. To avoid division-by-zero errors, all the diagonals in the input distance matrix are replaced by the mean of their neighbors. Figure 3. Reciprocating the distance matrix so that larger numbers represent shorter distances in the input distance matrix. The input distance matrix is shown in left and the reciprocated distance matrix is on the right. To avoid division-by-zero errors, all the diagonals in the input distance matrix are replaced by the mean of their neighbors. Methods For instance, if the highest probability bin for a residue pair is the range [6.5, 7], then the predicted distance becomes 6.75 Å. Real‑valued distance map prediction. Predicting continuous distance values, like many other regres- sion problems, is challenging. Considering the distance prediction as a regression problem, in particular, has a unique domain specific characteristic, i.e., it is more important to predict shorter distances more accurately than longer distances. This is because, from the perspective of structure prediction and binding-site prediction, it is more meaningful to predict inter-residue interactions than non-interactions (i.e. closer/smaller distances are more important). Andras Fiser’s group have referred to such interactions as ‘interaction hubs’27. It immediately follows that the commonly used machine learning loss functions such as mean squared error or mean absolute error are unfit for this purpose because they focus on optimizing the longer distance values before the shorter ones. Recently, ‘logcosh’ loss (logarithm of hyperbolic cosine) has been found to be highly effective for many problem domains. It behaves similar to the squared loss for smaller loss values and is also similar to absolute loss otherwise, i.e., the loss is not so strongly affected by the occasional incorrect predictions. However, this loss function also does not focus on optimizing shorter distances. As a solution, we propose a novel loss function that precisely focuses on optimizing the shorter distances first. The idea is to reciprocate the true and predicted distances separately and then apply the logcosh loss to the difference between the two. This ‘reciprocal’ log cosh loss is given by: Loss = log cosh K P + e − K T + e . Here, P is a predicted distance matrix, T is the true distance matrix, e is a small positive number (epsilon), and K is a scalar that scales the losses to prevent underflow. We empirically set K to 100. However, our initial implemen- tation of this loss function in Tensorflow did not converge quickly enough. We do not fully understand why such a loss function is so efficient when implemented. As a solution, we reciprocated our distance matrices (output Here, P is a predicted distance matrix, T is the true distance matrix, e is a small positive number (epsilon), and K is a scalar that scales the losses to prevent underflow. We empirically set K to 100. Results D Results Dataset. PDNET, when zipped, is only around 1 GB in size. All the scripts used to curate the dataset, gen- erate the input features and distance maps, as well as the scripts with deep learning architectures for training, validation and testing, are released along with the data. Evaluation of predicted distances and contacts. Here we present the evaluation of our deep learning methods that predict contacts (PDNET-Contact), distance intervals (PDNET-Binned), and real-valued distances (PDNET-Distance), on the 150 proteins in the test set. Overall, we determined that all methods performed rela- tively well on the test set because of the large and high-quality input alignments in the set, as shown in Table 2. PL and PNC for the PDNET-Contact method are 69.5% and 61.1%, respectively. The contact precision of our multi-class classification method, PDNET-Binned, is lower than our binary predictor. PNC for PDNET-Binned is 60.5% on the test dataset. These findings slightly contradict with the findings of Jinbo Xu’s group30 who found up to 4% improvement with their method based on binning. We believe that this slightly poor performance of a multi-class classification model on the mean absolute error and precision metrics is compensated by the pre- dicted probability information for each class, which can then be used to build distogram potentials3 for build- ing models. PDNET provides the platform to further improve this precision through improved architectures, hyper-parameters, and overall training techniques. For instance, we found that binning in a way such that more bins are below the 8 Å threshold can improve contact prediction precision. This is expected because, in contrast, equal-width binning implies that there will be more bins above the 8 Å threshold than below (assuming that the full range is 4–20 Å) forcing the deep learning models to equally and precisely predict the difficult-to-predict larger distance values. This, in turn, can hurt a model’s performance when the contact precision (distances only below 8 Å) is calculated. Our PDNET-Distance method predicts long-range distances with an MAE8 of 4.1 Å on the test set. Notably, this value seems higher because MAE8 is the evaluation of all true distances below 8 Å, i.e. some incorrect predic- tions can significantly impact the average error. Results in Table 2 show that PNC of PDNET-Distance is around 3.3% (2% points) less than that of PDNET-Contact, i.e., PDNET-Distance predicts contacts with slightly less precision than PDNET-Contact. Methods However, our initial implemen- tation of this loss function in Tensorflow did not converge quickly enough. We do not fully understand why such a loss function is so efficient when implemented. As a solution, we reciprocated our distance matrices (output https://doi.org/10.1038/s41598-020-70181-0 Scientific Reports \| (2020) 10:13374 \| www.nature.com/scientificreports/ Table 2. MAE and the precision of the contact prediction method (PDNET-Contact), the distance prediction method (PDNET-Distance), and binned distance prediction method (PDNET-Binned), on the 150 proteins in the test set. All MAEs are in Å, and the PL and PNC for the three PDNET methods are shown in percentage. DeepCov and DEEPCON methods’ performances, which are trained on the same dataset, are also reported. a As reported in the DeepCov16 paper. Methods MAE8 PL PNC PDNET-Contact – 69.5 61.1 PDNET-Distance 4.1 67.5 59.1 PDNET-Binned 3.8 67.8 60.5 DeepCova – 55.1 – DEEPCON – 63.4 55.8 Methods MAE8 PL PNC PDNET-Contact – 69.5 61.1 PDNET-Distance 4.1 67.5 59.1 PDNET-Binned 3.8 67.8 60.5 DeepCova – 55.1 – DEEPCON – 63.4 55.8 Table 2. MAE and the precision of the contact prediction method (PDNET-Contact), the distance prediction method (PDNET-Distance), and binned distance prediction method (PDNET-Binned), on the 150 proteins in the test set. All MAEs are in Å, and the PL and PNC for the three PDNET methods are shown in percentage. DeepCov and DEEPCON methods’ performances, which are trained on the same dataset, are also reported. a As reported in the DeepCov16 paper. Table 2. MAE and the precision of the contact prediction method (PDNET-Contact), the distance prediction method (PDNET-Distance), and binned distance prediction method (PDNET-Binned), on the 150 proteins in the test set. All MAEs are in Å, and the PL and PNC for the three PDNET methods are shown in percentage. DeepCov and DEEPCON methods’ performances, which are trained on the same dataset, are also reported. a As reported in the DeepCov16 paper. labels for the deep learning model) instead of reciprocating the loss function. In other words, we reciprocated the input distance matrices and used the standard logcosh loss (see Fig. 3). Such a setting made it easier for the deep learning setup to converge reliably. Results D The output of PDNET-Distance, PDNET-Contact, and PDNET-Binned are shown in the bottom row. Figure 4. True distance map, true contact map and the native structure of ‘1a6m-A’ in the test set are shown in the top row. The output of PDNET-Distance, PDNET-Contact, and PDNET-Binned are shown in the bottom row. Real‑valued distance predictions. Our real-valued distance prediction method (PDNET-Distance), trained by reciprocating the distance maps, helps the deep learning optimization focus on correctly predicting shorter distances before optimizing the longer ones. In other words, the model first attempts to predict shorter distances over others, i.e., focuses on the residue pairs that are closer in physical distance but not necessarily in sequence. To visually investigate the model’s focus on shorter distances, we randomly selected two proteins from the test set, and plotted the predicted long-range distances vs. the true long-range distances. Figure 5 shows that the model makes more precise predictions for shorter distances. These visualized examples validate that PDNET-Distance effectively focuses on correctly predicting the shortest distances over others. Evaluation on CASP13 and CAMEO hard targets. We further evaluated our methods on the two most difficult datasets—131 hard targets in the CAMEO competition, released after December 8, 2018, and the free- modeling targets of the CASP13 competition. Because of CASP’s agreements and policies, unlike the assessors of CASP13 and some of the top groups, we (referred to as the public) do not have access to all the native structures. Of the 32 free-modeling domains, we only had access to nine domains. Hence, we were limited in our ability to evaluate these nine domains without a way to compare with the other 23 domains. Consistent with the practice, we predicted distances and contacts for the full target and evaluated on the domains26, 27. For all our predictions, we used the alignments predicted by the trRosetta method, as our input. These alignment files are publicly avail- able at https://yanglab.nankai.edu.cn/. On these nine free-modeling domains our PDNET-Contact and PDNET- Distance methods achieve PL of 38.8% and 32.3% compared to 45.0% by the RaptorX method, the top group in the competition (see Table 3). To evaluate our methods on the 131 CAMEO hard targets, we used the align- ments generated using the same trRosetta method. Eight of these 131 targets’ native structure did not have any long-range contacts; thus, we skipped them and did not attempt any analysis of these targets in our evaluation. Results D The PDNET-Distance method, however, predicts granular information con- tained in real-valued distance predictions which can be potentially more informative than the binary contact/ non-contact prediction. As an example, in Fig. 4 we visualize and compare the predicted contacts, binned dis- tances, and real-value distances predicted by our three methods, for the protein chain ‘1a6mA’ in the test dataset. p y p Next, we compared our methods with other standard methods trained on the same dataset but using covari- ance matrix, a slightly different input feature. Table 2 summarizes our comparison with two existing methods, DeepCov16 and DEEPCON21. These two methods only accept a covariance matrix as input. Notably, both features sets, covariance matrix or PDNET features, are derived from the same multiple sequence alignments, i.e., the input information for deriving coevolutionary signals is the same. The superior performance of all three PDNET methods compared to the DeepCov and the DEEPCON method draws attention to the fact that the seven fea- tures we selected are more predictive than the covariance matrix alone. It is obvious that adding the covariance matrix to our list of features may improve the precision. However, since covariance matrices typically have 400+ channels, the input feature files become exceptionally large (roughly 200 MB per protein chain, on average); thus, integrating the covariance matrix could make PDNET unportable. Overall, our results show that the features we have selected are more informative than the standard covariance matrix only feature. y We also trained our models to predict inter-residue Cα (carbon-alpha) distances in addition to Cβ , and we did not find marked improvement in mean absolute error or contact precision. This can explain why methods such as the ones developed by Jinbo Xu’s group use separate models to predict Cα −Cα or Cγ −Cγ distances30. Furthermore, our PDNET-Binned method was extremely slow to train. On average, for one epoch of training with a batch size of 2, our PDNET-Binned method took around 10 hours to train, compared to our PDNET-Contact and PDNET-Distance methods, which only took 20 minutes. In general, we observed that the training time of our multi-class classifier was proportional to the number of classes or distance bins. https://doi.org/10.1038/s41598-020-70181-0 Scientific Reports \| (2020) 10:13374 \| www.nature.com/scientificreports/ Figure 4. True distance map, true contact map and the native structure of ‘1a6m-A’ in the test set are shown in the top row. Results D Performance of CASP13’s top group and the trRosetta method on the CAMEO-HARD dataset are provided as a reference. Results of the trRosetta method are copied from the Yang et al.18 since the predicted contacts are not publicly available. Dataset Methods PL PNC CASP13 FM PDNET-Contact 38.8 38.5 PDNET-Distance 32.3 30.3 Top CASP13 group 45.0 45.7 CAMEO-HARD PDNET-Contact 48.3 45.1 PDNET-Distance 46.7 43.7 trRosetta 48.0 – Table 3. Summary of the performance of PDNET-Contact and PDNET-Distance on the 9 CASP13 free- modeling domains (for which the native structures are publicly available) and on the 131 CAMEO hard- targets. Performance of CASP13’s top group and the trRosetta method on the CAMEO-HARD dataset are provided as a reference. Results of the trRosetta method are copied from the Yang et al.18 since the predicted contacts are not publicly available. Dataset Methods PL PNC CASP13 FM PDNET-Contact 38.8 38.5 PDNET-Distance 32.3 30.3 Top CASP13 group 45.0 45.7 CAMEO-HARD PDNET-Contact 48.3 45.1 PDNET-Distance 46.7 43.7 trRosetta 48.0 – Dataset Methods PL PNC CASP13 FM PDNET-Contact 38.8 38.5 PDNET-Distance 32.3 30.3 Top CASP13 group 45.0 45.7 CAMEO-HARD PDNET-Contact 48.3 45.1 PDNET-Distance 46.7 43.7 trRosetta 48.0 – Table 3. Summary of the performance of PDNET-Contact and PDNET-Distance on the 9 CASP13 free- modeling domains (for which the native structures are publicly available) and on the 131 CAMEO hard- targets. Performance of CASP13’s top group and the trRosetta method on the CAMEO-HARD dataset are provided as a reference. Results of the trRosetta method are copied from the Yang et al.18 since the predicted contacts are not publicly available. Table 3. Summary of the performance of PDNET-Contact and PDNET-Distance on the 9 CASP13 free- modeling domains (for which the native structures are publicly available) and on the 131 CAMEO hard- targets. Performance of CASP13’s top group and the trRosetta method on the CAMEO-HARD dataset are provided as a reference. Results of the trRosetta method are copied from the Yang et al.18 since the predicted contacts are not publicly available. Table 3. Summary of the performance of PDNET-Contact and PDNET-Distance on the 9 CASP13 free- modeling domains (for which the native structures are publicly available) and on the 131 CAMEO hard- targets. Performance of CASP13’s top group and the trRosetta method on the CAMEO-HARD dataset are provided as a reference. Results of the trRosetta method are copied from the Yang et al.18 since the predicted contacts are not publicly available. Results D When long-range contacts were evaluated, our results showed that our PDNET-Contact and PDNET-Distance methods predicted contacts with precision similar to the trRosetta method18. The PL for trRosetta, PDNET-Con- tact, and PDNET-Distance were 48.0%, 48.3% and 46.7% respectively. Overall, this comparable performance of PDNET models with other state-of-the-art methods that use additional strategies, such as using larger training sets and model ensembling, highlight the representativeness and overall value of PDNET. It is worth noting that compared to the methods such as trRosetta, which requires many GPUs and high-speed solid state disks for training, PDNET requires lesser resources and is highly scalable. Finally, as a minor additional experiment, we cleaned the trRosetta alignments for the CAMEO targets by removing the alignment rows which are composed only of gap characters and by removing duplicate rows. We then ran our PDNET-Distance method. There were 55 such alignments out of 131. Such an alignment cleaning, we observed, did not significantly improve the con- tact precision, except for one protein chain, 5YVQA, for which the top L long-range precision improved from 0 to 4%. Scientific Reports \| (2020) 10:13374 \| https://doi.org/10.1038/s41598-020-70181-0 www.nature.com/scientificreports/ Figure 5. Comparison of true long-range distances and the distances predicted by our PDNET-Distance method for two random examples from the test dataset: ‘1a6m-A’ (top row) and ‘1gmi-A’ (bottom row). True distance maps and predicted distance maps are shown in the first and second columns respectively. The plots in the last column visualize the comparison of predicted long-range distances and the corresponding true distances. In these two plots, top L long-range predictions are shown in red and all other long-range distances are shown in blue. Figure 5. Comparison of true long-range distances and the distances predicted by our PDNET-Distance method for two random examples from the test dataset: ‘1a6m-A’ (top row) and ‘1gmi-A’ (bottom row). True distance maps and predicted distance maps are shown in the first and second columns respectively. The plots in the last column visualize the comparison of predicted long-range distances and the corresponding true distances. In these two plots, top L long-range predictions are shown in red and all other long-range distances are shown in blue. Table 3. Summary of the performance of PDNET-Contact and PDNET-Distance on the 9 CASP13 free- modeling domains (for which the native structures are publicly available) and on the 131 CAMEO hard- targets. www.nature.com/scientificreports/ built using distance restraints was significantly more accurate with a much higher TM-score (see Supplementary Fig. 1). We translated the predicted real-valued distances to upper and lower bound constrains using an empirical rule. For a predicted real-valued distance d, we calculated error range, δ = 0.03 ∗d ∗d , where d is the predicted real-valued distance, lower bound l = d −δ/2 , and upper bound u = d + δ/2 (see Supplementary Fig. 2). This empirical rule of setting a larger error range for larger distances follows the design of our loss function, which focuses on learning to predict shorter (not short-range) distance values before longer ones, i.e. shorter distance predictions are more likely to be correct. We assert that the methods for calculating δ can be further optimized for a chosen 3D modeling protocol or even be predicted as an additional output channel of a deep learning model.f g p p p p g Next we investigated the effect of training dataset size on the precision of prediction and the importance of features used in PDNET. After selecting 11 subsets of the training set consisting of a random 100, 200, 300, 400, 500, 1,000, 1,500, 2,000, 2,500, 3,000, and 3,356 (i.e. full set) protein chains as the training dataset, we trained PDNET-Distance models and based our evaluation on the PSICOV150 test dataset. A careful analysis of the results revealed that the precision of the deep learning model starts to taper off after around 1,500 proteins in the training set (see Supplementary Fig. 3). Our results also revealed that the deep learning models trained using just a few hundred protein chains predicted remarkably precise distances. Next, to better understand the contribution from the input features used in PDNET, we grouped the seven features into five groups and reran our PDNET-Distance training by removing each feature or feature set each time. Our results showed that the CCMpred and FreeContact features are the most important input features. Removing these two features drops the top L long-range contact precision by 45% (see Supplementary Table 1). In addition to these experiments, we also tested deep learning architectures other than the DEEPCON type architecture used in the PDNET-Distance method which included: (a) a standard residual network, i.e., with no dilations and dropouts, and (b) a fully convolutional network (FCN). www.nature.com/scientificreports/ We observed that both residual networks perform substantially better than the FCN method, while the standard residual network performed just slightly worse than the architecture used in the PDNET-Distance method (see Supplementary Table 2).h ( pp y ) Other groups have also attempted to ‘democratize’ the deep learning of protein distance prediction. The Pro- teinNet dataset by Alquraishi32 was released as a standardized dataset for machine learning of protein structure. It consists of large and small subsets for learning many features. Although the full dataset is four terabytes in size, smaller subsets are available for download upon request. Benchmark results for contact prediction or distance prediction are not available for this dataset. Similarly, Yun Song’s group have released TAPE33, a dataset packaged for predicting secondary structures, inter-residue contacts, and remote homology detection. These datasets can also be extended to predict inter-residue distances. Our work here, however, serves as the first work, to not only to discuss the distance prediction problem as the primary focus but also to release a full deep learning framework to train and evaluate distance predictions. p We believe that PDNET will be particularly helpful for researchers with some machine learning background, who are interested in difficult problems like protein structure prediction. PDNET can be easily extended to test the significance of adding other features such as the covariance matrix16 and the precision matrix8; moreover, PDNET can be used to predict dihedral angles/orientations18. We also believe that significant future contribu- tions can be made by focusing on novel feature engineering techniques, loss functions, and architectures that may be particularly suitable for this specific problem of distance prediction. Data availability ll d y All scripts, training data, deep learning code for training, validation, and testing, as well as Python Notebo are available at https://github.com/ba-lab/pdnet/. Received: 1 May 2020; Accepted: 23 July 2020 References 1. Abriata, L. . A., Tamò, G. . E. & Dal Peraro, M. A further leap of improvement in tertiary structure prediction in casp13 prompts new routes for future assessments. Proteins: Struct. Funct. Bioinf. 87, 1100–1112 (2019). 1. Abriata, L. . A., Tamò, G. . E. & Dal Peraro, M. A further leap of improvement in tertiary structure prediction in casp13 prompts new routes for future assessments. Proteins: Struct. Funct. Bioinf. 87, 1100–1112 (2019). f 2. Kandathil, S. . M., Greener, J. . G. & Jones, D. . T. Prediction of interresidue contacts with deepmetapsicov in casp13. Proteins Struct. Funct. Bioinf. 87, 1092–1099 (2019). f 3. Senior, A. . W. et al. Protein structure prediction using multiple deep neural networks in the 13th critical assessment of protein structure prediction (casp13). Proteins: Struct. Funct. Bioinf. 87, 1141–1148 (2019). f 4. Li, Y., Zhang, C., Bell, E. W., Yu, D.-J. & Zhang, Y. Ensembling multiple raw coevolutionary features with deep residual neural networks for contact-map prediction in casp13. Proteins: Structure. Funct. Bioinf. 87, 1082–1091 (2019). 4. Li, Y., Zhang, C., Bell, E. W., Yu, D.-J. & Zhang, Y. Ensembling multiple raw coevolutionary features with deep residual neural networks for contact-map prediction in casp13. 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Improved protein structure prediction using predicted interresidue orientations. Proc. Natl. Acad. Sci. (2020). 19 D N L t l C h d d di i f i h h d N l i A id R 19. Dawson, N. L. et al. Cath: an expanded resource to predict protein function through structure and sequence. Nucleic Acid 45, D289–D295 (2017).f 20. AlQuraishi, M. End-to-end differentiable learning of protein structure. Cell Syst. 8, 292–301 (2019 f 21. Adhikari, B. Acknowledgementsi We acknowledge financial support from US National Science Foundation to B.A (award number 1948117), and the support from NVIDIA and Google. We thank Bikash Shrestha, Jamie Lea, Jacob Barger, Mrinal Rawool, David Richards, Amarilda Dyrmishi, Patrick Kong, and Anthony Ackah-Nyanzu at the University of Missouri-St. Louis for their contributions to interesting and beneficial discussions during this work. We are also extremely thankful to the reviewers of the International Conference on Machine Learning (ICML) 2020 conference, Dr. John Moult at University of Maryland, Dr. Andras Fiser at Albert Einstein College of Medicine, and Mark Chonofsky at University of Oxford, for providing many useful comments. Author contributions d h Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-70181-0. Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-70181- Correspondence and requests for materials should be addressed to B.A. 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 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) 2020 Scientific Reports \| (2020) 10:13374 \| https://doi.org/10.1038/s41598-020-70181-0
https://openalex.org/W4287840928	https://jurnal.unimed.ac.id/2012/index.php/gesture/article/download/36390/pdf	Indonesian	null	ANALISIS KEBUTUHAN PENINGKATAN MINAT PESERTA DIDIK LAKI-LAKI DALAM PEMBELAJARAN SENI TARI	Gesture: Jurnal Seni Tari/Gesture : Jurnal Seni Tari	2,022	cc-by-sa	4,471	Keywords: Male Interests, Learning, Improvement, Learners, art of dance Abstract: Kurangnya minat peserta didik laki-laki dalam mengikuti pembelajaran seni membuat proses belajar mengajar menjadi tidak efektif. Masalah yang diambil yaitu peningkatan minat peserta didik laki laki dalam pembelajaran seni tari. Tujuan pembelajaran adalah untuk mengetahui proses pembelajaran model inkuiri. Manfaat pembelajaran dapat diajukan untuk acuan pembelajaran model inkuiri. Metode yang digunakan adalah kualitatif dan R&D. Teknik pengumpulan data menggunakan observasi, wawancara, dan dokumentasi. Teknik validasi data menggunakan metode triangulasi. Teknik analisis data yang digunakan adalah reduksi data, penyajian ulang, dan penarikan kesimpulan. Berdasarkan enam tahap proses pembelajaran inkuiri peserta didik laki-laki pada awalnya tidak tertarik dengan tarian dan hanya diam saja pada saat pembelajaran seni tari, dengan diterapkannya model inkuiri siswa menjadi lebih aktif dan terlibat berkontribusi pada pembelajaran di kelas. Saran untuk lebih memperhatikan waktu selama penyampain karena membutuhkan waktu yang lama agar materi yang lain juga tersampaikan sesuai rencana. Kata Kunci: Laki-laki Minat, Pembelajaran, Peningkatan, Peserta Didik, Seni Tari Kata Kunci: Laki-laki Minat, Pembelajaran, Peningkatan, Peserta Didik, Seni Tari ANALISIS KEBUTUHAN PENINGKATAN MINAT PESERTA DIDIK LAKI-LAKI DALAM PEMBELAJARAN SENI TARI Harist Harjanti1, Sunarto2, Wahyu Lestari3 1Pendidikan Seni, Pascasarjana, Universitas Negeri Semarang, haristharjanti443@gmail.com 2Pendidikan Seni, Pascasarjana, Universitas Negeri Semarang, sunartofbs@mail.unnes.ac.id 3Pendidikan Seni, Pascasarjana, Universitas Negeri Semarang, wahyupyarlestari@mail.unnes.ac.id ANALISIS KEBUTUHAN PENINGKATAN MINAT PESERTA DIDIK LAKI-LAKI DALAM PEMBELAJARAN SENI TARI Harist Harjanti1, Sunarto2, Wahyu Lestari3 1Pendidikan Seni, Pascasarjana, Universitas Negeri Semarang, haristharjanti443@gmail.com 2Pendidikan Seni, Pascasarjana, Universitas Negeri Semarang, sunartofbs@mail.unnes.ac.id 3Pendidikan Seni, Pascasarjana, Universitas Negeri Semarang, wahyupyarlestari@mail.unnes.ac.id ANALISIS KEBUTUHAN PENINGKATAN MINAT PESERTA Harist Harjanti1, Sunarto2, Wahyu Lestari3 1Pendidikan Seni, Pascasarjana, Universitas Negeri Semarang, haristharjanti443@gmail.com 2Pendidikan Seni, Pascasarjana, Universitas Negeri Semarang, sunartofbs@mail.unnes.ac.id 3Pendidikan Seni, Pascasarjana, Universitas Negeri Semarang, wahyupyarlestari@mail.unnes.ac.id Abstrak: The lack of interest of male students in participating in art learning makes the teaching and learning process ineffective. The problem taken is the increase in the interest of male students in learning the art of dance. The purpose of learning is to determine the progress of learning the inquiry model. This research interest can be used as a reference for the next inquiry model learning process. The method used is qualitative and R&D. Data collection techniques using observation, interviews, and documentation. The data validation technique uses the triangulation method. The data analysis technique used is data reduction, restatement, and drawing conclusions. Based on the six stages of the learning process, boys were initially not interested in dance and just kept quiet, with the implementation of the question and answer model, students became more active and involved in contributing to class learning. It is recommended to pay more attention to time during the investigation as it takes time to convey other materials as well. I. PENDAHULUAN negara. Salah satu ujung tombak dalam sebuah pendidikan yaitu adanya seorang guru yang harus dibekali dengan kemampuan yang baik guna membelajarkan peserta didik. Selain dari pendidik, salah satu unsur Pendidikan merupakan salah satu unsur terpenting dalam kemajuan sebuah negara, dengan demikian pendidikan harus dikelola dengan baik demi mewujudkan cita-cita 129 tari, musik ataupun teater. Pemilihan aspek tersebut tergantung dari bapak/ibu guru yang ada di sekolah masing-masing. Banyak dari peserta didik yang menganggap bahwa matapelajaran seni budaya adalah matapelajaran yang menyenangkan. Hal tersebut tidak terlepas dari peran guru ketika proses belajar mengajar sehingga menumbuhkan minat peserta didik dalam belajar khususnya pada mata pelajaran seni budaya. Rast, Harmin dan Simon dalam (Mulyati, 2004: 46) menyatakan bahwa terdapat beberapa hal pokok yang berkaitan dengan minat diantaranya ada perasaan senang dalam diri yang memberikan perhatian kepada objek tertentu, adanya ketertarikan terhadap objek tertentu, adanya aktivitas atas objek tertentu dan adanya kecenderungan untuk mengarahkan dan mempengaruhi tingkah laku individu. Kecenderungan minat peserta didik terhadap pembelajaran seni tari peneliti temui pada saat melakukan observasi di SMA Laboratorium Universitas PGRI pada jenjang X MIPA. pendidikan yang menunjang proses pembelajaran yang baik yaitu terbentuknya proses pembelajaran yang baik antara guru dengan peserta didik (Kusumastuti, 2015: 1). Pembelajaran dapat dilihat sebagai perwujudan dari kurikulum formal, pada umumnya dalam proses belajar siswa harus ada pengembangan bagi guru untuk melaksanakan proses pembelajaran sesuai dengan tujuan (Ismianto, 1999). Pembelajaran dapat dilihat sebagai perwujudan dari kurikulum formal, pada umumnya dalam proses pembelajaran siswa perlu adanya pengembangan bagi guru untuk melaksanakan proses pembelajaran menuju tujuan yang baik (Martono, 2012: 4) Pembelajaran merupakan terjemahan dari kata “Instruction” dalam kegiatan pembelajaran disebut dengan instruksional adalah usaha untuk mengelola lingkungan dengan sengaja agar seseorang membentuk diri secara positif tertentu dalam kondisi tertentu (Miarso, 2004: 538). Untuk dapat mengubah siswa menjadi orang yang positif tentunya harus melalui proses dan cara tertentu. Metode ini diterapkan dalam pendidikan formal di sekolah atau di pendidikan nonformal, dalam penelitian ini akan diajukan pertanyaan- pertanyaan yang berkaitan dengan kajian seni tari di lembaga formal pada tingkat menengah atas. SMA Laboratorium Universitas PGRI merupakan salah satu sekolah swasta terbaik yang ada di Kota Semarang. Sekolah ini terletak di Jalan. Gajah Raya No. 40, Siwalan, Gayam Sari, Semarang. SMA ini lebih dikenal dengan istilah SMA Labschool UPGRIS sekolah ini adalah sekolah swasta dibawah naungan Universitas PGRI Semarang. I. PENDAHULUAN Sekolah ini termasuk sekolah yang baru namun fasilitasnya sangat lengkap mulai dari kelas ber AC sampai dengan tenaga pendidikan yang ada di SMA Labschool juga Proses pembelajaran seni yang ada di sekolah formal utamanya terletak pada matapelajaran seni budaya. Pada matapelajaran tersebut peserta didik dapat mempelajari berbagai seni yang ada baik rupa, 130 metode tersebut diharapkan peserta didik dapat aktif dalam, proses pembelajaran di kelas. Berdasarkan hal tersebut dapat ditarik masalah terkait bagaimana proses peningkatan minat peserta didik laki-laki dalam pembelajaran seni tari model inkuiri di SMA Laboratorium Universitas PGRI pada kelas X MIPA. Tujuan dari artikel yaitu mengetahui proses peningkatan minat peserta didik laki- laki dalam pembelajaran seni tari di SMA Lab Upgris dengan menggunakan metode inkuiri. Teori yang digunakan pada penelitian ini yaitu teori pembelajaran, proses pembelajaran, minat dan model pembelajaran inkuiri, dengan penjabaran sebagai berikut: berkualitas. Saat ini di SMA Lab terdiri dari 9 kelas paralel yang terdiri dari 3 kelas untuk jenjang X, 4 kelas untuk jenjang XI dan 2 kelas untuk jenjang XII. Matapelajaran yang diajarkan di SMA Lab Upgris sama seperti sekolah SMA yang lain yaitu mulai dari mata eksak dan non eksak. Untuk matapelajaran seni budaya aspek yang diajarkan yaitu seni tari untuk jenjang semester 1 dan seni musik untuk jenjang semester 2. Saat semester 1 peserta didik diberikan materi teori maupun materi praktek. Teori yang digunakan pada penelitian ini yaitu teori pembelajaran, proses pembelajaran, minat dan model pembelajaran inkuiri, dengan penjabaran sebagai berikut: Kemudian saat materi praktek semua peserta didik harus dituntut untuk bisa mempraktikan satu tarian yang telah disepakati bersama. Namun pada kenyataanya peserta didik laki laki tidak tertarik untuk mengikuti pelajaran menari, hal tersebut dibuktikan pada saat guru menyampaikan materi praktik peserta didik banyak yang ngobrol sendiri dan tidak fokus dalam mengikuti pelajaran. Mereka masih menganggap bahwa menari hanya untuk perempuan dan menganggap bahwa banyak pandangan negatif ketika seorang laki laki menari. Meskipun terdapat dalam proses pembelajaran materi antara laki laki dan perempuan dibedakan peserta didik laki laki tetap tidak antusias. Berdasarkan hal tersebut maka perlunya perubahan metode dalam proses pembelajaran yang ada di kelas. Salah satu metode yang dapat merangsang peserta didik untuk aktif dikelas yaitu metode inkuiri. Inkuiri berasal dari kata inquire yang artinya meminta keterangan dalam model ini peserta didik dituntut untuk berperan aktif dengan 3. Pembelajaran Inquiry. Pada penelitian ini proses pembelajaran akan menggunakan pembelajaran inquiry pembelajaran dilakukan melalui proses mengamati, menciba, menalar dan mengkomunikasikan, dari pembelajaran inquiry akan menghasilkan peserta didik yang produktif, kreatif, inovatif dan afektif. Inkuiri berasal dari kata inquire yang berarti menyelidiki. Siswa diprogram untuk tetap aktif secara mental dan fisik. Materi yang disampaikan guru tidak hanya disajikan dan diterima oleh siswa, tetapi siswa berusaha sedemikian rupa sehingga memperoleh berbagai pengalaman untuk “menemukan kembali” konsep-konsep yang dimaksudkan oleh guru. (Ahmadi, 1997). Model adalah instruksi yang meminta siswa untuk memproses pesan sehingga mereka memperoleh pengetahuan, keterampilan, dan nilai. Dalam model survei, siswa dirancang untuk berpartisipasi dalam melakukan penyelidikan. Model pengajaran tanya jawab adalah pengajaran yang berpusat pada siswa. Dengan cara ini, siswa lebih aktif dalam belajar. Tujuan utama dari model tanya jawab adalah untuk mengembangkan keterampilan intelektual, berpikir kritis dan kemampuan memecahkan masalah secara ilmiah. iah (Dimyati & Mudjiono, 1994). 1. Pembelajaran. Pembelajaran adalah proses interaksi antara peserta didik dengan lingkungan sehingga terjadi perubahan perilaku kearah yang lebih baik (Kunandar, 2007: 1). Belajar juga dikatakan sebagai proses pemberian bimbingan atau bantuan kepada siswa dalam proses belajar. Peran guru sebagai pembimbing dimulai dari banyaknya siswa yang mengalami masalah. Menurut Gagne dan Briggs (1979) dalam buku (Nyoman, 2008) menyatakan bahwa pengajaran adalah suatu sistem pendukung proses belajar yang dirancang agar siswa aktif belajar di dalam kelas. Belajar berasal dari kata instruction yang artinya instruksi. Pembelajaran kata sangat dipengaruhi oleh aliran psikologis, dan teknologi dikatakan memudahkan siswa untuk mempelajari sesuatu secara instan melalui media yang berbeda. (Lestari & Cahyono, 131 Agus, 2021: 3). Pembelajaran dapat dikatakan baik jika sesuai dengan rencana. Rencana dalam proses pembelajar tertuang dalam RPP dengan berpedomankan silabus (Kadir, 2017: 25) menyatakan bahwa proses pembelajaran adalah proses sistematis yang melibatkan tahapan desain, pelaksanaan, dan evaluasi. Belajar tidak terjadi begitu saja, tetapi telah melalui beberapa tahap persiapan. 25) Berdasarkan beberapa pendapat tersebut dapat disimpulkan bahwa pembelajaran merupakan suatu proses pembimbingan kepada peserta didik dengan menggunakan sistem yang telah dirancangan sedemikian rupa oleh guru dengan tujuan terjadi perubahan perilaku pada peserta didik. Pada penelitian ini proses pembelajaran akan menggunakan pembelajaran inquiry pembelajaran dilakukan melalui proses mengamati, menciba, menalar dan mengkomunikasikan, dari pembelajaran inquiry akan menghasilkan peserta didik yang produktif, kreatif, inovatif dan afektif. Berdasarkan beberapa pendapat tersebut dapat disimpulkan bahwa pembelajaran merupakan suatu proses pembimbingan kepada peserta didik dengan menggunakan sistem yang telah dirancangan sedemikian rupa oleh guru dengan tujuan terjadi perubahan perilaku pada peserta didik. Berdasarkan beberapa pendapat diatas dapat disimpulkan bahwa pembelajaran adalah suatu aktivitas yang sistematis yang melibatkan tahapan perencanaan, pelaksanaan dan evaluasi dalam proses tersebut memerlukan interkasi. 2. Proses Pembelajaran. Menurut Djamarah dalam (Adi & Hartati, 2015: 62) Dalam proses pembelajaran di kelas utama, harus ada interaksi antara guru dan siswa, yaitu interaksi dengan rasa tujuan, yaitu interaksi yang direncanakan untuk beberapa motivasi, setidaknya hasilnya. tentang tujuan pembelajaran atau memahami tujuan yang dituangkan dalam kegiatan satuan pembelajaran Pembelajaran guru merupakan interaksi terpadu antara pendidik dan siswa. Pendapat lain juga disampaikan oleh Knirk dan Gustafson dalam Rahayu (2016: 9) mengemukakan pandangan lain yang 132 132 Inkuiri adalah rangkaian kegiatan pembelajaran yang menekankan pada proses berpikir kritis untuk mencari dan menemukan jawaban dari suatu masalah yang diberikan. Proses berpikir ini dapat dilakukan melalui tanya jawab dari guru dan siswa (Jumanta Hamdayana 2014: 31). Langkah dalam proses pembelajaran inquiry adalah orientasi, merumuskan masalah, merumuskan hipotesis, menguji hipotesis dan menarik kesimpulan. Sasaran utama kegiatan inkuiri adalah keterlibatan siswa secara logis dan sistematis pada tujuan pembelajaran dan mengembangkan sikap percaya diri siswa tentang apa yang ditemukan dalam proses inkuiri (Tritanto, 2007: 135). Pada dasarnya, kepedulian adalah penerimaan hubungan antara diri sendiri dan sesuatu di luar diri sendiri. Semakin kuat atau intim hubungan tersebut, semakin besar minatnya. Hobi adalah kecenderungan untuk terus-menerus memperhatikan dan mengingat aktivitas tertentu (Slemato, 2015). Berdasarkan teori tersebut dapat disimpulkan bahwa minat adalah kecenderungan terhadap suatu hal yang muncul dari perasaan seseorang dan berkeinginan untuk mengingat secara terus menerus. 5. Metode. Penelitian minat peserta didik laki laki dalam proses pembelajaran seni tari SMA Laboratorium Universitas PGRI Semarang menggunakan metode kualitatif R n D secara umum, metode penelitian adalah sarana untuk memperoleh pengetahuan dan memecahkan masalah yang dihadapi. Metode penelitian juga dapat dipahami sebagai sarana ilmiah untuk mengumpulkan data yang valid dengan tujuan menemukan, mengembangkan, dan membuktikan pengetahuan tertentu yang dapat digunakan untuk memahami, memecahkan, dan mengantisipasi masalah dalam dunia pendidikan. (Sugiyono, 2015: 15). Penelitian ini menggunakan model pembelajaran inkuiri yang dimana dalam proses ini peserta didik dituntut untuk selalu aktif dalam proses pembelajaran di kelas. Berdasarkan beberapa pendapat diatas dapat disimpulkan bahawa proses pembelajaran inquiry adalah proses pembelajaran yang menuntut peserta didik untuk selalu berpikir kritis dan aktif secara mental dan fisik. Pada proses pembelajaran inquiry juga menuntut peserta didik untuk bisa memecahkan masalah secara mandiri yang terdiri atas 6 tahapan. 4. Minat. Minat (interest) adalah kecenderungan untuk selalu memperhatikan dan mengingat sesuatu secara terus menerus (Ashobah et al., 2019). Minat ini erat kaitannya dengan perasaan, terutama kesenangan, karena minat dapat dikatakan muncul dari sikap gembira terhadap sesuatu. Gagasan lain adalah bahwa hobi adalah perasaan cinta dan perasaan keterikatan pada suatu objek atau aktivitas tanpa ada yang memintanya. (Slemato, 2015) Lokasi penelitian peningkatan minat peserta didik laki laki dalam pembelajaran seni tari berada di Jalan. Rajah Raya No. 40, Gayam Sari, Semarang. Sasaran utama pada 133 penelitian ini yaitu fokus pada peningkatan minat peserta didik laki laki terhadap pembelajaran seni tari. Subjek penelitian yaitu peserta didik kelas X MIPA, Guru Seni budaya dan kepala sekolah. Narasumber yang dipilih sebagai informasi utama karena diyakini dapat memberikan infomasi yang akurat dan dapat dipercaya. data primer, dan teknik pengumpulan data lebih banyak pada observasi, pada observasi berperan serta dan dokumentasi (Sugiyono, 2015: 309) Penelitian ini menggunakan tiga teknik pengumpulan data yaitu observasi, wawancara dan dokumentasi dengan penjabaran sebagai berikut. Peneliti melakukan observasi satu kali secara langsung pada tanggal 17 Mei 2022 observasi pertama dilakukan untuk melihat gedung SMA Lab Upgris yang berada di Jalan. Gajah Raya No. 40. Tujuan observasi yaitu untuk mengetahui lokasi SMA Lab Upgris dan untuk mengetahui profil sekolah. Cara yang dilakukan oleh peneliti pada saat observasi yaitu dengan cara peneliti datang langsung ke tempat penelitian. Hasil dari observasi yang pertama yaitu peneliti mengetahui lokasi dan profil SMA Lab Upgris. Data merupakan bahan mentah yang diperoleh sehingga menghasilkan informasi atau keterangan (Putra, 2012: 17) Data yang harus dikumpulkan mungkin berupa data primer, data sekunder. Data primer pada penelitian meliputi data proses pembelajaran model inkuiri, perangkat pembelajaran dan hasil proses pembelajaran model inkuiri kelas X. Data Sekunder didapat dari profil sekolah SMA Lab Upgris. Sumber data adalah objek yang datanya dapat diperoleh jika peneliti melakukan wawancara sambil mengumpulkan data, sumber datanya disebut responden, yaitu orang yang menjawab atau menanggapi pertanyaan peneliti. (Putra, 2012: 29–30). Sumber data penelitian ada dua yaitu sumber data primer dan sekunder. Sumber data primer dalam penelitian ini yaitu peserta didik kelas X MIPA, data sekunder pada penelitian ini yaitu Ibu Nur Khoiriah selaku kepala sekolah SMA Lab Upgris. Teknik yang kedua adalah wawancara merupakan teknik pengumpulan data apabila peneliti ingin melakukan studi pendahuluan untuk menemukan permasalahan yang harus diteliti, tetapi jika peneliti ingin mengetahui hal-hal dari responden yang lebih mendalam. 4. Minat. Teknik pengumpulan data ini mendasarkan diri pada laporan tentang diri sendiri atau self- repot atau setidak tidaknya pada pengetahuan atau keyakinan pribadi .(Sugiyono, 2015: 194) Wawancara dilakukan berbarengan dengan observasi yaitu pada tanggal 17 Mei 2022 peneliti melakukan wawancara kepada Kepala Sekolah untuk menanyakan terkait beberapa hal yang berkaitan dengan profil sekolah, kurikulum, RPP silabus, peneliti juga Teknik pengumpulan data merupakan langkah yang paling penting dalam penelitian. Pengumpulan data dapat dilakukan dalam berbagai sumber, dan berbagai cara. Penelitian kualitatif, pengumpulan data dilakukan pada natural setting (kondisi yang alamiah), sumber 134 mewawancarai guru seni menanyakan terkait proses pembelajaran. Hasil dari wawancara peneliti mendapat informasi terkait profil SMA Lab. Triangulasi teori yaitu membandingkan data dengan teori yang digunakan. Contohnya data yang dapat melalui wawancara yang berkaitan dengan pembelajaran, bahwa dalam proses pembelajaran terdiri atas pembukaan isi dan penutup hasil dari teknik observasi tersebut kemudian dibandingkan dengan teori yang digunakan, sehingga terjadi kecocokan data. Teknik yang ketiga yaitu Dokumen merupakan catatan peristiwa yang sudah berlalu. Dokumen bisa berbentuk tulisan, gambar, atau karya-karya monumental dari seseorang. Dokumen yang berbentuk tulisan misalnya cacatata harian, sejarah kehidupan cerita, biografi, peraturan, kebijakan, saat peneliti observasi peneliti juga langsung mendokumentasikan beberahal yang dianggap penting seperti, visi misi sekolah dan lain lain. Teknik analisis data menggunakan teori Miles Huberman (1984) dalam bahwa aktivitas dalam analisis data kualitatif dilakukan secara interaktif dan berlangsung terus menerus sampai tuntas sehingga datanya sudah jenuh. Aktivitas dalam menganalisis data yaitu:(1) reduksi data, (2) penyajian data, dan (3) penarikan simpulan (Sugiyono, 2016: 337). Teknik keabsahan data yang digunakan yaitu teknik triangulasi. Terdapat 4 macam triangulasi sebagai teknik pemeriksaan yang memanfaatkan penggunaan sumber, teknik, penyidik, dan teori (Sugiyono, 2015: 330). Peneliti menggunakan 2 teknik trianggulasi teknik dan teori. Reduksi data dapat diartikan sebagai proses pemilihan, pemusatan perhatian pada penyederhanaan, pengabstrakan dan transformasi data yang muncul dari catatan- catatan di lapangan (Sumaryanto, 2007: 107). Pengaplikasian reduksi data yaitu data yang diambil dari teknik observasi, wawancara dan dokumentasi peneliti kumpulkan menjadi satu, kemudian peneliti pilih data yang sesuai dan data yang tidak sesuai dengan penelitian, kemudian peneliti klasifikasikan data tersebut sesuai dengan rumusan masalah. 2.2. Rancangan pembelajaran. Perencanaan pembelajaran merupakan tahap paling awal dalam sebuah pembelajaran, guru menyiapkan perangkat pembelajaran untuk proses belajar mengajar agar proses pembelajaran berjalan dengan baik. Rancangan pembelajaran RPP, Silabus dll. Saat ini SMA Laboratorium Universitas PGRI menggunakan kurikulum 2013 dengan 5 hari sekolah. Proses pembelajaran yang ada di SMA Laboratorium saat ini sudah tatap muka seperti biasa. Untuk sasaran yang dipilih dalam pelaksanaan pembelajaran model inkuiri ini fokus pada peserta didik laki laki kelas X MIPA. Kompetensi Dasar yang digunakan yaitu memahami konsep, teknik dan prosedur dalam ragam gerak tari tradisi dengan materi pokok tari tradisional tujuan 4. Minat. Triangulasi teknik yaitu peneliti menggunakan teknik pengumpulan data yang berbeda untuk mendapatkan data dari sumber yang sama salah satu contohnya ketika peneliti melakukan pengecekan data menggunakan data dengan teknik wawancara dan observasi yang aplikasikan pada saat peneliti menanyakan profil dari SMA Lab Upgris kemudian peneliti bandingkan hasil yang diperoleh dari wawancara dengan teknik observasi, hasil kedua teknik tersebut kemudian peneliti analisis sehingga menghasilkan data tentang profil sekolah yang kredibel. Penyajian data dan kumpulan informasi-informasi yang diperoleh dari hasil wawancara, observasi dan dokumentasi disajikan secara lengkap dan jujur. Pada tahap penyajian data, data dianalisis antara kategori 135 dan permasalahan yang ada agar sajian data dapat lebih jelas dan sistematis. berbagai permasalahan dalam sistem pendidikan yang semestinya sampai tahun 2019. Pada tahun 2019 SMA PGRI 1 Semarang mengalami perbaikan manajemen sekolah dengan merubah semua unsur di dalamnya, baik SDM maupun sarana dan prasarana pendukung lainnya. Di bawah kepemimpinan kepala sekolah yang baru dengan dukungan YPLP DM PGRI Kota Semarang bersama dengan Universitas PGRI Semarang mulai menata manajemen sekolah dalam rangka membangun kembali dengan mengaktifkan data pokok pendidikan (Dapodik) yang sudah mengalami soft delete. Saat ini SMA Laboratorium Universitas PGRI Semarang memiliki 9 kelas paralel dan 24 guru. Kesimpulan awal yang ditemukan masih bersifat tentatif dan akan berubah jika tidak ditemukan bukti yang kuat untuk mendukung tahap pengumpulan data selanjutnya. Jika kesimpulan yang ditarik pada tahap awal didukung oleh bukti yang valid dan konsisten ketika peneliti kembali ke lapangan untuk mengumpulkan data, kesimpulan yang disempurnakan itu kredibel (Sugiyono, 2016: 345). Pada tahap ini peneliti melakukan penarikan kesimpulan mengenai keterlibatan peserta didik laki laki dalam proses pembelajaran seni tari. II. HASIL DAN PEMBAHASAN Analisis kebutuhan keterlibatan peserta didik dalam proses pembelajaran seni tari di SMA Lab Upgris akan disajikan beberapa hasil data yang nantinya dapat menunjang penelitian, data yang akan disajikan dalam penelitian ini yaitu terkait profil SMA Lab Upgris, Perangkat pembelajaran dan penerapan metode inkuiri dalam proses pembelajaran seni tari dengan fokus siswa kelas X MIPA. Model Inkuiri. Inkuiri berasal dari kata inquire yang artinya meminta keterangan. Siswa dalam proses pembelajaran dituntut untuk selalu aktif dalam memecahkan masalah di kelas. Menurut Sanjaya (2006: 201) terdapat 6 langkah pembelajaran yang ada pada proses pembelajaran inkuiri orientasi, merumuskan masalah, mengajukan hipotesis, mengumpulkan data, menguji hipotesis dan merumuskan kesimpulan dengan penjabaran sebagai berikut: 2.1. Profil SMA Laboratorium Universitas PGRI SMA Laboratorium Universitas PGRI Semarang merupakan peralihan nama dari SMA PGRI 1 Semarang. Sejak tahun 2014 SMA PGRI 1 Semarang telah mengalami kondisi yang tidak stabil yang ditandai dengan tidak adanya jumlah peserta didik yang mendaftar, sehingga sekolah mengalami 136 Fungsi tari tradisional, pada tahap ini guru memerintah kepada peserta didik untuk mencari satu contoh tari tradisional beserta asalnya. Kelas X MIPA terdapat 30 peserta didik maka akan ada 30 contoh tari tradisional yang berasal dari nusantara, pada tahap observasi peserta didik telah mendapat materi tentang tari tradisional beserta contohnya. utama dalam proses pembelajaran ini yaitu peserta didik dapat memahami tari tradisional dan mempraktikan satu tari tradisional dengan benar. Untuk alokasi waktunya 8 Jam Pelajaran. Model pembelajaran yang digunakan yaitu inkuiri dengan 6 tahapan mulai dari merumuskan masalah, merumuskan hipotesis, pengumpulan data, menguji hipotesis dan merumuskan kesimpulan. a. Orientasi. Langkah yang pertama yaitu orientasi tujuan dari langkah ini yaitu agar peserta didik mempunyai gambaran terkait materi yang akan disampaikan dalam proses ini sesuai jenjang yang dipilih yaitu kelas X dengan KD Memahami konsep, teknik dan prosedur dalam ragam gerak tari tradisi fokus materi tentang tari tradisional. Langkah observasi guru memberikan pengantar penjelasan terkait tari tradisional, kemudian dalam langkah observasi guru juga menjelaskan tentang b. Merumuskan masalah. Langkah kedua yaitu perumusan masalah dalam langkah ini peserta didik akan diberi sebuah masalah oleh guru dan peserta didik akan memecahkan masalah tersebut bersama kelompok persoalan yang disajikan adalah persoalan yang kompleks. Pengaplikasian dalam tahap perumusan masalah yaitu peserta didik setelah mengetahui beberapa tarian yang ada di Indonesia kemudian dalam tahap ini guru membagi peserta didik kedalam beberapa kelompok yang satu anggotanya terdiri dari 4-5 anggota kemudian sesuai dengan materinya agar mereka lebih memahami tentang tari tradisional Jawa Tengah guru menugaskan peserta didik untuk mencari ciri gerak dari tari tradisional Jawa Tengah dan contoh tari tradisional yang ada di Jawa Tengah. d. Pengumpulan data. Mengumpulkan data adalah aktivitas menjaring informasi yang dibutuhkan untuk menguji hipotesis yang diajukan. Pada tahap ini peserta didik mencari satu contoh tari yang ada di Jawa Tengah yang sesuai dengan ciri ciri yang ada dihipotesis kemudian mereka belajar mandiri menghafal tarian tersebut serta harus memahami latar belakang hingga ragam gerak yang digunakan. Kesimpulan. Berdasarkan hasil penelitian dapat disimpulkan bahwa SMA Laboratorium Universitas PGRI Semarang merupakan salah satu sekolah terbaik yang ada di Kota Semarang.yang terdiri dari 9 kelas paralel. Penerapan model inkuiri di SMA Laboratorium Universitas PGRI Semarang dilakukan pada jenjang X MIPA dengan materi tari tradisional. Pada tahap awal peserta didik diberikan materi tentang pengertian tari tradisional, pada tahap ini peserta didik juga mencari satu tari tradisional yang ada di Indonesia. Langkah kedua guru membagi mereka menjadi beberapa kelompok kemudian guru memberikan memberikan satu masalah yaitu ciri-ciri dari tari tradisional Jawa tengah. Pada tahap guru ini memberikan saran agar mencari beberapa tarian Jawa Tengah kemudian hasil dari pengamatan mereka analisis. Langkah ketiga yaitu menyimpulkan jawaban sementara terkait masalah yang ada kemudian mereka akan buktikan dengan memahami satu tari tradisional mulai dari latar belakang hingga menampilkannya. Langkah empat yaitu mempelajari tarian yang sudah dipilih. Langkah kelima yaitu menampilkan sebagian tari yang sudah dipilih kemudian dikaitkan dengan ciri ciri tari tradisional Jawa Tengah yang sudah mereka tulis diawal. Langkah yang terakhir yaitu menampilkan satu tarian penuh dengan menggunakan kostum sederhana kemudian mereka simpulkan hasil dari penampilan tari yang disimpulkan bahwa Berdasarkan hasil penelitian dapat disimpulkan bahwa SMA Laboratorium Universitas PGRI Semarang merupakan salah satu sekolah terbaik yang ada di Kota Semarang.yang terdiri dari 9 kelas paralel. Penerapan model inkuiri di SMA Laboratorium Universitas PGRI Semarang dilakukan pada jenjang X MIPA dengan materi tari tradisional. Pada tahap awal peserta didik diberikan materi tentang pengertian tari tradisional, pada tahap ini peserta didik juga mencari satu tari tradisional yang ada di Indonesia. Langkah kedua guru membagi mereka menjadi beberapa kelompok kemudian guru memberikan memberikan satu masalah yaitu ciri-ciri dari tari tradisional Jawa tengah. Pada tahap guru ini memberikan saran agar mencari beberapa tarian Jawa Tengah kemudian hasil dari pengamatan mereka analisis. Langkah ketiga yaitu menyimpulkan jawaban sementara terkait masalah yang ada kemudian mereka akan buktikan dengan memahami satu tari tradisional mulai dari latar belakang hingga menampilkannya. Langkah empat yaitu mempelajari tarian yang sudah dipilih. Langkah kelima yaitu menampilkan sebagian tari yang sudah dipilih kemudian dikaitkan dengan ciri ciri tari tradisional Jawa Tengah yang sudah mereka tulis diawal. Langkah yang terakhir yaitu menampilkan satu tarian penuh dengan menggunakan kostum sederhana kemudian mereka simpulkan hasil dari penampilan tari yang disimpulkan bahwa c. Mengajukan hipotesis. Hipotesis merupakan dugaan sementara. Pada tahap ini peserta didik menyimpulkan hasil sementara terkait ciri ciri tari tradisional Jawa Tengah, kemudian pada tahap ini peserta didik untuk menguji hipotesis tersebut peserta didik akan 137 e. Menguji Hipotesis. Setelah mereka mencari beberapa data terkait tarian yang ada di Jawa Tengah, selanjutnya untuk menguji hasil dari hipotesis mereka menarikan sebagian dari tarian yang mereka pelajari dan mereka kaitkan dengan ciri ciri tradisional yang mereka buat. III. Kesimpulan dan Saran Kesimpulan. membuktikannya dengan menampilkan satu tarian yang ada di Jawa Tengah. Ucapan Terima Kasih. Peneliti mengucapkan terimakasih yang sebesar kepada Bapak Sunarto dan Ibu Wahyu Lestari yang telah memberikan banyak ilmu selama perkuliahan. Tidak lupa peneliti ucapkan terimakasih kepada SMA Laboratorium Universitas PGRI Semarang yang telah memberikan banyak infomasi terkait penelitian. Martono, H. (2012). Jurnal seni tari. Joged Jurnal Seni Tari, Vol. 3, no. 1, hal. 36– 48. Miarso, Y. (2004). Menyemai Benih Teknologi Pendidikan. Kencana. Mulyati. (2004). Minat Belajar Siswa. PT Rineka Cipta. Nyoman. (2008). Konsep Belajar dan Mengajar. PT Rajagrafindo Persada. Saran. Lebih diperhatikan lagi waktu dalam proses pembelajaran inkuiri karena membutuhkan waktu yang lama agar materi yang lain juga bisa tersampaikan. Lestari, W., & Cahyono, Agus, Y. (2021). Sikap Siswa Terhadap Pembelajaran Seni Tari Di Masa. Vol. 21, no.1, hal. 101–110. f. Merumuskan Kesimpulan. Merumuskan kesimpulan merupakan kegiatan mendeskripsikan temuan yang diperoleh berdasarkan hasil. Pada tahap ini peserta didik melakukan pementasan dengan menggunakan kostum yang sederhana, setelah mereka melakukan pementasan kemudian di akhir pertunjukan masing-masing kelompok menyimpulkan terkait ciri tari tradisional Jawa Tengah berdasarkan apa yang mereka pentaskan sebelumnya. Jadi dari hal tersebut mereka membuktikan ciri ciri dari tari Jawa Tengah dengan menampilkan tarian Jawa Tengah. 138 terdapat kesesuaian antara ciri ciri tari Jawa Tengah yang mereka tampilkan. Dari enam langkah tersebut peserta didik terutama peserta didik laki-laki mulanya pada saat pelajaran menari mereka tidak bersemangat dan hanya diam saja, dengan model inkuiri yang sudah diterapkan peserta didik menjadi lebih aktif dan mau ikut berkontribusi dalam pembelajaran kelas. Ismianto, P. S. P. (1999). “Creative Problem Solving dalam Pembelajaran Pendidikan Seni Rupa: Sebuah Penawaran Pendekatan Pembelajaran" dalam Lingua Artistika. Kadir, Abdul. (2017). Konsep Pembelajaran Kontekstual Di Sekolah. Dinamika Ilmu, Vol. 13, no. 1, hal. 21–38. Kunandar. (2007). Guru Profesional. Raja Grafindo Persada. Kusumastuti, E. (2015). Pendidikan Seni Tari Melalui Pendekatan Ekspresi Bebas, Disiplin Ilmu, Dan Multikultural Sebagai Upaya Peningkatan Kreativitas Siswa. Daftar Pustaka. Putra, E. (2012). Teknik Penyusunan RicikanPenelitian. Pustaka Pelajar. Adi, S., & Hartati, S. C. Y. (2015). 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https://openalex.org/W2945688641	https://europepmc.org/articles/pmc6566186?pdf=render	English	null	On Providing Multi-Level Quality of Service for Operating Rooms of the Future	Sensors	2,019	cc-by	15,624	Received: 15 March 2019; Accepted: 6 May 2019; Published: 18 May 2019 Received: 15 March 2019; Accepted: 6 May 2019; Published: 18 May 2019 Abstract: The Operating Room (OR) plays an important role in delivering vital medical services to patients in hospitals. Such environments contain several medical devices, equipment, and systems producing valuable information which might be combined for biomedical and surgical workﬂow analysis. Considering the sensibility of data from sensors in the OR, independently of processing and network loads, the middleware that provides data from these sensors have to respect applications quality of service (QoS) demands. In an OR middleware, there are two main bottlenecks that might suffer QoS problems and, consequently, impact directly in user experience: (i) simultaneous user applications connecting the middleware; and (ii) a high number of sensors generating information from the environment. Currently, many middlewares that support QoS have been proposed by many ﬁelds; however, to the best of our knowledge, there is no research on this topic or the OR environment. OR environments are characterized by being crowded by persons and equipment, some of them of speciﬁc use in such environments, as mobile x-ray machines. Therefore, this article proposes QualiCare, an adaptable middleware model to provide multi-level QoS, improve user experience, and increase hardware utilization to middlewares in OR environments. Our main contributions are a middleware model and an orchestration engine in charge of changing the middleware behavior to guarantee performance. Results demonstrate that adapting middleware parameters on demand reduces network usage and improves resource consumption maintaining data provisioning. Keywords: distributed systems; health informatics; middleware; operating room; quality of service Vinicius Facco Rodrigues 1,,† , Rodrigo da Rosa Righi 1 , Cristiano André da Costa 1 , Björn Eskoﬁer 2 and Andreas Maier 3 Vinicius Facco Rodrigues 1,,† , Rodrigo da Rosa Righi 1 , Cristiano André da Costa 1 , Björn Eskoﬁer 2 and Andreas Maier 3 1 Software Innovation Lab – SOFTWARELAB, Universidade do Vale do Rio dos Sinos – Unisinos, 93022-718 São Leopoldo, RS, Brazil; rrrighi@unisinos.br (R.d.R.R.); cac@unisinos.br (C.A.d.C.) p , , ; g ( ); ( ) 2 Machine Learning and Data Analytics Lab, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany; bjoern.eskoﬁer@fau.de p g 2 Machine Learning and Data Analytics Lab, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany; bjoern.eskoﬁer@fau.de g y j 3 Pattern Recognition Lab, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany andreas.maier@fau.de 3 Pattern Recognition Lab, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany; andreas.maier@fau.de * Correspondence: vfrodrigues@unisinos.br † Current address: Software Innovation Lab – SOFTWARELAB, Universidade do Vale do Rio dos Sinos – U i i A U i i 950 93022 718 Sã L ld RS B il † Current address: Software Innovation Lab – SOFTWARELAB, Universidade do Vale do Rio dos Sinos – Unisinos, Av. Unisinos, 950, 93022-718 São Leopoldo, RS, Brazil. † Current address: Software Innovation Lab – SOFTWARELAB, Universidade do Vale do Rio dos Sinos – Unisinos, Av. Unisinos, 950, 93022-718 São Leopoldo, RS, Brazil. ceived: 15 March 2019; Accepted: 6 May 2019; Published: 18 May 2019 sensors sensors sensors Article On Providing Multi-Level Quality of Service for Operating Rooms of the Future Vinicius Facco Rodrigues 1,*,† , Rodrigo da Rosa Righi 1 , Cristiano André da Costa 1 , Björn Eskoﬁer 2 and Andreas Maier 3 1. Introduction The adoption of Internet of Things (IoT) technologies in the healthcare ﬁeld has increased considerably in recent years [1]. Healthcare environments constantly demand improved quality of care and smaller operational costs [2]. In particular, the Operating Room (OR) plays an important role in delivering vital medical services for patients in the hospital [3]. In an OR setting, many sensors and applications are involved in the process of generating workﬂow data from procedures in real time [4]. OR settings contain many sensors and applications involved in the process of monitoring procedures, which produces valuable information for data fusion and complex analysis [5,6]. However, providing data from all sensors to user applications is challenging due to the increasing variety of Sensors 2019, 19, 2303; doi:10.3390/s19102303 www.mdpi.com/journal/sensors 2 of 27 Sensors 2019, 19, 2303 heterogeneous sensors which suffer from interoperability problems [1]. IoT service-based architecture is becoming trending approach which provides a layer between user applications and data from sensors to simplify data processing [1,7,8]. The service layer is able to provide such information to any application that might require it for further processing. In summary, a middleware is in charge of collecting data from sensors and delivering it to applications that previously requested it. The middleware concept deﬁnes an intermediary layer between two or more systems to mediate its integration [9,10]. Figure 1 depicts a representation of this conﬁguration showing a scenario where multiple applications access data from several sensors in the OR. The model shows three different levels: (i) User, which comprises the user applications; (ii) Service, which encompasses the middleware responsible for integrating the other levels; and (iii) Sensor, which includes all sensors and devices responsible for producing data. Different kind of sensors are responsible for producing information such as medical staff positions, patients physiological parameters, equipment status, and environment conditions during procedures [11]. These sensors can be radio frequency tags and readers, camera devices, medical devices, etc. Each sensor monitors particular information, and the middleware gathers data from all available sensors providing real-time data for users. Real-time is a very important concern in such environments since saving lives is a matter of seconds in surgical procedures and, besides decreasing time response to critical situations, it is also helpful to avoid medical errors, which is a common problem in ORs [12–14]. 1. Introduction These levels correspond to measurements of several network metrics, such as packet loss, throughput, transmission delay, etc., which describe the overall performance of a service. The growing number of sensors focusing on monitoring patients, medical staff, and equipment results in a need for device data interoperability [17]. That is, the more the number of sensors the more the different information data is produced to be accessed and interpreted. Improving patients’ safety depends on the middleware capacity to acquire and provide large amounts of data in real time [17] regardless the processing and network load. This capacity depends on the middleware performance in terms of network and computing power, which must respect Quality of Service (QoS) requirements of user applications. QoS is a common concept in computing networks that consists of control mechanisms aimed to guarantee that acceptable performance levels of given service are respected. These levels correspond to measurements of several network metrics, such as packet loss, throughput, transmission delay, etc., which describe the overall performance of a service. Middlewares for OR is a novel concept with the potential to be present in the most technological ORs. Data streaming in such environments has the potential to highlight anomalies in surgery workﬂows and support medical decisions. If the middleware fails to deliver data at some moment violating QoS requirements, these anomalies might go unnoticed. In this context, to maintain a desirable QoS levels for real-time applications that consume data from the surgical workﬂow is challenging since the number of sensors and user applications might dynamically change. For instance, in a surgical procedures there are many people are involved in the process which might go in or out the OR at any time. In these situations, wearable motion and indoor location sensors, such as radio-frequency tags, accelerometers, gyroscopes, and magnetometers, are detected only when the subject is within the room. The growth of the number of sensors and devices in the OR increases the complexity of real-time information monitoring since the more the number of sensors the higher the amount of information that the OR middleware must handle simultaneously. Besides handling network connections to sensors at the sensor level, the middleware also must manage user applications requests in the User level, which demand real-time data. 1. Introduction Therefore, it is required that an OR middleware is capable of identifying and handling such situations to keep a certain level of QoS, not impacting in the user experience. Currently, most studies aiming at providing medical systems for ORs focus on describing architectures for data monitoring [18–28]. However, they do not address QoS mechanisms in their solutions, which, to the best of our knowledge, indicates a lack of studies focusing on this signiﬁcant topic for patient safety. Therefore, to the best of our knowledge, there is no current research that explores QoS for OR middlewares. The current strategies focus mainly on developing monitoring systems for OR not addressing QoS. This highlights a knowledge gap that is a signiﬁcant topic for patient safety. In this context, this article presents the QualiCare model, which aims to provide multi-level QoS for OR middlewares in both User and Sensor levels. The scientiﬁc contributions of this research are twofold: 1. Introduction It is important to point out that the middleware provides not only real-time data but also historical data, characterized by two different models: (i) real-time; and (ii) ofﬂine. The former model provides the most updated data from the OR, while the second provides data from past events. Therefore, even if there are any user applications requesting data, the middleware remains gathering data for persistence. This can be achieved, for instance, by using an external database located in the Cloud, as in [15]. Cloud platforms enable easy database scalability through the cloud elasticity feature [16]. Data Collection and Analisys Service Sensor 0 Middleware Application 0 Application 1 Application m ... Sensor 1 Sensor 2 Sensor 3 Sensor n ... client’s requests data acquisition Patients, medical staff, equipment, resources, etc. Operating Room Hospital Administrators Stakeholders Physicians Client applications outside the Operating Room User Level Service Level Sensor Level Figure 1. A monitored Operating Room model with a middleware acquiring and providing information in real time for user applications. The middleware is present in the service level, which can be deployed in a server or datacenter. As the number m user applications and n sensors increase, the middleware may decrease performance and consequently QoS. Service Level Patients, medical staff, equipment, resources, etc. Figure 1. A monitored Operating Room model with a middleware acquiring and providing information in real time for user applications. The middleware is present in the service level, which can be deployed in a server or datacenter. As the number m user applications and n sensors increase, the middleware may decrease performance and consequently QoS. Sensors 2019, 19, 2303 3 of 27 The growing number of sensors focusing on monitoring patients, medical staff, and equipment results in a need for device data interoperability [17]. That is, the more the number of sensors the more the different information data is produced to be accessed and interpreted. Improving patients’ safety depends on the middleware capacity to acquire and provide large amounts of data in real time [17] regardless the processing and network load. This capacity depends on the middleware performance in terms of network and computing power, which must respect Quality of Service (QoS) requirements of user applications. QoS is a common concept in computing networks that consists of control mechanisms aimed to guarantee that acceptable performance levels of given service are respected. • a model of an adaptable QoS-enabled middleware for OR; • a model of an adaptable QoS-enabled middleware for OR; 2. Background Distributed system environments are characterized by resource sharing among many users and applications [29,30]. These systems rely on networked connections for communication, which may suffer instabilities and consequently impact on performance. QoS speciﬁcations deﬁne a set of parameters for different components in a distributed system [31,32]. QoS strategies are employed to guarantee that distributed systems services maintain a certain level of quality avoiding problems [29,30]. According to Wang [33], QoS is “the capability to provide resource assurance and service differentiation in a network”. In the Internet, for instance, there are many QoS requirements, including for real-time applications [34,35]. Their requirements deﬁne acceptable levels for metrics related to network, such as latency, jitter, and transfer rate. The transfer rate metric measures the amount of information that is possible to transmit in a given time interval. In general, to this time interval is considered the amount of one second and the information grain varies. Latency, in turn, is the time interval between the cause and effect of an action. More speciﬁcally, in computer networks the latency is the time interval between a sender dispatching a packet and the receiver receiving it. High latencies cause high response time of network systems and can deteriorate the quality of an online service, such as video streaming in real time. In computer networks, jitter measures the time delay between the arrival of two consecutive packets. As in distributed networks, routers might transmit different packets by different routes, and the receiver may face variations in the packet inter-arrival time due to the disparities of the paths which can vary in number of hops and network congestion load. Such a phenomenon causes variations in the data ﬂow arrival according to the network load and, depending on the application, strategies have to be implemented to mitigate the problem. For instance, applications that transmit and process video and voice data require the packets to arrive in a regular pace. In this scenario, packets which do not arrive in an expected time have to be discarded or the receiver must use a jitter buffer to store temporally incoming packets to smooth the arrival times. According to Shin and Ramanathan [36], real-time systems are characterized by three main components: (i) time; (ii) reliability; and (iii) environment. The ”time“ is the most important factor real-time systems must control. p Q • an orchestration engine to provide different services for user applications and sensors. p • an orchestration engine to provide different services for user applications and sensors. The model multi-level approach provides services to tackle QoS violations for both user applications at the User level and hardware devices in the Sensor level. QualiCare is an adaptable middleware model to provide multi-level QoS, improve user experience, and increase hardware utilization to middlewares in OR environments. The main focus is to guarantee that the middleware respects QoS levels by improving network and computing performance at both Sensor and User levels. The model proposes the combination of different methods to offer such features. It provides a manager module which controls QoS levels by monitoring different metrics from the sensors and middleware modules. The manager allows the parametrization of thresholds for different metrics as input, and it automatically performs conﬁguration adjustments in all middleware modules through a reactive rule-based strategy in a periodic monitoring fashion. Sensors 2019, 19, 2303 4 of 27 The remainder of the article is structured as follows. Section 2 introduces important concepts related to this study and Section 3 describes the literature review. Sections 4 and 5 present the core ideas of this document. The former introduces the design decisions and the architecture of the model The later presents the QoS model and strategies that QualiCare proposes. Then, Section 6 presents the preliminary results of the ﬁrst experiments. Finally, Section 7 describes some limitations of the study and Section 8 presents the ﬁnal remarks. 2. Background Cooperating tasks are required to be completed within a given deadline, otherwise the computation may be compromised. In addition, reliability is required since system failures might cause catastrophes and even loss of lives. Finally, the environment under which the system operates is also important since physical events trigger the system to perform tasks and process these events. Real-time systems are classiﬁed depending on the consequences of meeting or not a deadline: hard; ﬁrm; and soft. Not meeting a hard deadline leads to catastrophic situations, for instance, in a crash of a ﬂight due to delays in the aircraft system to computes readings of ﬂight sensors. In the case of ﬁrm deadlines, the produced results are useless after the deadline but do not cause any harmful situation. In turn, not respecting soft deadlines produces useful results that decrease in quality as the time goes by. However, like ﬁrm deadlines, it does not cause catastrophic situations. In particular, the real-time term we are using in OR environments refers to soft real-time deﬁnition since occasional delays in the data delivery do not trigger catastrophic situations. Sensors 2019, 19, 2303 5 of 27 3. Related Work The literature review in this study adopts the principles of systematic reviews [37] to achieve reproducibility and high-quality results. Its goal is to provide an overview of OR middleware strategies. The scope of the literature search encompasses the selection of literature databases. It is narrowed to sources that: (i) index articles from relevant conferences and journals from Computer Science and Medicine; and (ii) include a broad selection of venues to maximize the number of returned articles. Based on these criteria, the following ﬁve databases are queried: IEEE Xplore (https:// ieeexplore.ieee.org/); Google Scholar (https://scholar.google.com.br/); PubMed (https://www.ncbi. nlm.nih.gov/pubmed/); ScienceDirect (https://www.sciencedirect.com/); Springer Link (https://link. springer.com/). Real-time, safety, and cost are highly related to the OR, therefore, our focus is to evaluate the state-of-the-art that targets ORs. Thus, to limit the scope of the article search strategy, the search string is deﬁned as follows considering the OR term: “operating room” AND “middleware”. The combination of these strings as a search string to be used in the target databases represent the inclusion criteria. The main goal is to hit all kind of studies that in some way approach middleware in OR environments. The raw literature corpus from the inclusion criteria contains a set of 1005 articles. The ﬁnal set of studies is selected through exclusion criteria applied to the raw corpus. These criteria are formed by the following removal ﬁlters: • Removal ﬁlter I - Duplicate removal: The remaining studies from individual databases were grouped and duplicates were eliminated; • Removal ﬁlter I - Duplicate removal: The remaining studies from individual databases w • Removal ﬁlter I - Duplicate removal: The remaining studies from individual databases were grouped and duplicates were eliminated; grouped and duplicates were eliminated; • Removal ﬁlter II - Title and abstract review: The title and abstract from each study are review y and those that do not address sensing technologies applied to healthcare monitoring are removed; R l ﬁl III Y l i W id i l bli i i hi h l 10 y and those that do not address sensing technologies applied to healthcare monitoring are removed; • Removal ﬁlter III - Year analysis: We are considering only publications within the last 10 years. and those that do not address sensing technologies applied to healthcare monitoring are removed; • Removal ﬁlter III - Year analysis: We are considering only publications within the last 10 years. 3. Related Work The literature corpus after application of all ﬁlters includes 31 articles. The contents of these studies are further analyzed to identify the main focus of the studies, which are: 1. Patient monitoring: [38–42]; 2. Device integration and data interoperability: [43–49]; p 4. Operating Room monitoring (Intelligent OR): [18–28]; 4. Operating Room monitoring (Intelligent OR): [18–28]; 5. QoS-Aware Middlewares: [53–57]. Table 1 presents speciﬁc details from each one of them. In particular, the last two sections group the studies high related to the focus of the current research. The “Operating Room monitoring (Intelligent OR)” group presents studies that demonstrate attempts on employing tracking technologies in surgeries to improve procedures. Basically, they focus mainly on two different goals: (i) activity recognition [18–20]; and (ii) personnel and equipment tracking [20–28]. The former group is composed of articles focusing on identifying the actions, which medical staff perform during surgeries. In turn, the latter group is composed of studies employing Real-Time Location Systems (RTLS) to track the location of people and equipment present in surgeries. Both sets of strategies have the workﬂow monitoring as a primary goal, employing different procedures and technologies. Analyzing the technologies employed by the studies in the group 4,RFID (Radio Frequency Identiﬁcation) emerges as a common strategy present in more than half of the articles. Active RFID tags are most used since they produce accurate readings. These strategies employ tags for identiﬁcation purposes and to track the location of people and equipment to improve efﬁciency and avoid medical errors. Besides RFID, Computer Vision techniques are present in 18% of the studies. In particular, Vaccarella et al. [21] focus on robotic systems for neurosurgery in which they integrate RTLS systems and Computer Vision techniques in real time. Regardless the technology, the majority of studies present concerns about real-time issues. 6 of 27 Sensors 2019, 19, 2303 Table 1. Summary of articles resulting from the review methodology. Table 1. Summary of articles resulting from the review methodology. Table 1. Summary of articles resulting from the review methodology. 3. Related Work Focus Reference Year Middleware Real-time Goal Area Technologies Description Patient monitoring [41] 2013 ✓ Monitoring WSN Zigbee Telemedicine [39] 2017 Tracking RTLS RFID Postoperative abdominal surgery [40] 2017 Monitoring IoT Pub/Sub Ambient assisted living [38] 2017 ✓ ✓ Tracking RTLS RFID Medication error monitoring [42] 2018 Tracking IoT RFID Hospital facilities Device Integration and Data Interoperability [43] 2010 ✓ ✓ Monitoring - Pub/Sub Hospital device framework [44] 2012 Monitoring CPS Pub/Sub Device integration and data extraction [47] 2012 ✓ - - Pub/Sub Medical applications [48] 2014 ✓ ✓ Communi- cation Medical Systems LAN Eye surgery [46] 2016 ✓ Monitoring RTPS Pub/Sub Medical devices integration [49] 2016 ✓ ✓ - IoT Pub/Sub Data exchange [45] 2017 ✓ Monitoring IoT - Integrated clinical environment Smart Hospitals [50] 2012 ✓ Tracking Ubiquitous Computing RFID Hospital location and tracking [51] 2015 ✓ Tracking - RFID Review of concepts [52] 2016 ✓ ✓ Tracking RTLS RFID Ward management Operating Room Monitoring (Intelligent OR) [24] 2010 ✓ ✓ Monitoring WSN Sensors Sensor data acquisition in minimally invasive surgery [19] 2011 ✓ Activity recognition Ubiquitous Computing - Recommendation system for surgical procedures [25] 2010 ✓ ✓ Tracking RTLS RFID Medical error detection system [26] 2011 ✓ ✓ Tracking RTLS RFID Literature review and system for surgical sponges and personnel tracking in surgery [28] 2011 ✓ ✓ Tracking RTLS RFID Patient workﬂow monitoring in surgeries [21] 2011 ✓ ✓ Tracking Computer Vision and RTLS Proprie- tary trackers Neurosurgery tracking from heterogeneous sources [20] 2011 ✓ ✓ Tracking/ activity recognition RTLS RFID Surgical monitoring system to identify critical situations [23] 2012 ✓ ✓ Tracking RTLS RFID Surgical monitoring system to identify critical situations [27] 2012 ✓ ✓ Tracking RTLS RFID OR team and sponge real-time tracking [22] 2014 ✓ ✓ Tracking RTLS RFID Patient tracking and surgical workﬂow monitoring [18] 2017 Activity recognition Computer Vision RGB-D Camera Human pose estimation and activity recognition during surgery QoS-Aware Middlewares [53] 2018 ✓ Network monitoring Edge Computing MQTT Distributed QoS-Aware MQTT middleware for Edge Computing applications [54] 2018 ✓ Network trafﬁc IoT - QoS management modules [55] 2018 ✓ Network trafﬁc IoT - QoS management modules [56] 2019 ✓ Network monitoring IoT Pub/Sub and SDN Differentiation of services in SDN-like Pub/Sub middlewares for IoT [57] 2019 Literature Review IoT - Systematic literature review Sensors 2019, 19, 2303 7 of 27 On the other hand, the “QoS-Aware Middlewares” group presents studies that focus on QoS strategies fo IoT middlewares. 3. Related Work Rausch et al. [53] propose a solution to improve QoS in MQTT middlewares. Their strategy consists of migrating clients connections of Edge Computing applications to closer brokers. In the studies [55] and [54], the authors propose the addition of QoS management modules. The strategy adds a communication layer on top of protocols such asHTTP (Hypertext Transfer Protocol), COaP (Constrained Application Protocol) and MQTT (Message Queuing Telemetry Transport). Shi et al. [56] propose a Software-Deﬁned Networking (SDN) controlling method to deliver different QoS levels for different clients. The solution is composed by a controller nodes that monitor and manage OpenFlow switches to conﬁgure its queue priority. Finally, Ghanbari et al. [57] present a systematic literature review focusing resource allocation in IoT. The authors demonstrate the different resource allocation strategies in the IoT ﬁeld showing they belief that in the future self-adaption might become a trend for IoT systems. By analyzing the aforementioned studies, it is possible to point out two open issues: (i) they do not propose speciﬁc middlewares for OR with multi-sensors; and (ii) lack of QoS strategies on middlewares for ORs. First, although a total of 11 articles focus the OR, the authors do not propose speciﬁc middlewares for OR. Instead, they present efforts on employing some different kind of technologies to monitor surgeries in the OR. Second, ﬁve recent studies focus on strategies for QoS middlewares or resource allocation in the IoT ﬁeld. These studies present strategies focused mainly on a speciﬁc layer of the middleware, and are highly concerned in network management to decrease latency. This landscape depicts a lack of studies focusing speciﬁcally on providing QoS at both user and sensor levels of time critical middlewares. Therefore, the current document focuses on this gap by proposing its main contribution which is an adaptable middleware model to provide multi-level QoS on OR. The proposed model seeks to deﬁne the architecture and strategies necessary to guarantee QoS for user applications, and for hardware sensors. The major challenge relies on which strategies to apply and how to do it taking into account a large number of user application connections. 4. QualiCare Model This article focuses on the gap presented in the previous section by proposing as main contribution an adaptable middleware model to provide multi-level QoS on OR. The proposed model seeks to deﬁne the architecture and strategies necessary to guarantee QoS for user applications, and for hardware sensors. The major challenge relies on employing different strategies considering a large number of sensors and applications. Our main focus is on QoS for hybrid OR, which are composed by a multi-disciplinary team, however our middleware can be seen as generic since it works with requirements related to CPU and I/O [58]. Thus, the system is used on demands that require QoS for these resources, requiring very low latency rates, cadence, and jitter. Therefore, the Sensor level can be seen in a generic form with data ﬂow incoming from physical sensors, which can be a database, ﬁle stream, etc. The User level, in turn, presents applications that either request data deﬁning their QoS requirements or, knowing the application protocol, the middleware already has standardized QoS for them. In other words, there are two types of QoS characterization: (i) pre-deﬁned by the application; (ii) on-the-ﬂy deﬁned by the middleware. For instance, a video processing application may requests video frames from the middleware deﬁning its required frames per second (FPS). On more example is an application that only needs data for real-time feedback in a dashboard interface. Qualicare acts collecting data from sensors, storing it, and delivering it to user applications meeting QoS requirements. These requirements consists of a set of thresholds for speciﬁc metrics, called QoS metrics, that measures from them must be respected. Its main characteristic is its ability to provide QoS for both user applications and sensors regardless the system load. QualiCare provides strategies to meet soft real-time requirements, including a Manager module, which is in charge of monitoring and adaptation tasks to ensure QoS levels. Real-time is important since the capabilities of medical systems to produce data in real time enables the detection of critical situations [59]. The faster these situations are detected the higher the chances to avoid them. Sensors 2019, 19, 2303 8 of 27 4.1. Design Decisions The model has two main actors involved in the production and consumption of data, respectively: (i) sensors; and (ii) user applications. QualiCare provides services to meet QoS requirements by monitoring different metrics in both sensors and user applications levels. In the user level, applications that consume data from the middleware deﬁne QoS requirements that the middleware must respect. If the user does not provide its requirements, QualiCare sets default requirements depending on the data the application requested. On the other hand, at the sensors level, the middleware deﬁnes requirements to guarantee data acquisition even if there are no user applications. Figure 2 illustrates the QualiCare idea in comparison to a default middleware without QoS support. In each level, QualiCare monitors speciﬁcs metrics related to response time, sampling rate, and latency. Based on the results of such monitoring, QualiCare adds or removes services individually to user applications or sensors to meet the QoS requirements. This process consists of a set of threshold-based rules strategy. QualiCare Manager compares measures from metrics to lower and upper thresholds, which indicate metrics that are violating speciﬁc parameters. According to them, the decision process takes actions to adapt the middleware. Data Collection and Analisys Service Sensor 0 Middleware Application 0 Application m ... Sensor n ... Module 0 Module p ... Data Collection and Analisys Service Sensor 0 Middleware Application 0 Application m ... Sensor n ... ... QualiCare Manager QoS requirements Metrics Services Rules Module 0 Module p (a) (b) Figure 2. OR middleware general ideas: (a) default approaches; and (b) QualiCare main idea. Data Collection and Analisys Service Sensor 0 Middleware Application 0 Application m ... Sensor n ... ... QualiCare Manager QoS requirements Metrics Services Rules Module 0 Module p (b) Data Collection and Analisys Service Sensor 0 Middleware Application 0 Application m ... Sensor n ... Module 0 Module p ... (a) Application 0 Data Collection and Analisys Service (a) (b) Figure 2. OR middleware general ideas: (a) default approaches; and (b) QualiCare main idea. 4.2. Architecture Figure 3 depicts QualiCare architecture highlighting three different layers: (i) User; (ii) Service; and (iii) Sensor. User applications which consume data from the middleware compose the User layer. The components that extract and manage data compose the Sensor and Service layers, respectively. Lines connecting components represent reliable channels for data exchange or control messages. Additionally, arrows represent the sensor data ﬂow direction. Gray boxes represent components the model provides, and the remaining white forms represent physical sensors, APIs (Application Programming Interface), and user applications. Additionally, communication between Service and Sensor layers must be in a private wired network for two main reasons: (i) to improve performance; and (ii) due to security issues since OR data is sensitive. 9 of 27 Sensors 2019, 19, 2303 SENSORS SERVICE USER Data Acquisition Controller Data Access Handler Sensor Data Producer 1 Sensor Data Producer m ... PUBLIC NETWORK SENSOR API SENSOR API QualiCare Manager Middleware Coordinator SENSOR API Data Processing & Storage Unit Sensor Data Producer 0 PRIVATE NETWORK Sensor s Sensor 0 Sensor s Sensor 0 Sensor s Sensor 0 ... ... ... Application 0 Application 1 Application n ... Figure 3. QualiCare architecture presenting the middleware in the Service level and additional modules in the Sensor level inside the OR. Arrows represent the communication direction in the following form: (i) application to middleware; (ii) middleware to applications; (iii) middleware to sensors; (iv) sensors to middleware; and (v) middleware components to middleware components. Items i to iv regard the sensor data ﬂow, while item v regards conﬁguration and metrics transmission. Figure 3. QualiCare architecture presenting the middleware in the Service level and additional modules in the Sensor level inside the OR. Arrows represent the communication direction in the following form: (i) application to middleware; (ii) middleware to applications; (iii) middleware to sensors; (iv) sensors to middleware; and (v) middleware components to middleware components. Items i to iv regard the sensor data ﬂow, while item v regards conﬁguration and metrics transmission. Figure 3. QualiCare architecture presenting the middleware in the Service level and additional modules in the Sensor level inside the OR. Arrows represent the communication direction in the following form: (i) application to middleware; (ii) middleware to applications; (iii) middleware to sensors; (iv) sensors to middleware; and (v) middleware components to middleware components. Items i to iv regard the sensor data ﬂow, while item v regards conﬁguration and metrics transmission. 4.2. Architecture Description of the network header ﬁelds. QualiCare architecture is composed by six different components which will be discussed in the next sections in details: (i) Sensor Data Producer; (ii) Data Acquisition Controller; (iii) Data Processing & Storage Unit; (iv) Data Access Handler; (v) Middleware Coordinator; and (vi) QualiCare Manager. 4.2. Architecture There are three different messages QualiCare modules might transmit between them: (i) conﬁguration data; (ii) metrics measurements; and (iii) sensor data. The transmission occurs through TCP/IP messages including an 8-byte network header which identiﬁes the messages (details in Table 2), and a variable payload that contains one of the three data. Figure 4 depicts the ﬁelds of the network header and the composition of each type of message. While the header is ﬁxed for all messages, the payload of each one is different depending on the message type. Metric measurements messages (type 3) contain readings of metrics from modules. The QualiCare Manager consumes this type of message for QoS monitoring purposes. These values are used to evaluate the status of the middleware and monitor QoS violations. Based on that, adaptations are performed by QualiCare to guarantee QoS. In turn, conﬁguration data messages (type 2) contain module conﬁgurations and might be used for two reasons. First, to check the current conﬁguration of a speciﬁc module. Second, to change the parameters of the modules, which the Middleware Coordinator is the only module allowed to do. Finally, sensor data messages (type 1) represent the central information that QualiCare modules transmit, which contains the data extracted from physical sensors. This information varies depending on the sensor, which can produce different types of data depending on the sensor. For instance, a temperature sensor produce a ﬂoat value corresponding the room temperature while a RTLS produces two or three integer values corresponding the 2D or 3D position of a given tag in the environment. Sensors 2019, 19, 2303 10 of 27 Sensor Raw Data Data Attributes Network Header Information data from sensors Message type 1 8 bytes variable bytes Configuration Data Network Header Middleware modules’ parameters Message type 2 8 bytes variable bytes Measurements Network Header QoS measurements Message type 3 8 bytes variable bytes payload size type id remaining Network header 0 1 3 7 8 bytes Figure 4. QualiCare message types and its contents. All messages use the same network header, which identiﬁes the packets. Figure 4. QualiCare message types and its contents. All messages use the same network header, which identiﬁes the packets. Table 2. Description of the network header ﬁelds. Field Description Type The payload type. ID Identiﬁcation of the request. Payload Size The size of the payload in bytes. Remaining Number of messages remaining to answer the request. Table 2. 4.2.1. Sensor Data Producer In particular, the Sensor Data Producer component might have from 1 to m instances running in the architecture. This component extracts information directly from 1 to s sensors using their APIs, respecting a speciﬁc sampling rate. When running the ﬁrst time, the process detects the sensors by testing the implemented APIs, and tries to reach the physical sensors using a pre-deﬁned conﬁguration. It includes the reachable sensors in its monitoring list, and the unreachable ones it keeps trying to reach them in periodic observations. To produce data from sensors, it extracts raw data respecting a sampling rate and builds a package with several pieces of information that characterize a sensor data. The module generates a single sensor data package for each sensor returned from the API. For instance, several RTLS tags might be acquired through the RTLS middleware API at once. The API always returns the available tags in the environment when requested. By transforming different types of information provided by different physical sensors, this module transforms all data to a common type of information that can be interpreted by all modules without needing to implement their APIs. It allows data interoperability between the modules and between the middleware and user applications that request these data. This process consists of gathering information from physical hardware sensors through either their API or a provided service by the vendor, and transforming it into a byte array that can be transmitted over network or stored in the disc or database. The module packs the byte array in a data structure, called sensor data package, which contains six speciﬁc attributes: (i) sensor ID; (ii) device ID; (iii) data producer ID; (iv) sample counter; 11 of 27 Sensors 2019, 19, 2303 (v) timestamp; and (vi) type. Table 3 organizes the details of each one of them brieﬂy. The Sensor ID, Device ID, and Data Producer ID ﬁelds identify the source of the sensor data. Sensor Data Producer instances might extract data from different physical sensors. Therefore, this set of IDs identify the sources individually. The Sample Counter deﬁnes the sample sequence of the sensor data, and the Timestamp is the instant of time that the data was extracted from the physical sensor. Finally, the Type deﬁnes the kind of data, which can be, for instance, a sample of the room temperature or the heart rate of a patient. 4.2.1. Sensor Data Producer Jointly to these ﬁelds, the raw sensor information data (byte array) is attached, composing a sensor data package. Table 3. Description of the data attributes from sensor data packages. Field Description Type The data type. Timestamp The time in milliseconds that the data is collected from the sensor. Sample Count The sequence number of the collected data. Data Producer ID The identiﬁcation of the module that collected the data. Device ID The identiﬁcation of the device from which the data was collected. Sensor ID The identiﬁcation of the sensor from which the data was collected. 4.2.2. Data Acquisition Controller Table 3. Description of the data attributes from sensor data packages. Table 3. Description of the data attributes from sensor data packages. Field Description Type The data type. Timestamp The time in milliseconds that the data is collected from the sensor. Sample Count The sequence number of the collected data. Data Producer ID The identiﬁcation of the module that collected the data. Device ID The identiﬁcation of the device from which the data was collected. Sensor ID The identiﬁcation of the sensor from which the data was collected. 4.2.2. Data Acquisition Controller 4.2.2. Data Acquisition Controller The Data Acquisition Controller component receives sensor data packages from all Sensor Data Producers respecting speciﬁc parameters for each Sensor Data Producer, which are deﬁned in a JSON-like conﬁguration ﬁle. This ﬁle contains a list of tuples deﬁning the network address and connection port for each Sensor Data Producer, the sampling rate per second, and the type of data to be acquired. It dispatches m threads, one for each Sensor Data Producer, which establish a TCP/IP (Transmission Control Protocol/Internet Protocol) connection using sockets. Once the connection is established, the thread starts a new thread to send sample data requests of given type mask at each time interval. The time interval is deﬁned by dividing 1000 ms (respective to 1 s) by the sampling rate parameter, and using the result as sleep time between intervals. The data type mask is an enumeration value for different data types which can be extended for new types of data according to the available type of sensors. This parameter is important since a given Sensor Data Producer instance might extract information from more than one types of sensors. Therefore, the data type mask deﬁnes from which sensor(s) the request aims to acquire data. 4.2.1. Sensor Data Producer Sensor data replies received by each thread are stored in a shared buffer to be accessed by the Data Processing and Storage Unit component. The thread responsible for establishing connection with the Sensor Data Producer, after dispatching the data request thread, starts to read replies in the socket. When a data replies arrives, it removes the data from the socket, populates the buffer, and triggers a signal to the Data Processing and Storage Unit informing that there is new sensor data in the buffer. Each thread has its own buffer so that write operations are performed only by one process. 4.2.5. Middleware Coordinator The Middleware Coordinator has administrative assignments mainly concerned to components parametrization. This component has access to change conﬁgurations of all components in the architecture, except the QualiCare Manager. For instance, it is possible to change the sampling rate per second a speciﬁc Sensor Data Producer extracts from a physical sensor. Another example regards network and compression conﬁgurations, such as TCP port to listen for connections and the activation or not of compressing algorithms in the data. The Coordinator does not request or transmit any sensor data. Instead, it acquires components conﬁgurations being able to modify them. Therefore, this is the only module capable of changing conﬁgurations of the middleware. These operations are performed through messages type 2 (see Figure 4). The Coordinator has its own conﬁguration ﬁle that deﬁnes all available modules and their network connection information. In addition, the Coordinator conﬁguration ﬁle also has the conﬁgurations for each module so that, when the Coordinator starts, it updates the conﬁgurations of all modules sending a message type 2. After this process the Coordinator starts listening network connections. The module is able to receive message type 2 from the QualiCare Manager and it interprets the messages in two different ways: (i) if the message payload is the size of 0 bytes, then the module replies the message with the current conﬁguration off all modules; (ii) if the message has a payload > 0, then the payload must be a conﬁguration ﬁle in a JSON string of a given module that must be updated. 4.2.4. Data Access Handler The Data Access Handler component manages user application connections and provides sensor data to them. It provides a web service interface which implements two different HTTP methods that the applications can use to access data: (i) REST (Representational State Transfer) API for retrieving ofﬂine data; and (ii) MQTT API for real-time data. Applications have to supply their requests with ﬁltering parameters and QoS requirements in terms of accepted delay to reply the request and accepted latency from the time the data is extracted to the time the request is replied. Through the REST API, the module provides a set of HTTP methods the application might call to access the data. The methods receive, through the calls, the ﬁltering parameters, which are sensor data ﬁelds, and replies a JSON-like string to the application. The MQTT API, in turn, provides topics for each physical sensor so that applications can subscribe to receive data. At each new sensor data information available, the Data Access Handler module publishes it in the respective topic. 4.2.3. Data Processing & Storage Unit This module is responsible for storing each new sensor data package in a database and to make it available to the Data Access Handler. The sensor data is converted to a JSON (JavaScript Object Notation) string so that it is possible to use the full string for NoSQL-like (Non Structured Query Language) databases. We opted by NoSQL systems due to its prevailing adoption in Big Data environments [60]. NoSQL distributed systems present advantages to IoT systems due to its capacity of scalability and storing multiple data types that can change over time [61]. Besides storing data in the database, the component provides sensor data in two models: (i) real-time; and (ii) ofﬂine. For the ﬁrst case, each new available reading of a sensor data provided by the Data Acquisition Controller is made available to the Data Access Handler so that it can be send to user applications as soon as possible. For the second, the module retrieves data from a database according to request parameters provided Sensors 2019, 19, 2303 12 of 27 by the Data Access Handler. The module employs data ﬁltering and aggregation strategies to improve the middleware performance. It accepts all ﬁelds presented in Table 3, and uses it as ﬁlters in the database to reply the request. Besides, repeated sensor data information, in which only the Timestamp changes, are transformed to only one sensor data information to avoid transmitting duplicated data. 5. Quality of Service Model QualiCare is designed as a closed feedback-loop architecture [62], involving two main components: the QualiCare Manager and the OR middleware. Control theory is an engineering and mathematics branch focused on dynamical systems behavior, and how they are affected by feedback [63]. Therefore, service provisioning decision should be made based on the system performance according to applications requirements. QualiCare Manager presents three main functions which characterize control systems: a sensor to acquire monitoring data, a controller to evaluate measurements, and an actuator to provide services. Figure 5 illustrates the architecture components and their organization showing the main control tasks of the QualiCare Manager. Each middleware component is represented as a Qualicare process, which can be distributed among computing nodes in a cluster within the hospital facilities or in a single server. It must be running only one instance of the Manger process to avoid concurrence of operations performed in the middleware resources. Otherwise, the same operation may be required to the Coordinator more than once. Furthermore, opposite operations might be required to the Coordinator in a short period of time if more than one Manager is running with different conﬁgurations. The Manager has access to each Qualicare process regardless of their locations, either among servers or clusters. The architecture is composed by a server and n nodes in which the QualiCare processes run performing the roles depicted in the previous Figure 3. The service provisioning is obtained by an orchestration model (detailed in Section 5.5), which evaluates a series of QoS metrics and deﬁnes the set of services for each component of the middleware. Operating Room Middleware QualiCare Manager ... Node n-1 Node 0 Middleware Server Database Sensor: Monitoring Controller: Metrics load and Service Orchestration Actuator: QoS actions and service provision Network APP 0 APP 1 APP 2 APP 3 APP a-1 Workload ... ... Users Caption APP User application switch P QualiCare process Sensor Computing node P P API API ... PPP Figure 5. QualiCare closed-loop model with a Manager in charge of monitoring and adapting the middleware according to the workload. At the user perspective, a denotes the number of user applications. At the middleware perspective, n denotes the number of nodes running a QualiCare process that acquires data from sensors in the OR. Network APP 0 APP 1 APP 2 APP 3 APP a-1 ... ... Users Figure 5. 5. Quality of Service Model QualiCare closed-loop model with a Manager in charge of monitoring and adapting the middleware according to the workload. At the user perspective, a denotes the number of user applications. At the middleware perspective, n denotes the number of nodes running a QualiCare process that acquires data from sensors in the OR. 4.2.6. QualiCare Manager Finally, the QualiCare Manager is the main component being responsible for managing QoS by providing modiﬁcations in the Service and Sensor levels. The Manager acquires measures from deﬁned QoS metrics from all components and applies algorithms to these data to verify if modiﬁcations are needed. The module collects the measures all metrics from each module through messages type 3 (see Figure 4). To request measures, the module sends a message with payload size of 0 bytes. All modules reply messages type 3 with the measures of all metrics it generates as a JSON string in the payload ﬁeld. Therefore, the Manager receives the measures to apply its algorithms in the monitoring process. In addition, the module is able to change parameters from all modules through messages type 2 which it sends to the Middleware Coordinator. Section 5.4 describes each metric and introduces the QualiCare Manager in more detail. Sensors 2019, 19, 2303 13 of 27 13 of 27 5.1. QoS Taxonomy In the OR scope, there are two different levels to which QualiCare taxonomy speciﬁes QoS parameters. User parameters inﬂuence the ﬁnal user perception of the performance of the middleware. These parameters are speciﬁcally related to the real-time data consumption ﬂow by user applications. On the other hand, Sensor parameters impact the middleware data acquisition for both persistence operations and user requests. Data consumption is a continuous task, which the middleware performs Sensors 2019, 19, 2303 14 of 27 for data persistence. Considering that user requests may consume data from past events (historical data), it is necessary to ensure that the middleware is able to acquire sufﬁcient data from all sources for persistence. Lacking data in some periods of time might impact critically in the workﬂow analysis, making it impossible in those periods. Therefore, Sensor parameters are critical to guarantee that the middleware is able to provide both real-time and no real-time data. Figure 6 depicts QualiCare QoS taxonomy presenting the metrics and services that QualiCare provides. Metrics are observed values that support the decision-making process. Services refer to methods available to tackle QoS situations based on the monitored metrics. These services impact directly in the metrics’ measures, that is, providing services changes the results of the metrics. Therefore, QualiCare Manager is in charge of monitoring such metrics and applying a suitable service to address problems. The Manager can combine a metric with one or more services or vice versa. A combination of metrics and services forms the QoS parameters. OR QoS Specification Metrics Services Parameters Adaptation Elasticity Data Compression CPU Load Sample Rate Memory Load Jitter Transmission Rate Latency User Sensor Figure 6. QualiCare QoS taxonomy presenting its the metrics and services. OR QoS Specification User Sensor Memory Load Figure 6. QualiCare QoS taxonomy presenting its the metrics and services. 5.3. Deﬁnition of Qos Services QualiCare provides three different services for each QualiCare process depending on monitoring aspects: (i) resource elasticity; (ii) data compression; and (iii) parameters adaptation. The services are represented by the functions Elast(), Compress(), and Adapt(), respectively. Services can be provided individually or combined depending on the middleware status. QualiCare might provide each service through the different architecture components. Compress() is provided only at the Sensor level since it is the source of the data. The Elast() service employs vertical elasticity strategies to increase or decrease the CPU and memory capacities without impacting in the processes operation. In particular, as the architecture modules are running in computing nodes, which might be physical or virtual machines, the service delivers new computing power to these instances. Elasticity is a popular concept in cloud platforms, which refers to the capacity of a system to automatically provision and de-provision resources according to workload changes [64]. QualiCare focuses on the vertical model of elasticity since it does no impose the modules to deal with duplicate instances which require load balancing strategies. The Compress() service focuses on improving the network performance of nodes running the QualiCare processes. However, it might increase the need for more computing resources, resulting in the need for the Elast() service. Compress() is available in the Sensor Data Producer, and Data Acquisition Controller processes. These components employ compression algorithms to compress sensor data before packing it for transmission. This feature can be enable or disabled through a parameters which is part of the conﬁguration of these modules. Enabling it allows the modules to apply the compression before sending it over the network. This decreases the network trafﬁc, which improves communication between processes. Depending on some situations, the amount of data requested for a module can be higher than the transmission capacity of the module. Thus, compressing data is a possible way to decrease the amount of data and guarantee that requests are replied. Finally, the Adapt() service consists of changing processes parameters, which affect the middleware behavior. Through this strategy, it is possible to change the sampling rate of data extraction from sensors or data acquisition the middleware performs. Additionally, it is also possible to deﬁne the data size in the Sensor level so that the number of bytes to be transmitted decreases. These adaptations provide hardware utilization improvements and modules parameters balancing. 5.2. Deﬁnition of Qos Metrics 5.2. Deﬁnition of Qos Metrics QualiCare processes calculate individual functions to extract metrics measures depending on the component role, which the QualiCare Manager gathers in its monitoring procedure. Table 4 shows these functions and the corresponding metrics. Components that calculate metrics CPU Load and Memory Load compute them for each process. The Data Acquisition Controller process computes the metrics Latency, Sampling Rate, and Transmission Rate for each Sensor Data Producer connection. In turn, the Data Access Handler process computes Jitter, Sampling Rate, and Transmission Rate for the sensor data transmitted for each user application connection. Table 4. QoS metric deﬁnition and their corresponding functions. Metric Function Source Latency Lat(si) Data Acquisition Controller Jitter Jit(uj) Data Access Handler Sampling Rate Sam(an) Data Acquisition Controller and Data Access Handler Transmission Rate Tra(an) Data Acquisition Controller and Data Access Handler CPU Load CPU(pq) All components Memory Load MEM(pq) All components Table 4. QoS metric deﬁnition and their corresponding functions. 15 of 27 Sensors 2019, 19, 2303 5.3. Deﬁnition of Qos Services For instance, if modules are working on different sampling rates the Data Acquisition Controller process may request information in a lower sampling rate than a particular Sensor Data Producer process is generating. Decreasing the sampling rate of the Producer avoids wasting resources. In particular, when changing the sampling rate, each parameter adaption evaluates if the new conﬁguration violates a upper or lower threshold. The threshold will be introduced in Section 5.5.1. 5.4. Qualicare Manager Figure 7 details the components of the QualiCare Manager depicting the inputs, and outputs. The Middleware Interface interacts with the middleware to collect metrics and send updates. Metric Monitoring is in charge of collecting each QoS metric measurement periodically at a given time interval. Thus, the main component, called Orchestration Engine, analyzes these measurements, including the feedback of previous decisions by comparing the variations in metrics measurements. The Engine contains the main strategies that the Manager applies to adapt the middleware. The Data and Performance Analyzer evaluates the QoS metrics from the middleware comparing them with QoS requirements to generate violation events. Thus, the User and Sensor Orchestration deﬁne actions that might be necessary to apply to tackle these events. After deﬁning the actions, the Engine calls the Service Provider component to deliver the needed services. This component is able to call either the Middleware Interface, to provide the Adapt() and Compress() services, or the Resource Management to deliver the Elast() service. 16 of 27 Sensors 2019, 19, 2303 Service Provider Metric Monitoring Middleware Inferface Middleware measures Hardware measures Change parameters Orchestration Engine QoS Services QoS Metrics Data and Performance Analyzer User Orchestration Hardware Orchestration Resource reconfigurations Input Output Caption Resource Management QualiCare Manager Figure 7. QualiCare Manager components, inputs, and outputs. The module receives measurements collected from the middleware and evaluates them based on metrics and services. The output are resource and parameters reconﬁgurations, if necessary, to guarantee QoS. User Orchestration Figure 7. QualiCare Manager components, inputs, and outputs. The module receives measurements collected from the middleware and evaluates them based on metrics and services. The output are resource and parameters reconﬁgurations, if necessary, to guarantee QoS. The QualiCare Manager process is in charge of monitoring metrics and delivering services for the different module processes. Each process has particular metrics and services that the Manager evaluates individually. Figure 8 depicts this task showing the main monitoring cycle. Additionally, Algorithm 1 details the Manager operations and procedures that occur periodically. First, the procedures from lines 4 and 5 collect data from the middleware and compute the QoS metrics. Then, the Orchestration Engine, through SensorOrchestration() and UserOrchestration(), deﬁne the actions the Service Provider must perform to tackle QoS violations through the procedure ProvideServices(). QualiCare Manager Collect metric measures. Analyse measures. Decide needed services. QoS violations? Wait until next monitoring cicle. Algorithm 1 QualiCare Manager Main Tasks. Algorithm 1 QualiCare Manager Main Tasks. Input: measures from metrics. Output: middleware adaptions. 1: running ←true; 2: cycle ←0; 3: while running do 4: CollectMonitoringData(); 5: ComputeMetrics(qos_metrics,cycle); 6: sensor_orchestration_actions[] ←SensorOrchestration(qos_metrics,qos_services); 7: user_orchestration_actions[] ←UserOrchestration(qos_metrics,qos_services); 8: if sensor_orchestration_actions > 0 then 9: ProvideServices(sensor_orchestration_actions); 10: sensor_orchestration_actions.clear(); 11: end if 12: if user_orchestration_actions > 0 then 13: ProvideServices(user_orchestration_actions); 14: user_orchestration_actions.clear(); 15: end if 16: sleep(); 17: cycle++; 18: end while 5.4. Qualicare Manager Data Acquisition Controller Data Access Handler Data Processing & Storage Unit Sensor Data Producer 0 Sensor Data Producer m ... Reorganize services. CPU Load Memory Load CPU Load Memory Load CPU Load Memory Load CPU Load Memory Load CPU Load Memory Load Sample Rate Jitter Transmission Rate Sample Rate Latency Transmission Rate Parameters Adaptation Elasticity Data Compression Parameters Adaptation Elasticity Data Compression Parameters Adaptation Elasticity Elasticity Elasticity YES NO output Start Measures from metrics Services QualiCare modules input Figure 8. QualiCare Manager main monitoring cycle showing the service distribution and the metrics it evaluaves. The idea is to monitor module metrics and organize services according to the measures. QoS violations? Figure 8. QualiCare Manager main monitoring cycle showing the service distribution and the metrics it evaluaves. The idea is to monitor module metrics and organize services according to the measures. 17 of 27 Sensors 2019, 19, 2303 5.5. Service Orchestration In cloud computing environments, resource orchestration consists of a set of operations that cloud providers offer to dynamically adjust hardware and software resources to guarantee QoS [65]. Deriving from these concepts, the QualiCare service orchestration is a decision process that selects and delivers the stack of services for each user application and sensor connected to the middleware. QualiCare adopts a rule-based strategy to choose which services are suitable to address QoS situations. This solution follows a Service Level Agreement (SLA) strategy which consists of a set of rules that monitors limits, called thresholds, for a given metric. SLA-base strategies are common in self-adaptable solutions which employ rule-based strategies, as in Hanif et al. [66]. By starting the QualiCare Manager, it receives as input an SLA ﬁle in a JSON format, according to the RFC 2647 [67], containing the default rules and thresholds for each metric. It is possible to change the behavior of the system just by editing the SLA ﬁle and running the QualiCare Manager again. Figure 9 demonstrates an SLA ﬁle example containing the values for the thresholds of the model. The ﬁle deﬁnes the name of the threshold and its value. 1 { 2 "Name": "SLAThresholds", 3 "Thresholds": [ 4 {"Name": "LatencyUpperThreshold", "Value": "1000"}, 5 {"Name": "CPUUpperThreshold", "Value": "90" }, 6 {"Name": "MEMUpperThreshold", "Value": "90" }, 7 {"Name": "TransmissionUpperThreshold", "Value": "512" }, 8 {"Name": "SamplingRateUpperThreshold", "Value": "24" }, 9 {"Name": "JitterUpperThreshold", "Value": "500" }, 10 {"Name": "LatencyLowerThreshold", "Value": "100" }, 11 {"Name": "CPULowerThreshold", "Value": "10" }, 12 {"Name": "MEMLowerThreshold", "Value": "10" }, 13 {"Name": "TransmissionLowerThreshold", "Value": "128" }, 14 {"Name": "SamplingRateLowerThreshold", "Value": "12" }, 15 {"Name": "JitterLowerThreshold", "Value": "100" } 16 ] 17 } Figure 9. SLA ﬁle containing the default values for each threshold used in the Orchestration Engine algorithms. Sensors 2019, 19, 2303 18 of 27 At each monitoring cycle, the Data and Performance Analyzer component accesses the current measures for each module/connection and evaluates them against the upper and lower thresholds by a multi-level rule set. Therefore, the service orchestration is performed for both User and Sensor levels. The User Orchestration and Sensor Orchestration deﬁne the ﬁnal stack of services for each module based on the results of the threshold analysis. Figure 10 depicts an example of the multi-level orchestration model showing that user applications and sensors have individual service stacks. 5.5. Service Orchestration QualiCare adopts a rule-based strategy to choose which service(s) is suitable to address situations, which impact the middleware QoS. At each monitoring cycle, the Data and Performance Analyzer accesses the current measures for each module/connection and evaluates them against upper and lower thresholds by a multi-level rule set. The User Orchestration and Sensor Orchestration component deﬁne the ﬁnal stack of services for each module based on the results of the threshold analysis. SENSOR LEVEL SERVICE LEVEL Application 0 Service Stack Service A Application m-1 Service Stack Sensor 0 Service Stack Service A Service B Service C Sensor n-1 Service Stack Service B Service C SERVICE ORCHESTRATION Available Services Service C Service B Service A Figure 10. Example of the service orchestration process performed by the Orchestration Engine. Figure 10. Example of the service orchestration process performed by the Orchestration Engine. 5.5.1. Sensor Orchestration 1: service_list ←new empty list; 2: for each si Sensor Data producer process do 3: if Lat(si) > LatencyUpperThreshold then 4: if CPU(pq) > CPUUpperThreshold & MEM(pq) > MEMUpperThreshold then 5: service_list.add(si,Elast(“increase”)); 6: end if 7: if Tra(ai) > TransmissionUpperThreshold then 8: service_list.add(si,Compress()); 9: end if 10: if Sam(ai) > SamplingRateUpperThreshold then 11: service_list.add(si,Adapt(“decrease”)); 12: end if 13: end if 14: if Lat(si) < LatencyLowerThreshold then 15: if CPU(pq) < CPULowerThreshold & MEM(pq) < MEMLowerThreshold then 16: service_list.add(si,Elast(“decrease”)); 17: end if 18: if Tra(ai) < TransmissionLowerThreshold then 19: service_list.add(si,Compress()); 20: end if 21: if Sam(ai) < SamplingRateLowerThreshold then 22: service_list.add(si,Adapt(“increase”)); 23: end if 24: end if 25: end for 26: return service_list; si: a Sensor Data Producer process; pq: the QualiCare process identiﬁcation corresponding to si; ai: a user application or Sensor Data Producer process; g Input: SLA threshold ﬁle and metrics measures. Output: QoS services. 1: service_list ←new empty list; 2: for each si Sensor Data producer process do 3: if Lat(si) > LatencyUpperThreshold then 4: if CPU(pq) > CPUUpperThreshold & MEM(pq) > MEMUpperThreshold then 5: service_list.add(si,Elast(“increase”)); 6: end if 7: if Tra(ai) > TransmissionUpperThreshold then 8: service_list.add(si,Compress()); 9: end if 10: if Sam(ai) > SamplingRateUpperThreshold then 11: service_list.add(si,Adapt(“decrease”)); 12: end if 13: end if 14: if Lat(si) < LatencyLowerThreshold then 15: if CPU(pq) < CPULowerThreshold & MEM(pq) < MEMLowerThreshold then 16: service_list.add(si,Elast(“decrease”)); 17: end if 18: if Tra(ai) < TransmissionLowerThreshold then 19: service_list.add(si,Compress()); 20: end if 21: if Sam(ai) < SamplingRateLowerThreshold then 22: service_list.add(si,Adapt(“increase”)); 23: end if 24: end if 25: end for 26: return service_list; si: a Sensor Data Producer process; pq: the QualiCare process identiﬁcation corresponding to si; ai: a user application or Sensor Data Producer process; 1: service_list ←new empty list; 5.5.1. Sensor Orchestration In the Sensor Orchestration, the Engine manages the service stack for each Producer individually. One main rule, based on the Lat(si) metric, and a set of sub-rules, one for each available service, compose the rule-based approach. After collecting all metrics from the connected producers, for each Producer si, the Engine veriﬁes if its current latency Lat(si) is violating an upper threshold or a lower threshold. It uses the latency metric since a high latency when acquiring data from sensors causes delays on delivering sensor data to the database and user applications that are expecting such information. Therefore, if the latency of a Sensor Data Producer is violating the upper threshold, the Engine evaluates three different metrics to choose between services to deliver: (i) high CPU and Memory loads, which may delay the processing of requests; (ii) high transmission rate, which can identify network congestion; and (iii) high sampling rate, which may cause high processing loads and network congestion. On the other hand, if the latency is below a given lower threshold, then the same metrics are evaluated to verify if they also may be violating a lower threshold. When violating lower thresholds, it is possible to release resources or increase the sampling rate to produce more data which results in better resource utilization. Algorithm 2 details the main operations the Orchestration Engine performs to orchestrate services in the Sensor level. This algorithm corresponds to the SensorOrchestration(qos_metrics,qos_services) function from Algorithm 1 (line 6). Lines 3 and 14 compose the main rules, which the algorithm checks for each si Sensor Data Producer process. The service_list stores the actions that the function returns, which the Service Provider delivers. 19 of 27 Sensors 2019, 19, 2303 Algorithm 2 Sensor Orchestration Exectution Tasks. Algorithm 2 Sensor Orchestration Exectution Tasks. Input: SLA threshold ﬁle and metrics measures. Output: QoS services. si: a Sensor Data Producer process; pq: the QualiCare process identiﬁcation corresponding to si; 5.5.2. User Orchestration 1: service_list ←new empty list; 2: for each uj user application do 3: if Jit(uj) > JitterUpperThreshold then 4: if CPU(pq) > CPUUpperThreshold & MEM(pq) > MEMUpperThreshold then 5: service_list.add(uj,Elast(“increase”)); 6: end if 7: if Tra(ai) > TransmissionUpperThreshold then 8: service_list.add(uj,Compress()); 9: end if 10: end if 11: if Jit(uj) < JitterLowerThreshold then 12: if CPU(pq) < CPULowerThreshold & MEM(pq) < MEMLowerThreshold then 13: service_list.add(uj,Elast(“decrease”)); 14: end if 15: if Tra(ai) < TransmissionLowerThreshold then 16: service_list.add(uj,Compress()); 17: end if 18: end if 19: end for 20: return service_list; uj: a user application connection; pq: the QualiCare process identiﬁcation corresponding to uj; ai: a user application or Sensor Data Producer process; Algorithm 3 User Orchestration Execution Tasks. Algorithm 3 User Orchestration Execution Tasks. 5.5.2. User Orchestration In the User Orchestration, the Engine employs a different set of rules. Algorithm 3 deﬁnes the operations the user orchestration performs in its execution process. This algorithm corresponds to the function UserOrchestration(qos_metrics,qos_services) from Algorithm 1 (line 7). Each user application may deﬁne its QoS requirements with respect to jitter. The requirements deﬁne upper (JTu) and lower (JTl) thresholds for the metric Jit(uj), which is used in the main rules of the model. If the application does not provide its requirements, the thresholds are initialized with default values. In contrast to the Sensor Orchestration which uses the latency as the main metric, in the User Orchestration the Engine veriﬁes, for each user application connection uj, if its current jitter Jit(uj) is violating an upper threshold or a lower threshold. It uses this metric since a high jitter when acquiring data from sensors causes instabilities and delays on delivering sensor data to the applications. Therefore, if the jitter of a uj user connection managed by the Data Access Handler is violating the upper threshold, the Engine veriﬁes two different metrics to choose between services to deliver: (i) high CPU and Memory loads, which causes delays on processing the replies to be sent to the applications; and (ii) high transmission rate, which causes network congestion if the number of applications is high. The same metrics are evaluated to verify if they also may be violating a lower threshold. When violating lower thresholds, the Engine may decrease the allocated resources and deactivate compression algorithms which may decrease response time to requests. As in the sensor orchestration algorithm, the service_list stores the actions the function returns to the Service Provider. 20 of 27 20 of 27 Sensors 2019, 19, 2303 Algorithm 3 User Orchestration Execution Tasks. Input: SLA threshold ﬁle and metrics measures. Output: QoS services. 6. Results and Discussion The model evaluation methodology consists on deploying the middleware in a simulated OR at the Unisinos Softwarelab (http://www.unisinos.br/softwarelab/en/) and performing experiments with the Sewio indoor RTLS solution (https://www.sewio.net/real-time-location-system-rtls-on-uwb/). We installed in the room a wired Gigabit Ethernet network, and a computing node (4GB RAM, quad-core Core i5) running a Sensor Data Producer instance acquiring tag positions from the RTLS system at 30 FPS. We are using the computing node with such conﬁguration since in future works we are planning to employ Computer Vision strategies and perform experiments that consider the extraction of depth and color images from camera devices to estimate human poses. We consider this kind of experiments due to these strategies be present in healthcare environments [68]. We deployed the remaining QualiCare components, including the QualiCare Manager, in an additional server node. At this research stage, we are evaluating the Sensor Data Producer performance and communication. By combining the number of tags in the room and the number of connections the Producer receives, we designed four different execution scenarios with 10 min of duration: 1. 1 tag with 1 connection (1T1C); 2. 1 tag with 3 connections (1T3C); 3. 3 tags with 1 connection (3T1C); 4. 3 tags with 3 connections (3T3C). Additionally, the same scenarios were executed with QualiCare Manager delivering the Adapt() service (Algorithm 2, line 11) decreasing the FPS by half. Although this can decrease the sampling rate a user application may be expecting, the Adapt() service always respect the Sensors 2019, 19, 2303 21 of 27 SamplingRateLowerThreshold value so that the minimum FPS is the value of this threshold. In our experiments, as we are evaluating the Sensor Data Producer performance and communication, and as we are starting from an FPS equal to 30, we deﬁned the SamplingRateLowerThreshold to 15 FPS. Figure 11 depicts the results without performing adaptions in the FPS. The network load is highly impacted by changing the number or connections or tags since the amount of data to be transmitted increases in these scenarios. Additionally, the processing load suffer more impact serving more clients than extracting more tag positions from the room. On the other hand, memory load does not suffer critical impacts in all scenarios. It occurs due to the size of information from tags being low, compared with the available memory. 6. Results and Discussion CPU Load Memory Load Transmission Rate 0 8 16 24 32 40 48 56 64 0% 5% 10% 15% 20% 25% 30% 35% 40% 0 60 120 180 240 300 360 420 480 540 600 660 Transfer Rate (KB/s) Load Time (seconds) (a) 1 tag with 1 connection 0 8 16 24 32 40 48 56 64 0% 5% 10% 15% 20% 25% 30% 35% 40% 0 60 120 180 240 300 360 420 480 540 600 660 Transfer Rate (KB/s) Load Time (seconds) (b) 1 tag with 3 connections 0 8 16 24 32 40 48 56 64 0% 5% 10% 15% 20% 25% 30% 35% 40% 0 60 120 180 240 300 360 420 480 540 600 660 Transfer Rate (KB/s) Load Time (seconds) (c) 3 tags with 1 connection 0 8 16 24 32 40 48 56 64 0% 5% 10% 15% 20% 25% 30% 35% 40% 0 60 120 180 240 300 360 420 480 540 600 660 Transfer Rate (KB/s) Load Time (seconds) (d) 3 tags with 3 connections Figure 11. Results without parameters adaptation. CPU Load Memo 0 8 16 24 32 40 48 56 64 0% 5% 10% 15% 20% 25% 30% 35% 40% 0 60 120 180 240 300 360 420 480 540 600 660 Transfer Rate (KB/s) Load Time (seconds) y Load Transmission Rate 0 8 16 24 32 40 48 56 64 0% 5% 10% 15% 20% 25% 30% 35% 40% 0 60 120 180 240 300 360 420 480 540 600 660 Transfer Rate (KB/s) Load Time (seconds) Time (seconds) (b) 1 tag with 3 connections 0 8 16 24 32 40 48 56 64 0% 5% 10% 15% 20% 25% 30% 35% 40% 0 60 120 180 240 300 360 420 480 540 600 660 Transfer Rate (KB/s) Load Time (seconds) (d) 3 tags with 3 connections (a) 1 tag with 1 connection 0 8 16 24 32 40 48 56 64 0% 5% 10% 15% 20% 25% 30% 35% 40% 0 60 120 180 240 300 360 420 480 540 600 660 Transfer Rate (KB/s) Load Time (seconds) Figure 11. Results without parameters adaptation. Figure 12, on the other hand, depicts the results with QualiCare Manager adjusting the FPS. In all graphs, it is possible to visualize the impact of the adaption, which occurs in the second 330. 6. Results and Discussion CPU Load Memo 0 8 16 24 32 40 48 56 64 0% 5% 10% 15% 20% 25% 30% 35% 40% 0 60 120 180 240 300 360 420 480 540 600 660 Transfer Rate (KB/s) Load Time (seconds) ry Load Transmission Rate 0 8 16 24 32 40 48 56 64 0% 5% 10% 15% 20% 25% 30% 35% 40% 0 60 120 180 240 300 360 420 480 540 600 660 Transfer Rate (KB/s) Load Time (seconds) (b) 1 tag with 3 connections 0 8 16 24 32 40 48 56 64 0% 5% 10% 15% 20% 25% 30% 35% 40% 0 60 120 180 240 300 360 420 480 540 600 660 Transfer Rate (KB/s) Load Time (seconds) (d) 3 tags with 3 connections (a) 1 tag with 1 connection 0 8 16 24 32 40 48 56 64 0% 5% 10% 15% 20% 25% 30% 35% 40% 0 60 120 180 240 300 360 420 480 540 600 660 Transfer Rate (KB/s) Load Time (seconds) (d) 3 tags with 3 connections Figure 12. Results with QualiCare Manager adapting the FPS. 6. Results and Discussion This demonstrates that changing FPS from Sensor Data Producer instances has two main advantages. First, the hardware utilization is improved, which might also impacts in energy consumption. Second, it is possible to serve more clients simultaneously since the adaption frees network and processing to new connections. Sensors 2019, 19, 2303 22 of 27 , , CPU Load Memory Load Transmission Rate 0 8 16 24 32 40 48 56 64 0% 5% 10% 15% 20% 25% 30% 35% 40% 0 60 120 180 240 300 360 420 480 540 600 660 Transfer Rate (KB/s) Load Time (seconds) (a) 1 tag with 1 connection 0 8 16 24 32 40 48 56 64 0% 5% 10% 15% 20% 25% 30% 35% 40% 0 60 120 180 240 300 360 420 480 540 600 660 Transfer Rate (KB/s) Load Time (seconds) (b) 1 tag with 3 connections 0 8 16 24 32 40 48 56 64 0% 5% 10% 15% 20% 25% 30% 35% 40% 0 60 120 180 240 300 360 420 480 540 600 660 Transfer Rate (KB/s) Load Time (seconds) (c) 3 tags with 1 connection 0 8 16 24 32 40 48 56 64 0% 5% 10% 15% 20% 25% 30% 35% 40% 0 60 120 180 240 300 360 420 480 540 600 660 Transfer Rate (KB/s) Load Time (seconds) (d) 3 tags with 3 connections Figure 12. Results with QualiCare Manager adapting the FPS. 8. Conclusions Many different sensors are in charge of monitoring subjects in the OR, which might be things or persons. Since data from surgical procedures are critical, its continuous ﬂow is essential to avoid critical situations and evaluation of the room setup. In addition, as the OR represents one of the highest costs in hospitals, improving procedures efﬁciency is also important. Several research focus OR environments proposing medical and monitoring systems to improve service performance and increase patient safety. The literature also presents developments of IoT middlewares which support QoS in several ﬁelds. However, they do not present the combination of QoS middlewares for OR environments, which demand monitoring of several equipment and people. In this context, this article presented an adaptable middleware model to provide multi-level QoS on ORs called QualiCare. The scientiﬁc contributions of this research are twofold: (i) we propose a model of an adaptable QoS-enabled middleware for OR;and (ii) we deﬁne an orchestration engine to provide different services for user applications and sensors. The model offers a set of services to guarantee the middleware scalability regardless the number of sensors and user applications producing and consuming data, respectively. QualiCare guarantees that one or more applications consume data from the middleware turning it scalable according to the applications load. QualiCare acts in both user applications and sensors levels in order to guarantee real-time data. To offer these features, QualiCare employs a rule-based service orchestration process based on a lower and upper thresholds. QualiCare monitors speciﬁc metrics in the different levels to combine its measures in the orchestration process for decision-making. Additionally, the model encompasses a Manager process which is charge of QoS monitoring and service provisioning. This component performs parameters adaptation, compression, and vertical elasticity in the middleware resources to tackle QoS violations. Besides the scientiﬁc contributions, this research also presents a social contribution related to the patients safety. By guaranteeing QoS on delivering medical information for hospital administrators and physicians, QualiCare ensures that sensitive data is properly delivered to its destinations regardless the system load. This might improve medical outcomes and consequently save lives. Results show that adapting the sampling rate of sensors within acceptable levels decreases the transmission rate consequently. Although our evaluation is preliminary, the experiments are promising and demonstrate the system signiﬁcance, even more considering hospitals with many surgical rooms and long duration procedures. 7. Limitations It is worth noting some limitations which might be explored in future research. Currently, the Orchestration Engine from the QualiCare Manager deﬁnes a threshold rule-based strategy in the user and sensor levels of the middleware. We intend to change the service orchestration strategy by a different heuristic to select the services for user applications and sensors. On the other hand, we also intend to adapt the current strategy so that the user can also conﬁgure the system rules through an SLA conﬁguration ﬁle like the SLA threshold ﬁle. Moreover, we are considering explore the effect of the previous decision by comparing the variation in metrics measurements. This feedback allows the employment of techniques to learn from previous actions and consider past events in the decision-making process. Considering the services, it is possible to explore the horizontal model of resource elasticity. In this regard, load balancing strategies and new strategies for communication reconﬁguration are the main challenges. At the current stage of this research, we have evaluated only the Sensor Data Producer module with the sampling rate metric. The next steps include the evaluation of the remaining modules and metrics in a real OR environment. Currently, we are deploying the infrastructure in a OR so that we can conduct more detailed experiments. Although we do not present deep evaluation of the prototype, our current results demonstrate the impact in network and processing resources. In future work, we also intend to evaluate the model with data from larger health databases and focus on increasing the possibilities of beneﬁts for patients and healthcare providers. Other important aspects to discuss are data distribution, scalability, security, and privacy. In addition, the prototype can expand to integrate with other open and proprietary health standards. Sensors 2019, 19, 2303 23 of 27 Author Contributions: Conceptualization, V.F.R., R.d.R.R. and C.A.d.C.; Investigation, V.F.R.; Methodology, V.F.R. and R.d.R.R.; Software, V.F.R.; Supervision, R.d.R.R.; Writing—original draft, V.F.R.; Writing—review & editing, V.F.R., R.d.R.R., C.A.d.C., B.E. and A.M. Conﬂicts of Interest: The authors declare no conﬂict of interest. Funding: This work was supported in part by the Coordination for the Improvement of Higher Education Personnel—Brazil (CAPES)—Finance Code 001 and by the National Counsel of Technological and Scientiﬁc Development—Brazil (CNPq)—Grant numbers 405354/2016-9 and 303640/2017-0. Acknowledgments: The authors would like to thank the Coordination for the Improvement of Higher Education Personnel—Brazil (CAPES) and the National Counsel of Technological and Scientiﬁc Development—Brazil (CNPq) for partially supporting this work. Bjoern Eskoﬁer gratefully acknowledges the support of the German Research Foundation (DFG) within the framework of the Heisenberg professorship programme (grant number ES 434/8-1). References 1. Zgheib, R.; Conchon, E.; Bastide, R. Semantic Middleware Architectures for IoT Healthcare Applications. In Enhanced Living Environments: Algorithms, Architectures, Platforms, and Systems; Ganchev, I., Garcia, N.M., Dobre, C., Mavromoustakis, C.X., Goleva, R., Eds.; Springer International Publishing: Cham, Switzerland, 2019; pp. 263–294. pp . Rothstein, D.H.; Raval, M.V. Operating room efﬁciency. Semin. Pediatr. Surg. 2018, 27, 79–85. [CrossRe , ; , p g y g , , [ ] 3. Phillips, N. Berry & Kohn’s Operating Room Technique; Elsevier Health Sciences: Amsterdam, The Netherlands, 2016. 4. Spangenberg, N.; Wilke, M.; Franczyk, B. A Big Data architecture for intra-surgical remaining time 3. Phillips, N. 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In future work, we intend to evaluate the model running the Sensor Data Producer instances in a private virtual environment which enables replication and different elasticity models. This will require load balancing strategies and new strategies for communication reconﬁguration in the service and sensor levels. Author Contributions: Conceptualization, V.F.R., R.d.R.R. and C.A.d.C.; Investigation, V.F.R.; Methodology, V.F.R. and R.d.R.R.; Software, V.F.R.; Supervision, R.d.R.R.; Writing—original draft, V.F.R.; Writing—review & editing, V.F.R., R.d.R.R., C.A.d.C., B.E. and A.M. Author Contributions: Conceptualization, V.F.R., R.d.R.R. and C.A.d.C.; Investigation, V.F.R.; Methodology, V.F.R. and R.d.R.R.; Software, V.F.R.; Supervision, R.d.R.R.; Writing—original draft, V.F.R.; Writing—review & editing, V.F.R., R.d.R.R., C.A.d.C., B.E. and A.M. Funding: This work was supported in part by the Coordination for the Improvement of Higher Education Personnel—Brazil (CAPES)—Finance Code 001 and by the National Counsel of Technological and Scientiﬁc Development—Brazil (CNPq)—Grant numbers 405354/2016-9 and 303640/2017-0. Funding: This work was supported in part by the Coordination for the Improvement of Higher Education Personnel—Brazil (CAPES)—Finance Code 001 and by the National Counsel of Technological and Scientiﬁc Development—Brazil (CNPq)—Grant numbers 405354/2016-9 and 303640/2017-0. Acknowledgments: The authors would like to thank the Coordination for the Improvement of Higher Education Personnel—Brazil (CAPES) and the National Counsel of Technological and Scientiﬁc Development—Brazil (CNPq) for partially supporting this work. 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). c⃝2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4366278500	https://www.mdpi.com/2076-2615/13/8/1386/pdf?version=1681804778	English	null	Nutritional Strategies to Improve Meat Quality and Composition in the Challenging Conditions of Broiler Production: A Review	Animals	2,023	cc-by	17,861	Citation: Choi, J.; Kong, B.; Bowker, B.C.; Zhuang, H.; Kim, W.K. Nutritional Strategies to Improve Meat Quality and Composition in the Challenging Conditions of Broiler Production: A Review. Animals 2023, 13, 1386. https://doi.org/10.3390/ ani13081386 Keywords: broilers; meat quality; meat composition; feed composition; bioactive compounds; plant polyphenol compounds Academic Editor: Sylwester ´Swi ˛atkiewicz Received: 13 March 2023 Revised: 14 April 2023 Accepted: 16 April 2023 Published: 18 April 2023 Review 2 , Byungwhi Kong 1, Brian C. Bowker 1, Hong Zhuang 1 and Woo Kyun Kim 2,* Janghan Choi 1,2 , Byungwhi Kong 1, Brian C. Bowker 1, Hong Zhuang 1 and Woo Kyun K 1 US National Poultry Research Center, USDA-ARS, Athens, GA 30605, USA; choij@uga.edu (J.C.); byungwhi.kong@usda.gov (B.K.) 2 Department of Poultry Science, University of Georgia, Athens, GA 30602, USA * Correspondence: wkkim@uga.edu; Tel.: +1-706-542-1346 1 US National Poultry Research Center, USDA-ARS, Athens, GA 30605, USA; choij@uga.edu (J.C.); byungwhi.kong@usda.gov (B.K.) 2 Department of Poultry Science, University of Georgia, Athens, GA 30602, USA * Correspondence: wkkim@uga.edu; Tel.: +1-706-542-1346 Simple Summary: Intense genetic selection and improvements in nutrient programs have enhanced the growth performance, feed efﬁciency, and meat yield of broiler chickens. The quality characteris- tics and nutrient composition of poultry meat are also crucial factors in poultry production. Many challenging conditions, including fast growth, pathogen infection, high rearing temperature, and feed contamination, can negatively inﬂuence poultry meat quality and composition. Studies have demonstrated that modulating the nutrient composition of feed and supplementing bioactive com- pounds, including vitamins, probiotics, prebiotics, exogenous enzymes, polyphenol compounds, and organic acids, have positively inﬂuenced the meat quality and body composition of broiler chickens. Abstract: Poultry meat is becoming one of the most important animal protein sources for human beings in terms of health beneﬁts, cost, and production efﬁciency. Effective genetic selection and nutritional programs have dramatically increased meat yield and broiler production efﬁciency. However, modern practices in broiler production result in unfavorable meat quality and body composition due to a diverse range of challenging conditions, including bacterial and parasitic infection, heat stress, and the consumption of mycotoxin and oxidized oils. Numerous studies have demonstrated that appropriate nutritional interventions have improved the meat quality and body composition of broiler chickens. Modulating nutritional composition [e.g., energy and crude protein (CP) levels] and amino acids (AA) levels has altered the meat quality and body composition of broiler chickens. The supplementation of bioactive compounds, such as vitamins, probiotics, prebiotics, exogenous enzymes, plant polyphenol compounds, and organic acids, has improved meat quality and changed the body composition of broiler chickens. animals animals 1. Introduction Poultry meat is one of the most inexpensive and popular protein sources in the world [1]. Furthermore, poultry meat is known to have a low fat content and high concen- trations of omega-3 fatty acids, which can be beneﬁcial for humans’ vascular health [2]. In 2019, the consumption rates of poultry meat, per capita in the world and the US, were approximately 14.7 kg (OECD-FAO) [3], which were higher than those for any other meat sources, including pork, beef, sheep, and goat meat [4]. It is expected that worldwide poultry meat consumption will continue to increase annually due to an increase in the global population and preference [5]. To meet this growing demand for poultry meat, there have been signiﬁcant enhancements in broiler meat production. For instance, while 42-day-old broilers weighed around 586 g with a feed conversion ratio of 2.8 in 1957 [6], modern broilers weigh around 3278 g with a feed conversion ratio of 1.55 at 42-days-old, Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/animals Animals 2023, 13, 1386. https://doi.org/10.3390/ani13081386 Animals 2023, 13, 1386 2 of 20 according to Cobb500 broiler performance and nutrition supplement 2022 [7]. Broiler meat is considered the most cost-effective and sustainable animal protein source because of the birds’ efﬁciency in converting feed to meat [8,9]. While effective genetic selection and nutrition programs have dramatically improved the productivity and efﬁciency of the broiler industry, fast-growing broilers more frequently show unfavorable meat quality compared to slow-growing broilers [10]. Poultry produc- tion continues to face a diverse range of challenges, including feed cost issues, bacterial and parasitic infection, heat stress, mycotoxins, etc., which can result in altered body composi- tion, increased food safety concerns, and reduced meat yield and quality [11]. Negatively affected meat quality parameters, including water holding capacity (WHC), myopathies, texture, and ﬂavor can inﬂuence consumer acceptance [12]. Nutrition is known to have a substantial inﬂuence on the meat quality and body composition of broilers [13]. This review summarizes (1) the effects of the diverse, challenging conditions in modern broiler produc- tion on meat quality and body composition and (2) the effects of nutritional strategies on the meat quality and body composition of broilers. 2. Challenging Conditions in Broiler Production Are Associated with Meat Quality 2.1. Fast Growth Rate Animals 2023, 13, 1386 3 of 20 Table 1. Differences in meat quality in slow- and fast-growing broilers. Comparison Strains Observations in Fast-Growing Broilers Replicates per Treatments References Fast-growing (FG) vs. slow-growing (SG) chickens from La Gamme Hubbard (Sedar Co., Mi˛edzyrzec Podlaski, Poland) A higher portion of breast and thigh yields Lower abdominal fats Higher fat and lower protein compositions Less yellowness 8 [29] Medium slow-growing broilers (SG, Hubbard Red JA) vs. Ross308 Higher breast and lower thigh yields 10 [30] Strains from S & G Poultry, Clanton, AL vs. Cobb-Vantress Inc., Siloam Spring, AR Lower protein and higher fat contents Lighter, more redness, and less yellowness Higher pH Lower drip loss and higher thaw and cooking loss 80 [31] Xueshan (Chinese local chickens) vs. Ross308 Lower pH Lighter and less redness and yellowness Lower protein and higher fat contents 6 [32] Table 1. Differences in meat quality in slow- and fast-growing broilers. 2.2. Bacterial Infection Bacterial infections, which can be caused by unfavorable rearing conditions and high stocking density, would be critical in broiler production [33]. The common pathogenic and infectious bacteria in broiler production include Staphylococcus aureus, Campylobacter jejuni, Salmonella spp., Clostridium perfringens, Escherichia coli, etc. [34]. Those bacterial infections can negatively affect the meat production and quality of broiler chickens, potentially via decreasing nutrient utilization, inducing inﬂammation, and inﬂuencing food safety [35–37]. For example, invasive food-borne pathogens, including Salmonella spp., C. jejuni, and S. aureus have been detected in chicken meat [38–40]. A previous study by Wang et al. [41] demonstrated that Salmonella Enteritidis infection increased fat accumulation by increasing lipid synthesis and reducing lipid transportation in the liver, which can potentially affect fat accumulation within the bodies of the chickens. Sadeghi et al. [42] demonstrated that S. Enteritidis infection increased the concentration of plasma malondialdehyde (MDA), an indicator for lipid peroxidation, and reduced the plasma total antioxidant capacity, thus potentially resulting in increased lipid peroxidation in the chicken meat. Likewise, many studies have shown that Campylobacter infection can induce systemic oxidative stress, which has the potential to induce lipid peroxidation in muscle [43–45]. However, more studies are needed to investigate the interactions between bacterial infection during the pre-harvest period and the meat quality of broiler chickens. 2. Challenging Conditions in Broiler Production Are Associated with Meat Quality 2.1. Fast Growth Rate Through more than 70 years of intense genetic selection and effective nutritional programs, increased body weight, growth rates, and feed efﬁciency have been achieved among modern broiler chickens [14]. Genetic selection programs and nutritional programs have been designed to produce broilers with higher breast meat yields and body weight without considering fat accumulation, resulting in high levels of fat accumulation in modern broilers [15–18]. The increased production of breast meat has resulted in a decrease in leg meat production in modern broilers due to a change in body shape [19]. Modern broilers contain approximately 15 to 20% fat, and the majority (>85%) of the fat is not necessary for broilers’ body function [20]. In fast growth, the imbalanced ratio between oxidants and antioxidants in the body and the limited oxygen supply compared to the demand required for fast growth can lead to oxidative stress and hypoxia, respectively, resulting in lipid peroxidation and woody breast (WB) in modern broiler chickens [21–24]. Differences in meat quality have been observed between slow- and fast-growing broilers, as shown in Table 1. Overall, fast-growing broilers showed lower protein and higher fat contents compared to the slow-growing broilers. However, the differences in meat quality parameters, such as the pH, color, and WHC, in slow- and fast-growing broilers were not consistent—potentially because the slow-growing broiler strains varied across studies. According to Che et al. [25], the incidence rates of spaghetti meat (SM), severe WB, and mild white striping (WS) were nearly 36, 12, and 96%, respectively, and approximately 85% of breast meat showed several myopathies in Ontario, Canada. While there were no surveys that compared the incidence of breast myopathies between fast-growing and slow- growing broilers, the prevalence of breast myopathies would be greater in fast-growing modern broilers, which may be due to higher breast meat yield and higher oxidative stress compared to slow-growing broilers [26]. In addition, Zhang et al. [27] and Popova et al. [28] suggested that fast-growing broilers, which are slaughtered at a young age (D 42), may contain lower polyunsaturated fatty acids compared to the slow-growing chickens (raised past D 42) because older chickens tend to have higher polyunsaturated fatty acid contents in their breast meat. Hence, whereas modern broilers have higher yields of meat compared to slow-growing broilers, the meat of modern broilers may have higher fat content, lower omega-3 polyunsaturated fatty acid proportions, and more muscle myopathies. 2.3. Coccidiosis Coccidiosis is an enteric disease caused by Eimeria spp. in chickens, and it is a prevalent disease all over the world [46]. Cornell et al. [47] reported that approximately 95% of ﬂocks in the western US using antibiotic-free production were infected with Eimeria spp. The seven species of Eimeria, including Eimeria acervulina, E. maxima, E. tenella, E. brunetti, E. necatrix, E. mitis, and E. praecox, colonize different regions of the gastrointestinal tract [48]. The most common Eimeria spp. found in chickens include E. tenella (ceca), E. necatrix (ileum), E. acervulina (duodenum), and E. maxima (jejunum) [49]. Many studies have reported that infection with E. acervulina and E. maxima reduced nutrient digestibility, especially amino acid (AA) digestibility, in broiler chickens [50,51]. Reduced AA digestibility may lead to decreased meat yield because AAs are building blocks for proteins in meat [52]. Our previous study demonstrated that E. tenella infection reduced the production of cecal short- chain fatty acids (SCFA; an important energy source for the host), which can lead to energy imbalances and reduce fat accumulation, resulting in decreased meat yields among broiler chickens [53,54]. Induced inﬂammation and oxidative stress caused by Eimeria infection can negatively affect meat quality in broilers [55,56]. Moreover, increased gut permeability Animals 2023, 13, 1386 4 of 20 4 of 20 caused by Eimeria infection can aggravate the negative effects of bacterial infection on meat safety and quality in broiler chickens [57]. Several studies have demonstrated the effects of Eimeria infection on meat quality in broiler chickens (Table 2). Eimeria infection induced lipid peroxidation in the breast meat, according to Cha et al. [58] and Partovi et al. [59]. Though conducted under identical experimental conditions, Qaid et al. [60] reported E. tenella infection increased lightness, while Qaid et al. [61] showed that E. tenella infection decreased lightness in the breast meat. Nonetheless, reduced growth performance due to Eimeria infection could both negatively and positively affect meat quality, because the fast growth rate of modern broilers can negatively inﬂuence meat quality and Eimeria infection may slow the growth rate. Table 2. Effects of Eimeria infection on breast meat quality in broiler chickens. Eimeria Species and Oocysts Observations in the Breast Meat Replicates per Treatments References 1 × 104 Eimeria tenella Reduced redness Increased thiobarbituric acid reactive substances, a byproduct of lipid peroxidation 10 [58] 5 × 105 mixture of E. acervulina, E. maxima, and E. 2.3. Coccidiosis tenella Increased drip loss and cooking loss Decreased protein content Reduced redness Increased the formation of malonaldehyde, a ﬁnal product of lipid peroxidation 10 [59] 4 × 104 Eimeria tenella Reduced lightness Reduced drip loss 5 [61] 4 × 104 Eimeria tenella Increased lightness 5 [60] Table 2. Effects of Eimeria infection on breast meat quality in broiler chickens. 2.4. Heat Stress Heat stress, referred to as an imbalance between body heat production and loss, induces metabolic disruption and oxidative stress, which leads to physiological and be- havioral alterations in broiler chickens [62]. Global warming is expected to accelerate the heat stress issues in broiler production [63]. Heat-stressed chickens have impaired growth performance along with poor meat quality, resulting in lower production efﬁciency and consumer acceptance [64]. The impact of heat stress on broiler meat quality is summarized in Table 3. According to Tavaniello et al. [65], heat stress reduced protein content and increased fat content in broiler chicken breast meat. Zhang et al. [66] and Zaboli et al. [64] demonstrated that heat stress increased the secretion of corticosterone, which is a critical stress hormone, reducing protein accumulation via protein degradation and increasing fat accumulation potentially via modulating the expression of fatty acid transport proteins and insulin receptors in broiler chickens. Heat stress increased the pH of breast meat, according to some studies [67,68]. It is hypothesized that heat stress induces the exhaus- tion of muscle glycogen antemortem and reduces postmortem glycolysis, which results in increased meat pH levels [69]. There were inconsistent results regarding the color of breast meat in heat-stressed broilers, as shown in Table 3. The potential reasons would be (1) increased pH due to the depletion of glycogen in the heat stress conditions resulting in a darker color of meat [70]; and (2) heat stress can denature sarcoplasmic proteins, which leads to the scattering of light, and this increases the lightness of meat [66]. However, a study by Zhang et al. [66] reported that heat stress could induce pale, soft, exudative (PSE)-like breast meat (e.g., low pH levels and pale coloring) in broilers. The differences might originate from different experimental conditions, such as diets, broiler strains, and management. Animals 2023, 13, 1386 5 of 20 Table 3. Impact of heat stress on broiler meat quality. 2.4. Heat Stress Observations in the Breast Meat Replicates per Treatments References Increased pH 10 [67] Decreased cooking loss Increased thiobarbituric acid reactive substances, a byproduct of lipid peroxidation 12 [71] Increased pH Increased lightness and decreased redness Increased cooking loss Decreased shear force 16 [68] Increased pH Decreased lightness Increased yellowness Increased total lipid and reduced collagen Increased omega-3 polyunsaturated fatty acids 6 [65] Reduced protein content and increased fat content Decreased pH Increased cooking loss Increased lightness and reduced redness Increased shear force 6 [66] 2.5. Mycotoxins Mycotoxins, the secondary metabolites of fungi, such as the genera Fusarium, Aspergillus, etc., are frequently found in poultry feed [72,73]. There are more than 400 mycotoxins with different absorption rates and effects on broiler chickens, as summarized in Table 4. Table 4. Diverse kinds of mycotoxins, their absorption rates, and their effects on broiler chickens. Mycotoxins Absorption Rate in the Gastrointestinal Tract Observations Aﬂatoxins Highly absorbable (80 to 90%) [74] Induced mild negative effects on the gastrointestinal tract [75] Induced oxidative stress, damaged liver, damaged RNA and DNA, and suppressed immune system [76] Deoxynivalenol Low absorption rate [77] Increased intestinal permeability [78] Damaged intestinal morphology and induced intestinal inﬂammation [79] Induced severe intestinal damage [80] Fumonisins Low absorption rate with quick plasma elimination but considerate accumulation rate [81,82] Damaged ileum morphology and negatively affected gut microbiota [83] Induced mild inﬂammation in the gastrointestinal tract [84] Ochratoxin A High absorption rate [85] Damaged RNA and DNA [86] Induced oxidative stress, lipid peroxidation, and cell apoptosis and inhibited protein synthesis [87] Suppressed immune system [88] Zearalenone High absorption rate [89] Induced liver and kidney damage [90] Induced oxidative stress and suppressed immune system by damaging spleen [91,92] Mycotoxins with high absorption and accumulation rates can remain in edible tissues (meat and liver). Hort et al. [93] showed that fumonisins were detected in the liver and ﬁllet muscles. While zearalenone is known to have a higher absorption rate in the gastrointestinal tract of chickens [89], it was not detected in the liver or muscles, potentially due to rapid elimination after absorption [93]. However, Iqbal et al. [94] reported that 35, 41, and 52% of poultry meat samples were contaminated with aﬂatoxins, ochratoxin A, and zearalenone, ti l i P ki t M t i i t d t i d d th Table 3. Impact of heat stress on broiler meat quality. 2.4. Heat Stress Proposed effects of low- and high- accumulating mycotoxins on the meat quality of broiler chickens. Low-accumulating mycotoxins negatively affect the gut health and gut microbiota of Figure 2. Proposed modes of action of polyphenols depending on their molecular weight and ab- sorption rate in the gastrointestinal tract. Figure 1. Proposed effects of low- and high- accumulating mycotoxins on the meat quality of broiler chickens. Low-accumulating mycotoxins negatively affect the gut health and gut microbiota of broiler chickens [96], and this can indirectly affect meat composition and quality by decreasing nutrient accumulation and inducing oxidative stress. In addition to indirect effects, such as low-accumulating mycotoxins, high-accumulating mycotoxins can negatively inﬂuence meat composition and quality by directly inhibiting protein metabolism, inducing protein degradation, and damaging RNA and DNA in the muscle [83,97]. Table 5 summarizes the meat quality of broiler-fed feeds containing mycotoxins. Myco- toxins induce oxidative stress, which can lead to lipid peroxidation and negatively modulate the fatty acid composition and color of meat. However, results from Armanini et al. [98] may suggest that mycotoxins improve the meat quality of broiler chickens, potentially because growth retardation due to mycotoxins ameliorated the meat quality among fast- growing broilers [26]. Table 5. Effects of mycotoxins on the meat quality of broiler chickens. Mycotoxins in the Feed Observations in the Breast Meat Replicates per Treatments References 72 to 200 µg/kg ochratoxin A Reduced pH Increased drip loss Reduced fat content Increased short-chain fatty acids and reduced polyunsaturated fatty acids Increased omega-6 to omega-3 fatty acid ratio 10 [87] 70 µg/kg aﬂatoxins B1 Increased yellowness 12 [99] Figure 1. Proposed effects of low- and high- accumulating mycotoxins on the meat quality of broiler chickens. Low-accumulating mycotoxins negatively affect the gut health and gut microbiota of Figure 1. Proposed effects of low- and high- accumulating mycotoxins on the meat quality of broiler chickens. Low-accumulating mycotoxins negatively affect the gut health and gut microbiota of broiler chickens [96], and this can indirectly affect meat composition and quality by decreasing nutrient accumulation and inducing oxidative stress. In addition to indirect effects, such as low-accumulating mycotoxins, high-accumulating mycotoxins can negatively inﬂuence meat composition and quality by directly inhibiting protein metabolism, inducing protein degradation, and damaging RNA and DNA in the muscle [83,97]. Table 5 summarizes the meat quality of broiler-fed feeds containing mycotoxins. 2.4. Heat Stress Mycotoxins with high absorption and accumulation rates can remain in edible tissues (meat and liver). Hort et al. [93] showed that fumonisins were detected in the liver and ﬁllet muscles. While zearalenone is known to have a higher absorption rate in the gastrointestinal tract of chickens [89], it was not detected in the liver or muscles, potentially due to rapid elimination after absorption [93]. However, Iqbal et al. [94] reported that 35, 41, and 52% of poultry meat samples were contaminated with aﬂatoxins, ochratoxin A, and zearalenone, respectively, in Pakistan. Mycotoxins in meat products can induce cancer and suppress the Animals 2023, 13, 1386 6 of 20 immune system in humans [95]. As shown in Figure 1, depen rate of mycotoxins, the effects of mycotoxins on meat quality c Figure 1. Proposed effects of low- and high- accumulating mycotoxins chickens. Low-accumulating mycotoxins negatively affect the gut heal Figure 2. Proposed modes of action of polyphenols depending on the sorption rate in the gastrointestinal tract. Figure 1. Proposed effects of low- and high- accumulating mycotoxin chickens. Low-accumulating mycotoxins negatively affect the gut healt chickens [96], and this can indirectly affect meat composition and q accumulation and inducing oxidative stress. In addition to indirect eff mycotoxins, high-accumulating mycotoxins can negatively inﬂuence by directly inhibiting protein metabolism, inducing protein degrada DNA in the muscle [83,97]. Table 5 summarizes the meat quality of broiler-fed feeds co toxins induce oxidative stress, which can lead to lipid peroxidati the fatty acid composition and color of meat. However, resul may suggest that mycotoxins improve the meat quality of b because growth retardation due to mycotoxins ameliorated th growing broilers [26]. Table 5. Effects of mycotoxins on the meat quality of broiler chickens Mycotoxins in the Feed Observations in the Breast Meat Re T 172 to 200 µg/kg ochratoxin A Reduced pH Increased drip loss Reduced fat content Increased short-chain fatty acids and reduced polyunsaturated fatty acids Increased omega-6 to omega-3 fatty acid ratio 70 µg/kg aﬂatoxins B1 Increased yellowness immune system in humans [95]. As shown in Figure 1, depending on the accumulating rate of mycotoxins, the effects of mycotoxins on meat quality can be different. immune system in humans [95]. As shown in Figure 1, depending on the accumulating rate of mycotoxins, the effects of mycotoxins on meat quality can be different. Figure 1. Mycotoxins in the Feed 2.6. Oxidized Oils and Fat Oil and fat sources are important energy sources, and they are normally included, at levels of up to 5%, in broiler feed [103]. The freshness of oil and fat sources is important in poultry feed because oxidized oil and fat can induce lipid peroxidation and oxidative stress in animals [104]. Dong et al. [105] showed that the inclusion of oxidized oils induced oxidative stress and increased MDA in broiler chickens. Zhang et al. [106] demonstrated that broilers fed with oxidized oil had higher protein and lipid oxidation levels, increased WHC, and decreased pH in the breast meat post-mortem. Appropriate levels of fresh oil and fat sources should be included in broiler diets. 2.4. Heat Stress Myco- toxins induce oxidative stress, which can lead to lipid peroxidation and negatively modulate the fatty acid composition and color of meat. However, results from Armanini et al. [98] may suggest that mycotoxins improve the meat quality of broiler chickens, potentially because growth retardation due to mycotoxins ameliorated the meat quality among fast- growing broilers [26]. Table 5. Effects of mycotoxins on the meat quality of broiler chickens. Table 5. Effects of mycotoxins on the meat quality of broiler chickens. Figure 2. Proposed modes of action of polyphenols depending on their molecular weight and ab- sorption rate in the gastrointestinal tract. Table 5. Effects of mycotoxins on the meat quality of broiler chickens. Mycotoxins in the Feed Observations in the Breast Meat Replicates per Treatments References 172 to 200 µg/kg ochratoxin A Reduced pH Increased drip loss Reduced fat content Increased short-chain fatty acids and reduced polyunsaturated fatty acids Increased omega-6 to omega-3 fatty acid ratio 10 [87] 70 µg/kg aﬂatoxins B1 Increased yellowness 12 [99] Animals 2023, 13, 1386 7 of 20 Table 5. Cont. Mycotoxins in the Feed Observations in the Breast Meat Replicates per Treatments References 451 µg/kg aﬂatoxin B1, 684 µg/kg ochratoxin A, and 320 µg/kg of T-2 toxin Decreased lightness Increased redness and yellowness 6 [100] 0.05 µg/kg aﬂatoxin and 20 µg/kg fumonisin Increased lightness Reduced cooking loss Modulated fatty acid composition Stimulated antioxidant system as a defensive reaction 4 [98] 0.5 mg/kg aﬂatoxin Increased thiobarbituric acid reactive substances, a byproduct of lipid peroxidation 15 [101] 43.2 mg/kg deoxynivalenol Decreased lightness 8 [102] Table 5. Cont. Table 5. Cont. 3.2. Amino Acids (Aas) Not only are AA important building blocks in the de novo synthesis of muscle pro- teins, but each AA also plays speciﬁc roles in modulating metabolic pathways, antioxidant systems, and enzymatic processes, which can potentially inﬂuence meat production and quality in animals [121]. Moreover, adding speciﬁc Aas above the recommended require- ments became a common practice to exhibit beneﬁcial effects in broiler chickens [122,123]. Methionine, the ﬁrst limiting AA in corn–soybean meal diets for broilers, also plays an important role in improving the antioxidant system by being involved in the synthesis of glutathione (endogenous antioxidant) [124,125]. Moreover, Wen et al. [124] showed that the supplementation of methionine decreased the cooking loss and increased the pH in the breast meat, potentially by decreasing pyruvate kinase activity. Consistent with those ﬁndings, Albrecht et al. [126] reported that the supplementation of methionine (0.04, 0.12, and 0.32% in the feed) reduced cooking loss with increased pH levels in the breast meat and, in turn, a decrease in lightness. The supplementation of arginine has been known to reduce fat accumulation in chickens by altering de novo lipogenesis via regulating genes related to fat accumulation [20]. Our previous study showed that arginine at the requirement level improved lean muscle accumulation without affecting fat accumulation in broilers [127]. Arginine can be converted into nitric oxide, which can increase blood ﬂow and can mitigate the incidence and severity of WS and SM abnormalities, potentially via reducing hypoxia in broiler chickens [128]. Moreover, nitric oxide has antioxidant effects, which have the potential to improve meat shelf life and attenuate lipid peroxi- dation in chicken meat [129]. Branched-chain amino acids (BCAAs), including valine, leucine, and isoleucine, play an important role in muscle protein synthesis because BCAAs are major essential AA in muscles [122,130,131]. Zhang et al. [132] demonstrated that the supplementation of BCAAs in low CP diets increased WHC, improved tenderness by reducing shear force, and increased intramuscular fat in ﬁnishing pigs. In chickens, Imanari et al. [133] showed that the supplementation of BCAA (isoleucine at 150% of the requirement and valine at 150% of the requirement) enhanced the free glutamine (e.g., the main taste-active component in meat) concentration in the breast meat of broiler chickens. A previous study by Kop-Bozbay et al. 3. Effects of Nutritional Interventions on the Meat Quality of Broiler Chickens 3.1. Energy and Crude Protein Recently, decreasing the energy levels and CP levels in poultry diets have drawn a lot of attention as options for reducing feed costs and improving energy and nutrient utilization in poultry production [107,108]. Energy and CP levels are key factors in determining the body composition of chickens [109]. The low- and high-energy levels of broiler diets can decrease and increase fat accumulation, respectively [110,111]. In contrast, a previous study by Lipi´nski et al. [112] reported that low-energy diets (−23.9 vs. −59.75 kcal/kg from the control feed) increased fat accumulation in the breast muscles, potentially as a defensive mechanism to cope with energy deﬁciency, while low energy diets reduced MDA concentrations in the breast meat. Nonetheless, Rosa et al. [113] reported that high-energy diets (2950 vs. 3200 vs. 3450 kcal/kg in the feed) induced greater fat accumulation in broiler chickens. Lipi´nski et al. [112] reported that low-energy diets increased WHC natural drip loss, pH, and lightness and reduced redness in the breast meat. In broilers fed low-energy diets, increased pH levels might be due to a lack of glycogen, which produces lactic acid and H+ ions post-mortem in the breast muscle [114]. Increased pH could lead to increased WHC in breast meat [115]. However, Arshad et al. [116] showed that low-energy diets (−100 kcal/kg in the starter feed and ﬁnisher feed) did not modulate pH, WHC, or lightness in the breast meat. According to Ciftci and Ceylan [117], the low CP levels (19.1% (normal) or 18.0% (low) for starter feed and 17.7% (normal) or 16.5% (low) for ﬁnisher feed) of the diets reduced the fat content of breast and thigh meat, potentially by reducing the energy available for fat accumulation. Brink et al. [118] reported that low CP diets (20.5% (high), 18.7% (medium), and 17.5% (low) in the grower phase and 19.5% (high), 18.0% (medium), and 16.6% (low) in the ﬁnisher phase feed) increased thawing loss, potentially by decreasing the myoﬁber density due to the bigger size of the myoﬁber. Consistently, Chodová et al. [119] demonstrated that low CP diets (21.6% (normal) and 20.39% (low) in the starter phase; 19.7% (normal) and 18.6% (low) in the grower phase; and 18.0% (normal) and 17.3% (low) in the ﬁnisher phase feed) increased drip loss in the breast meat. 3.2. Amino Acids (Aas) [134] showed that the dietary supplementation of BCAA ((1.0, 0.25, and 0.75 g/kg of L-leucine, L-isoleucine, and L-valine compared to the control (non-supplementation)) was detrimental for the meat yield of broilers under heat stress conditions, potentially due to antagonistic interactions between valine and isoleucine [134]. Zhang and Kim [135] reported that the supplementation of threonine (0.65, 0.70, 0.75, and 0.80 threonine to lysine ratios) did not inﬂuence meat quality in broiler chick- ens. While Aas are important building blocks and their speciﬁc functions have the potential to improve meat quality, the supplementation of Aas does not guarantee improvements in the meat yield or quality of broiler chickens. 3. Effects of Nutritional Interventions on the Meat Quality of Broiler Chickens 3.1. Energy and Crude Protein However, Park and Animals 2023, 13, 1386 8 of 20 8 of 20 Kim [120] showed that different CP levels (23% (normal) and 21% (low) in the starter phase and 20% (normal) and 18% (low) in the ﬁnisher phase) did not alter the pH, cooking loss, drip loss, or color of breast meat. The differences might originate from different animals, diets, experimental periods, etc. 3.3. Vitamins The supplementation of vitamins is essential for broiler nutrition [68]. Whereas the supplementation of vitamin C is not required in broiler feed because the liver can synthesize enough, heat stress dramatically decreases vitamin C synthesis, and the sup- plementation of vitamin C can alleviate the negative effects of heat stress by fulﬁlling its requirement [136]. Vitamin E is an essential vitamin that should be supplemented in diets because it is not synthesized by chickens [137]. Both vitamins C and E are strong antioxidants in chickens and are known to alleviate oxidative stress induced by heat stress [24]. Gao et al. [138] and Peˇcjak et al. [139] reported that vitamin E supplementation (20 mg/kg feed (normal) vs. 200 mg/kg feed (high vitamin E)) improved antioxidative Animals 2023, 13, 1386 9 of 20 status and reduced lipid peroxidation in the breast meat of broiler chickens. The sup- plementation of vitamin E (10 mg/kg (normal) vs. 200 mg/kg (high vitamin E)) in the early-phase feed decreased the severity of WB and shear force in broiler chickens [140]. Mazur-Ku´snirek et al. [141] showed that the supplementation of vitamin E (200 mg/kg feed) decreased drip loss and enhanced WHC in broilers reared under high temperatures. However, Zeferino et al. [68] showed that the supplementation of vitamin C (257 mg/kg (normal) vs. 288 mg/kg (supplemented)) and vitamin E ((93 mg/kg (normal) vs. 109 mg/kg (supplemented)) did not inﬂuence the meat quality of broilers raised under heat stress conditions. Savaris et al. [142] reported that different levels of vitamin A (0, 6000, 16,000, 26,000, 36,000, and 46,000 IU/kg) and its supplementation period (D 0 to 21 vs. D 0 to 42) modulated the incidence and severity of WS in the breast meat and meat yield in broiler chickens, potentially via modulating cell metabolism in the muscle. Different forms of vitamin D, including 25-OHD3, 1,25(OH)2D3, and 1α(OH)D, modulated the color of the breast meat in broiler chickens [143]. Different rearing conditions can alter requirements (optimal doses), and different concentrations and forms of vitamins can affect the meat yield and quality of broiler chickens. 3.4. Omega-3 Fatty Acid Sources A number of studies have been conducted to increase the content of omega-3 fatty acids in chicken meat products [144]. Providing omega-3 fatty acid-rich sources in diets, including ﬂaxseed oil, rapeseed oil, canola oil, ﬁsh oil, and marine algae, has been known to enhance the omega-3 fatty acid content in poultry meat products [145]. The supplementation of ﬁsh oil with conjugated linoleic acid at 2% in the feed reduced omega-6 fatty acids in the breast meat [146]. The supplementation of ﬁsh oil at 3% in the feed enhanced omega-3 fatty acids (docosahexaenoic and eicosatetraenoic acid) in the breast meat [147]. Moreover, Konieczka et al. [148] showed that the supplementation of ﬁsh, rapeseed, and ﬂaxseed oils at 1% in the ﬁnisher phase increased the levels of omega-3 fatty acids in the breast meat. Moreover, Tompkins et al. [149] showed that material supplementation of ﬁsh oil at 2.3% in the feed enhanced the lean mass of offspring broiler chickens on D 42. Omega-3 fatty acids can improve blood ﬂow in animals [150], which indicates that its supplementation can reduce hypoxia in fast-growing broilers and it has the potential to reduce the incidence and severity of WS and SM abnormalities. A previous study by Yang et al. [150] showed that dietary supplementation with ﬁsh oil at 2.5% in the feed instead of soybean oil reduced both fat accumulation and the ratio of omega-6 to omega-3 fatty acids and the ratio of polyunsaturated to saturated fatty acids, while it decreased lightness and increased drip loss in the breast meat of broilers. However, the supplementation of omega-3 fatty acid-rich oils in the feed is not cost-effective and can induce lipid peroxidation during storage [145]. Therefore, more studies are required to determine speciﬁc supplementation period and levels of omega-3 fatty acid sources in broiler productions. 3.5. Probiotics and Prebiotics Probiotics and prebiotics are deﬁned as beneﬁcial live microorganisms and non- digestible substances that can be food for beneﬁcial bacteria, respectively [151,152]. The inclusion of probiotics and prebiotics in poultry feed became common to improve gut health and growth performance by enhancing gut microbiota in the poultry industry [153,154]. The supplementation of probiotics and prebiotics is known to reduce the incidence and severity of bacterial infection in the gastrointestinal tract of broiler chickens, which can improve meat safety [155–157]. Furthermore, the supplementation of Bacillus cereus var. toyoi (1 × 109 CFU/g) and B. subtilis PB6 (2 × 107 CFU/g) at 0.1% in the feed attenuated oxidative stress in broilers infected with Salmonella spp. [158]. Aluwong et al. [159] also showed that the oral administration of 1 mL of a yeast probiotic (Saccharomyces cerevisiae; 4.125 × 104 CFU) improved the activities of glutathione peroxidase in the serum of broiler chickens. Improved antioxidant capacity has the potential to reduce lipid peroxidation in the breast meat of broiler chickens. Bai et al. [160] reported that the supplementation of Animals 2023, 13, 1386 10 of 20 10 of 20 B. subtilis fmbJ (4 × 1010 CFU/kg feed) improved endogenous antioxidant capacity and reduced MDA in the serum and liver, resulting in reduced drip loss and cooking loss in the breast meat. Probiotics may improve antioxidant capacity by reducing the colonization of pathogenic bacteria [161]. Mohammed et al. [162] reported that the supplementation of B. subtilis at 0.5 g/kg of feed reduced the cooking loss and pH and increased the taste of the leg meat of broiler chickens. The microbiota of the gastrointestinal tract of broiler chickens plays an important role in producing SCFA, which is an important energy source for the host [53]. Increased SCFA production levels, due to beneﬁcially modulated microbiota, can affect the fat accumulation and body composition of broiler chickens [54]. However, a pre- vious study by Wang et al. [163] showed that the supplementation of Lactobacillus johnsonii BS15 (1 × 106 CFU/kg feed) reduced fat accumulation by downregulating fat accumulation- related genes (e.g., lipoprotein lipase) in adipose tissue and by upregulating hepatic genes related to fatty acid β-oxidation in broiler chickens. However, whether the mode of action of probiotics affects hepatic fat accumulation or oxidation is still unknown and demands more research. B. 3.5. Probiotics and Prebiotics subtilis fmbJ (4 × 1010 CFU/kg feed) improved endogenous antioxidant capacity and reduced MDA in the serum and liver, resulting in reduced drip loss and cooking loss in the breast meat. Probiotics may improve antioxidant capacity by reducing the colonization of pathogenic bacteria [161]. Mohammed et al. [162] reported that the supplementation of B. subtilis at 0.5 g/kg of feed reduced the cooking loss and pH and increased the taste of the leg meat of broiler chickens. The microbiota of the gastrointestinal tract of broiler chickens plays an important role in producing SCFA, which is an important energy source for the host [53]. Increased SCFA production levels, due to beneﬁcially modulated microbiota, can affect the fat accumulation and body composition of broiler chickens [54]. However, a pre- vious study by Wang et al. [163] showed that the supplementation of Lactobacillus johnsonii BS15 (1 × 106 CFU/kg feed) reduced fat accumulation by downregulating fat accumulation- related genes (e.g., lipoprotein lipase) in adipose tissue and by upregulating hepatic genes related to fatty acid β-oxidation in broiler chickens. However, whether the mode of action of probiotics affects hepatic fat accumulation or oxidation is still unknown and demands more research. 3.6. Exogenous Enzymes There are diverse sorts of exogenous enzymes, including phytase, protease, carbohy- drase, polysaccharidase, and lipase, and they have been supplemented in broiler feed to improve the growth performance, gut health, and nutrient utilization of broilers [164,165]. Exogenous enzymes have the potential to inﬂuence body composition by enhancing energy and protein utilization in broilers, but limited data are available thus far. Hajati [166] reported that the supplementation of enzyme cocktails (500 mg/kg of feed) containing arabinoxylanase, beta-glucanase, protease, and amylase increased the carcass yield and relative portion of thighs and drumsticks. Lu et al. [167] showed that the supplementation of protease (0.0125% of feed) reduced drip loss in the breast meat compared to the con- trol group. Phytase, which became an essential feed additive for non-ruminant animals, hydrolyzes phytate-bound phosphorus (e.g., phytic acid; antinutritional factor) into free phosphate [168]. The supplementation of phytase can enhance energy and AA digestibility in broiler chickens [169]. However, many studies have reported that the supplementation of phytase did not inﬂuence the meat quality of broiler chickens [170–172]. The supplemen- tation of exogenous enzymes may not signiﬁcantly alter the meat quality or composition in broiler chickens. 3.7. Plant Polyphenolic Compounds (e.g., Plant Extracts and Essential Oils) Plant extracts and essential oils, which contain diverse polyphenolic compounds (e.g., secondary plant metabolites), have obtained a lot of attention as alternative antibiotic growth-promoters in broiler production due to their antimicrobial, antioxidative, and anti-inﬂammatory effects [37,173,174]. Their antioxidative and anti-inﬂammatory effects have the potential to improve the meat quality of broiler chickens. Depending upon the molecular weight and absorption rate of polyphenols, their effects varied among broiler chickens, as shown in Figure 2. Soldado et al. [175] suggested that high molecular weight polyphenolic compounds have the potential to exhibit systemic antioxidative effects by improving antioxidant status in the gastrointestinal tract and interacting with other antioxidants in animals. The ben- eﬁcial effects of diverse polyphenols (e.g., plant extracts and essential oils) are shown in Table 6. The antioxidative effects of polyphenols would account for their beneﬁcial effects on improving meat quality and decreasing lipid peroxidation in chicken meat [176]. How- ever, some studies showed that the supplementation of polyphenol compounds did not inﬂuence the meat quality of broiler chickens [177,178]. Appropriate polyphenol sources (e.g., molecular weight) should be determined depending on the broilers’ raising conditions. 11 of 20 11 of 20 Animals 2023, 13, 1386 g chic Animals 2023, 13, 1386 11 of 20 2, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/animals chickens. Low-accumulating mycotoxins negatively affect the gut health and gut microbiota of Figure 2. Proposed modes of action of polyphenols depending on their molecular weight and ab- sorption rate in the gastrointestinal tract. Figure 2. Proposed modes of action of polyphenols depending on their molecular weight and absorption rate in the gastrointestinal tract. Table 6. Effects of the supplementation of plant polyphenolic compounds (e.g., plant extracts and essential oils) on the meat quality of broiler chickens under different raising conditions. Sources Doses in the Feed Bird Conditions/Replicates Observations in the Breast Meat References Polyphenols originated from onions (quercetin and ﬂavonols) and grape seeds (catechins, ﬂavonols, pro-cyanidins, and anthocyanidins). 3.7. Plant Polyphenolic Compounds (e.g., Plant Extracts and Essential Oils) 100 or 200 mg/kg Heat-stressed/8 Reduced natural drip loss and water-holding capacity Increased crude ash Increased monounsaturated fatty acids and reduced omega-6 polyunsaturated fatty acids [141] Fed rancid oils/10 Increased lightness Modulated fatty acid composition Increased crude fat and ash [179] Fed diets contaminated with ochratoxin A/10 Enhanced water-holding capacity Modulated fatty acid composition (C12:0) [87] Bamboo leaf extract 1 to 5 g/kg Normal condition/6 Improved antioxidant capacity and alleviated lipid peroxidation [176] Eucalyptus leaf polyphenol extract 500 mg/kg Normal condition/4 Improved antioxidant capacity and increased redness [180] Cinnamaldehyde 50 mg/kg Normal condition/8 Increased pH [181] A blend of essential oils: carvacrol, thymol, and cinnamaldehyde and curcumin 150 mg/kg Normal condition/3 Improved antioxidant capacity and reduced thiobarbituric acid reactive substances, a byproduct of lipid peroxidation Reduced thawing loss Increased yellowness Reduced short-chain fatty acids and increased monounsaturated fatty acids and polyunsaturated fatty acids [182] Thymol and carvacrol 150 mg/kg Normal condition/3 Reduced lipid peroxidation during storage [183] 2, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/animals Figure 2. Proposed modes of action of polyphenols depending on their molecular weight and ab- sorption rate in the gastrointestinal tract. Figure 2. Proposed modes of action of polyphenols depending on their molecular weight and absorption rate in the gastrointestinal tract. Table 6. Effects of the supplementation of plant polyphenolic compounds (e.g., plant extracts and essential oils) on the meat quality of broiler chickens under different raising conditions. Sources Doses in the Feed Bird Conditions/Replicates Observations in the Breast Meat References Polyphenols originated from onions (quercetin and ﬂavonols) and grape seeds (catechins, ﬂavonols, pro-cyanidins, and anthocyanidins). 3.8. Organic Acids Organic acids, considered to be alternatives for antibiotic growth promotors, are deﬁned as organic compounds with acidic properties [186]. Most organic acids contain a carboxylic group (-COOH) including SCFA (≤C6), such as formic, acetic, propionic, fumaric, lactic, etc., but there are medium-chain fatty acids (C7 to C12) and long-chain fatty acids (>C12) [187]. Not only can organic acids improve food safety by showing antimicrobial effects [188] but animals also readily use organic acids as energy sources, which can affect the meat quality of broilers [189]. Galli et al. [190] showed that the supplementation of organic acids (blends of formic, phosphoric, lactic, acetic, butyric, and propionic acids; 0.75 to 3 g/kg of feed) reduced yellowness and improved the antioxidant status in the breast meat of broiler chickens. Ma et al. [191] showed that the supplementation of blends of formic acid, ammonium formate, acetic acid, propionic acid, and lactic acid (3 to 6 g/kg of feed) increased the meat pH and increased the ratio of polyunsaturated to saturated fatty acids in the breast meat. The supplementation of organic acids has the potential to alter the meat quality of broiler chickens. 3.7. Plant Polyphenolic Compounds (e.g., Plant Extracts and Essential Oils) 100 or 200 mg/kg Heat-stressed/8 Reduced natural drip loss and water-holding capacity Increased crude ash Increased monounsaturated fatty acids and reduced omega-6 polyunsaturated fatty acids [141] Fed rancid oils/10 Increased lightness Modulated fatty acid composition Increased crude fat and ash [179] Fed diets contaminated with ochratoxin A/10 Enhanced water-holding capacity Modulated fatty acid composition (C12:0) [87] Bamboo leaf extract 1 to 5 g/kg Normal condition/6 Improved antioxidant capacity and alleviated lipid peroxidation [176] Eucalyptus leaf polyphenol extract 500 mg/kg Normal condition/4 Improved antioxidant capacity and increased redness [180] Cinnamaldehyde 50 mg/kg Normal condition/8 Increased pH [181] A blend of essential oils: carvacrol, thymol, and cinnamaldehyde and curcumin 150 mg/kg Normal condition/3 Improved antioxidant capacity and reduced thiobarbituric acid reactive substances, a byproduct of lipid peroxidation Reduced thawing loss Increased yellowness Reduced short-chain fatty acids and increased monounsaturated fatty acids and polyunsaturated fatty acids [182] Thymol and carvacrol 150 mg/kg Normal condition/3 Reduced lipid peroxidation during storage [183] posed modes of action of polyphenols depending on their molecular weight and ab- in the gastrointestinal tract. Figure 2. Proposed modes of action of polyphenols depending on their molecular weight and absorption rate in the gastrointestinal tract. Table 6. Effects of the supplementation of plant polyphenolic compounds (e.g., plant extracts and essential oils) on the meat quality of broiler chickens under different raising conditions. Animals 2023, 13, 1386 12 of 20 12 of 20 Plant polyphenol compounds are known to affect fat accumulation in broiler chickens. Park and Kim [184] reported that the supplementation of Achyranthes asper (0.025% to 0.1% in the feed) reduced abdominal fat and increased breast meat portions in broiler chickens, and this could be due to its component, saponin, which might reduce lipid absorption in the gastrointestinal tract. Huang et al. [185] demonstrated that the oral administration (50 or 100 mg/kg of body weight) of green tea polyphenols decreased fat accumulation via downregulating genes related to fat accumulation and upregulating genes related to fat metabolism and transportation. Our previous study showed that the supplementation of tannic acid (0.5 to 2.5 g/kg of feed) reduced fat accumulation in broiler chickens by reducing the production of SCFA (e.g., microbial metabolites; important energy sources for the host) [54]. Therefore, the supplementation of polyphenolic compounds can positively affect the meat quality and body composition of broiler chickens. Data Availability Statement: No new data was generated in this manuscript. 4. Conclusions Producing favorable-quality and lower-fat meat is important in broiler production to meet consumers’ desires and to be more competitive among other meat sources. However, a variety of different challenging conditions in poultry production have reduced meat yield and quality by reducing nutrient utilization and metabolism and inducing oxidative stress in broiler chickens. The supplementation of AA (e.g., arginine and BCAA) showed the potential to enhance the meat quality and body composition of broiler chickens by changing their metabolism. The supplementation of bioactive compounds, such as vitamins, probiotics, prebiotics, polyphenol compounds, and organic acids, improved the meat quality parameters, such as WHC, antioxidant capacity, and body composition, of broiler chickens. Potentially, plant polyphenolic compounds that have antioxidative, antimicrobial, and anti-inﬂammatory effects may be an effective nutritional strategy to improve meat quality and meat yield by improving nutrient utilization and reducing lipid peroxidation in broiler production. The development of nutritional strategies based on challenging conditions to meet market demands could play a key role in the improvement of the meat quality and composition of broiler chickens. Author Contributions: Conceptualization, J.C. and W.K.K.; writing—original draft preparation, J.C.; writing—review and editing, J.C., B.K., B.C.B., H.Z. and W.K.K.; supervision, W.K.K. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: No new data was generated in this manuscript. 13 of 20 13 of 20 Animals 2023, 13, 1386 Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Daniel, C.R.; Cross, A.J.; Koebnick, C.; Sinha, R. Trends in meat consumption in the USA. Public Health Nutr. 2011, 14, 575–583. 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https://openalex.org/W2166346004	https://www.pure.ed.ac.uk/ws/files/28382771/Cadar_et_al_2016_International_Journal_of_Geriatric_Psychiatry.pdf	English	null	The role of cognitive reserve on terminal decline: a cross‐cohort analysis from two European studies: OCTO‐Twin, Sweden, and Newcastle 85+, UK	International journal of geriatric psychiatry	2,015	cc-by	8,602	Digital Object Identifier (DOI): 10.1002/gps.4366 Digital Object Identifier (DOI): 10.1002/gps.4366 Link: Link to publication record in Edinburgh Research Explorer Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record Published In: International Journal of Geriatric Psychiatry Publisher Rights Statement: (c) 2015 The Authors. International Journal of Geriatric Psychiatry Published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited Publisher Rights Statement: (c) 2015 The Authors. International Journal of Geriatric Psychiatry Published by John Wiley & So This is an open access article under the terms of the Creative Commons Attribution License, whic distribution and reproduction in any medium, provided the original work is properly cited General rights g Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. The role of cognitive reserve on terminal decline A cross-cohort analysis from two European studies: OCTO-Twin, Sweden, and Newcastle 85+, UK A cross-cohort analysis from two European studies: OCTO-Twin, Sweden, and Newcastle 85+, UK Citation for published version: Cadar, D, Stephan, BCM, Jagger, C, Johansson, B, Hofer, SM, Piccinin, AM & Muniz-Terrera, G 2015, 'The role of cognitive reserve on terminal decline: A cross-cohort analysis from two European studies: OCTO- Twin, Sweden, and Newcastle 85+, UK', International Journal of Geriatric Psychiatry, vol. 31, no. 6, pp. 601- 610. https://doi.org/10.1002/gps.4366 Edinburgh Research Explorer Edinburgh Research Explorer The role of cognitive reserve on terminal decline: a cross- cohort analysis from two European studies: OCTO-Twin, Sweden, and Newcastle 85+, UK Dorina Cadar1, Blossom C. M. Stephan2, Carol Jagger2, Boo Johansson3, Scott M. Hofer and Graciela Muniz-Terrera1 1MRC Unit for Lifelong Health and Ageing at University College London, London, UK 2Institute of Health and Society, Newcastle University, Newcastle, UK 3Department of Psychology, University of Gothenburg, Gothenburg, Sweden 4Department of Psychology, University of Victoria, Victoria, Canada Correspondence to: D. Cadar, E-mail: d.cadar@ucl.ac.uk 1MRC Unit for Lifelong Health and Ageing at University College London, London, UK 2Institute of Health and Society, Newcastle University, Newcastle, UK 3Department of Psychology, University of Gothenburg, Gothenburg, Sweden 4Department of Psychology, University of Victoria, Victoria, Canada Correspondence to: D. Cadar, E-mail: d.cadar@ucl.ac.uk 1MRC Unit for Lifelong Health and Ageing at University College London, Lon 2Institute of Health and Society, Newcastle University, Newcastle, UK 3Department of Psychology, University of Gothenburg, Gothenburg, Sweden 4Department of Psychology, University of Victoria, Victoria, Canada Correspondence to: D. Cadar, E-mail: d.cadar@ucl.ac.uk Objective: Cognitive performance shows a marked deterioration in close proximity to death, as postu- lated by the terminal decline hypothesis. The effect of education on the rate of terminal decline in the oldest people (i.e. persons 85+ years) has been controversial and not entirely understood. In the current study, we investigated the rate of decline prior to death with a special focus on the role of education and socioeconomic position, in two European longitudinal studies of ageing: the Origins of Variance in the Old-Old: Octogenarian Twins (OCTO-Twin) and the Newcastle 85+ study. Methods: A process-based approach was used in which individuals’ cognitive scores were aligned according to distance to death. In a coordinated analysis, multilevel models were employed to examine associations between different markers of cognitive reserve (education and socioeconomic position) and terminal decline using the mini-mental state examination (MMSE), controlling for age at baseline, sex, dementia incidence and time to death from the study entry to the time of death within each cohort. Results: The current ﬁndings suggest that education was positively associated with higher MMSE scores prior to death in the OCTO-Twin, but not in the Newcastle 85+ study, independent of socioeconomic position and other factors such as baseline age, sex and time to death from the study entry. However, education was not associated with the rate of terminal decline in both of these studies. Conclusions: Our results offer only partial support to the cognitive reserve hypothesis and cognitive performance prior to death. The role of cognitive reserve on terminal decline: a cross- cohort analysis from two European studies: OCTO-Twin, Sweden, and Newcastle 85+, UK # 2015 The Authors International Journal of Geriatric Psychiatry Published by John Wiley & Sons Ltd. Key words: cognition; death; dementia; MMSE; education; terminal decline; cognitive reserve; longitudinal study; mixed multilevel History: Received 17 January 2015 Accepted 10 September 2015; Published online 15 October 2015 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/gps.4366 # 2015 The Authors. International Journal of Geriatric Psychiatry Published by John Wiley & Sons Ltd. Int J Geriatr Psychiatry 2016; 31: 601–610 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduc- tion in any medium, provided the original work is properly cited. Take down policy Th U i i f Ed p y 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 RESEARCH ARTICLE RESEARCH ARTICLE Study population OCTO-Twin, Sweden Study population OCTO-Twin, Sweden Study population OCTO-Twin, Sweden The ﬁrst sample used in these analyses was from the comprehensive longitudinal Origins of Variance in the Old-Old: Octogenarian Twins (known as the OCTO-Twin study) based on the oldest cohort of the Swedish Twin Registry. The sample includes 702 participants, with 351 complete twin pairs born in 1913 and earlier, who were, or became, 80years of age during the ﬁrst wave of data collection (1991– 1993). Participants have been assessed on ﬁve occa- sions at two-year intervals, for a total of up to eight years of follow-up. The average rate of attrition from one testing wave to the next was 20% (10% per year) and was primarily because of death (Cederlof and Lorich, 1978, McClearn et al., 1997, Pedersen et al., 2002). g Often used as a proxy for cognitive reserve, educa- tion has been shown to be protective against faster rates of cognitive decline from middle to later life, in healthy individuals (Richards and Sacker, 2003, Stern, 2002), and related to both a delayed onset of dementia (Stern, 2009, Stern et al., 1999) and a delayed onset of terminal decline (Muniz-Terrera et al., 2014). How- ever, from a life-course perspective, neuropathological burden could be considered as either a potential medi- ating factor between intelligence and mortality or a prior cause, if a suboptimal neurodevelopment was the antecedent for both the neurological condition and early mortality (Batty et al., 2007). In light of this possibility, the curvilinear age trends for cognitive function in late life could actually be an artefact of ac- cumulation over time, in individuals with different neuropathological burden and cognitive symptom- atology. The aggregate function could be inﬂuenced by the increasing risk of terminal decline and its cur- vature, reﬂecting an average terminal decline within persons who are still cognitively stable (Singer et al., 2003). As a result, it is less clear, whether education, or other markers of cognitive reserve such as occupa- tion, could moderate terminal decline in the oldest people, because the nature of this decline may or may not follow the assumptions of ﬂexible and efﬁ- cient use of pre-existing cognitive reserve (Stern, 2002). In addition, it is unclear if cognitive reserve will differentially inﬂuence the rates of decline prior to death for individuals who remain non-demented and those diagnosed with dementia. Introduction from a preterminal phase of relative stability into more rapid decline that ends with death (Kleemeier, 1962, Riegel and Riegel, 1972). However, this theoretical con- ceptualisation lacks speciﬁcity distinguishing those experiencing serious neurobiological compromise such as dementia from those who remain free of dementia before death. A better understanding of this differentia- tion could carry distinct theoretical and clinical implica- tion (e.g. Anstey et al., 2006, Laukka et al., 2006). Signiﬁcant evidence suggests that cognitive perfor- mance shows a marked deterioration in close proximity of death, known as ‘terminal decline’ (e.g. Sliwinski et al., 2006, Wilson et al., 2007, Thorvaldsson et al., 2008, MacDonald et al., 2011, Muniz-Terrera et al., 2011, Piccinin et al., 2011, Muniz-Terrera et al., 2013). This emphasises a within-person process of change D. Cadar et al. 602 From many key modiﬁable risk factors, education received substantial interest in the ﬁeld of dementia research, but its predictability of terminal decline is not entirely understood. The association between edu- cation and terminal decline remains highly controver- sial. For example, there are some evidence suggesting that education may modify the association between levels of cognitive function in proximity to death and Alzheimer’s pathology assessed post-mortem (Bennett et al., 2003), while other studies support the associa- tion between education and level of cognitive perfor- mance, but not the association with the rate of cognitive decline prior to death (Johansson et al., 2004, Laukka et al., 2006, MacDonald et al., 2006, Piccinin et al., 2011). Furthermore, education appears to delay the onset of terminal decline in the oldest people (Muniz-Terrera et al., 2014) and to impact dif- ferently the rate of terminal decline according to vari- ous cognitive domains (Batterham et al., 2011). (SEP) on these trajectories, using a coordinated analy- sis of two European population-based samples of the oldest people from Sweden and the UK. In addition, we explored these trajectories among healthy individ- uals and those who developed dementia during the study period. In this respect, this cross-cohort investi- gation represents a unique evaluation of consistency in patterns of associations for education and SEP as sug- gested markers of cognitive reserve. We analysed per- formance prior to death and the rate of change from study entry to time of death, reducing potential sources of heterogeneity that could emerge from dif- ferences in the analytical methodologies employed. Methods Study population OCTO-Twin, Sweden Cognitive assessments In both studies, global cognitive function was assessed with the MMSE (Folstein et al., 1975), one of the most commonly used screening instruments for cognitive impairment. The test measures various domains in- cluding the following: orientation, registration (imme- diate memory), short-term memory and attention, the ability to follow verbal and written commands, writing and copying. The MMSE scores range from 0 to 30, with high values indicating healthier cognitive status. The MMSE was administered at each of the ﬁve waves in OCTO-Twin and only at three waves in Newcastle 85+ (baseline, phase 3 and phase 4 at approximately 18-month intervals). y A series of mixed models were ﬁtted to MMSE to estimate trajectories of decline in global cognitive per- formance prior to the time of death, by employing regular mixed models and Tobit models indepen- dently within each study. Tobit models are often used to model trajectories of outcome variables that have ﬂoor or ceiling effects, such as MMSE. Bayesian Infor- mation Criteria (BIC), an index that combines model parsimony and goodness of ﬁt (Raftery 1986), was used for model selection, and based on this we selected to report the results from the Tobit models. Study population Newcastle 85+, UK The second sample was drawn from the Newcastle 85+ study; a longitudinal population-based study of health and ageing. The sampling frame comprised all people born in 1921, recruited at age 85 during 2006–2007 (phase 1) when recruitment commenced and was broadly representative of 85years old in En- gland and Wales in terms of gender, residence in a care home and living alone. Participants have been re-tested on three occasions (phases 2, 3 and 4) at approximately 18-month intervals; however, the cog- nitive data were not available at phase 2, because of time restrictions. All assessments were conducted by a trained research nurse in the participants’ resi- dence (for full details see Collerton et al. (2007) and Collerton et al. (2009)). Most of post-baseline The aim of our study was to examine rates of termi- nal decline in mini-mental state examination (MMSE) and the role of education and socio-economic position nal Journal of Geriatric Psychiatry Published by John Wiley & Sons Ltd. Int J Geriatr Psychiatry 2016; 31: 601–610 603 A cross-cohort analysis on education and terminal decline attrition was because of death and participants’ with- drawal (Davies et al., 2014). within each study and classiﬁed into three occupational categories (low, medium and high SEP). For more in- formation see Supporting Information. These categories represent general and well-differentiated employment occupations in modern societies (Chandola, 2000). Ethics p Other covariates included sex, baseline age, incident dementia and the distance to death from the study entry. The OCTO-Twin study received approval from the Ethics Committee at the Karolinska Institute in Stock- holm and from the Swedish Data Inspection Authority in Sweden. The Newcastle 85+ study received ethical approval from the North Tyneside 1 Research Ethics Committee in the UK. Informed written consent was obtained from all participants in each study, or their carer, where capacity to consent was questionable, in case of severe cognitive impairment or dementia. Dementia diagnosis In both studies, dementia was diagnosed by consensus according to the revised third edition of the Diagnostic and Statistical Manual of Mental Disorders. In OCTO- Twin, dementia prevalence was identiﬁed by a multi- disciplinary team consisting of a physician, a research nurse and two neuropsychologists, who reviewed the cognitive test results and medical records at each new wave (van den Kommer et al., 2009). In the New- castle 85+, dementia prevalence was determined at each wave, based on a review of data from general practice records alone (Collerton et al., 2009). All models used maximum likelihood estimation. Missing observations were assumed to be missing at random (Little and Rubin, 2002), and model assump- tions were veriﬁed by examining residuals computed from the predicted values. For more information see Supporting Information. Cognitive change was modelled as a linear function of time to death in both datasets and as a quadratic function in the OCTO-Twin study given the larger number of measurements available in the Swedish study. The linear slopes represent annual rate of change (i.e. increase and decrease) per year closer to death and when a quadratic model is ﬁtted (in OCTO-Twin Data analyses and analytic approach We undertook a coordinated analysis across the two cohort studies, employing the same statistical method- ology within each study, to allow comparisons be- tween the results and to examine consistency of patterns in terminal decline in these studies. The ana- lytical sub-samples included only data from individ- uals who died during the study and who were free of dementia at study entry. By excluding dementia cases at the study entry, we examined and compared the rates of terminal decline between those who developed dementia during the study period and those who remained free of dementia over the study period within each study. We derived an indicator variable (0=no dementia, 1=diagnosed with dementia during study follow up time) to account for individuals with dementia, and we obtained distinct estimates within each study. nal Journal of Geriatric Psychiatry Published by John Wiley & Sons Ltd. Int J Geriatr Psychiatry 2016; 31: 601–610 # 2015 The Authors. International Journal of Geriatric Psychiatry Published by John Wiley & Sons Ltd. Int J Geriatr Psychiatry 2016; 31: 601–610 Covariates Educational attainment was recorded in each cohort as a continuous measure, deﬁned as the number of years of education. Information related to SEP were ob- tained during the home interview or via questionnaires D. Cadar et al. 604 who remained free of dementia during the study period, in OCTO-Twin, ranged from 0 to 23 years at baseline and from 0 to 18 years at the last follow-up (wave 5). In contrast, for the incident de- mentia cases in this study, education ranged from 2 to 17years at baseline and from to 2 to 13 years at the last follow-up. In the Newcastle 85+ study, ed- ucation ranged from 6 to 20 years at baseline and from 8 to 17years at the last wave for non-cases. For those with dementia in this study, education ranged from 8 to 17 years at baseline and from to 9 to 12 years at the last follow-up. study), the linear term represents rate of change at the intercept and the quadratic term represents change (i.e. increase or acceleration and decrease or deceleration) in rate of change. g Level and rate of change were adjusted for demo- graphic characteristics including sex (male partici- pants used as reference category), years of education (centred at the mean value of 7 (OCTO-Twin) or 10 (Newcastle 85+) years), age at study entry (centred at 83 (OCTO-Twin) or 85 (Newcastle 85+) years) and time to death from the study entry (centred at 6years to death (OCTO-Twin) or 2.6years to death (Newcastle 85+)). Given this model speciﬁca- tion, the intercept represents the level of cognitive per- formance at this centred distance from death, for an individual with values of zero on all covariates in each study investigated. Estimates and standard errors for the MMSE inter- cept and slope across the two cohorts are shown in Table 3. Longitudinal changes in MMSE scores were modelled as a linear function in both studies and also as a quadratic function in the OCTO-Twin study. We used the lowest BIC scores to select the best model ﬁt (BIC regular model=10121.96 and BIC Tobit model=9442.56 in OCTO-Twin, BIC regular model=2885.10 and BIC Tobit model=2727.63 in Newcastle 85+ study); therefore, we reported the re- sults from the Tobit models. Covariates y g Although the age range at the study entry of the in- dividuals in each study is relatively narrow (mean age=83.5, SD=3.0 in the OCTO-Twin study and mean age=85.4, SD=0.44 in the Newcastle 85+), those who joined the study at the younger age are ex- pected to perform differently than those who joined the study at older age. Similarly, the performance of individuals who entered the study at a closer time to death was expected to differ from those who entered the study at a time further from death. To account for these differences (between-person differences in age at study entry and distance to death), we adjusted our models for distance to death and age at study entry (Piccinin et al., 2011). The results of these analyses suggest that the refer- ence OCTO-Twin individual, a Swedish man who en- tered the study aged 83, had an average of 7years of education and was 6years from death, showed an an- nual decline of 1.60 (SE=0.21) MMSE points per year. The decline accelerated by 0.12 (SE=0.02) points per year closer to the actual time of death. In contrast, the reference Newcastle 85+ partici- pant, a British man of 85years, with an average of 10years of education, and entering the study at around 2.6years before death, had a linear rate of decline of 1.08 (SE=0.23) MMSE points per year. Data analyses were conducted using the following statistical programmes: Mplus (version 6.11), Com- puter Software Los Angeles, CA (Muthén and Muthén, 2007) for the Regular Mixed and Tobit regression models (see the Mplus script as Supporting Informa- tion); STATA (StataCorp, 2013) Stata Statistical Soft- ware: Release 13. College Station, Texas: StataCorp LP for descriptive analyses and R Studio (R Develop- ment Core Team, 2010) for ﬁgures. Figure 1 illustrates the MMSE trajectories plotted against time to death for non-cases and dementia cases in each of the two studies for different values of distance to death and education. Additional model estimated mean curves are plotted for the upper and lower bands of 3years education. In both studies, those who developed dementia during the study period had signiﬁcantly lower MMSE scores prior to the time of death compared with those who remained dementia free, with approximately 12.14 (SE=0.90) and 10.68 (SE=1.48) points on MMSE in OCTO-Twin and Newcastle 85+ respec- tively. Covariates Incident dementia cases also experienced a steeper rate of decline with 2.31 (SE=0.25) in OCTO-Twin and 1.97 (SE=0.28) units per year in Newcastle 85+. The acceleration in the rate of terminal decline in OCTO-Twin study was estimated at 0.14 (SE=0.02) units per year closer to death. Results Demographics and cognitive scores at baseline and each follow-up wave for the two studies (OCTO-Twin and Newcastle 85+) are presented in Tables 1 and 2 respectively. Participants diagnosed with dementia at baseline (98 in OCTO-Twin; 66 in Newcastle 85+) were excluded from the current analyses. y The majority of participants in each study were women (64% in OCTO-Twin; 56% in Newcastle 85+). The number of years of education for participants nal Journal of Geriatric Psychiatry Published by John Wiley & Sons Ltd. Int J Geriatr Psychiatry 2016; 31: 601–610 605 y p p OCTO-Twin study waves of testing Wave 1 Wave 2 Wave 3 Wave 4 Wave 5 1991–1993 1993–1995 1995–1997 1997–1999 1999–2001 Study samplea 556 457 351 256 174 Cumulative dementia cases 98b 42 80 100 126 Age (Mean/SD) 83.5 (3.0) 85.4 (3.0) 87.2 (2.7) 89.1 (2.8) 90.9 (2.4) Female (%) 63.5% 62.8% 63.8% 68.7% 72.4% MMSE included sample (mean/SD) 26.4 (4.0) 25.3 (5.4) 24.4 (6.0) 23.1 (7.7) 21.6 (7.7) MMSE non-dementia cases (mean/SD) 26.7 (3.9) 26.4 (4.4) 26.7 (4.2) 25.7 (5.6) 23.9 (5.7) MMSE dementia cases (Mean/SD) 25.2 (3.8) 22.2 (6.7) 18.6 (8.5) 16.9 (8.4) 13.7 (8.3) Education range 0–23 years 0–20 years 0–20 years 0–18 years 0–18 years (mean/SD)-total included sample 7.2 (2.3) 7.2 (2.3) 7.2 (2.3) 7.1 (2.1) 7.1 (2.2) Education range 0–23 years 0–20 years 0–20 years 0–18 years 0–18 years (Mean/SD) non-dementia cases 7.2 (2.3) 7.3 (2.4) 7.3 (2.4) 7.2 (2.2) 7.1 (2.2) Education range 2–17 years 2–17 yrs 2–13 years 2–13 years 2–13 years (Mean/SD) dementia cases 7.0 (1.8) 7.0 (1.8) 6.8 (1.6) 6.8 (1.7) 6.7 (1.8) Average time to death (years) from each wave (mean/SD) 6.0 (3.8) 4.8 (3.4) 3.9 (2.8) 3.1 (2.2) 2.1 (1.7) aSample of individuals who died during the study period in OCTO-Twin, representing those included in the current analyses. bDementia cases at baseline (N = 98 in OCTO-Twin study) have been excluded from these analyses. aSample of individuals who died during the study period in OCTO-Twin, representing those included in the current analyses. bDementia cases at baseline (N = 98 in OCTO-Twin study) have been excluded from these analyses. Results Table 2 Characteristics of study participants in the Newcastle 85+ Table 2 Characteristics of study participants in the Newcastle 85+ Newcastle 85+ study waves of testing Wave 1 Wave 3a Wave 4 2006–2007 2009–2010 2011–2012 Study sampleb 845 485 344 Cumulative dementia cases 66c 116 139 Age (Mean/SD) 85.4 (0.44) 88.4 (0.39) 90.5 (0.40) Female (%) 55.6% 55.6% 53.7% MMSE included sample (Mean/SD) 25.8 (5.3) 25.4 (5.4) 24.8 (6.3) MMSE non-dementia cases (mean/SD) 26.0 (4.6) 26.3 (4.2) 26.9 (3.7) MMSE dementia cases (mean/SD) 15.3 (8.5) 15.6 (8.2) 13.1 (9.0) Education range 6–20 years 7–19 years 9–15 years (Mean/SD) total included sample 9.9 (1.8) 10.1 (1.9) 10.3 (1.6) Education range 6–20 years 7–19 years 9–15 years (Mean/SD) non-dementia cases 9.9 (1.8) 10.2 (2.1) 10.4 (2.0) Education range 8–17 years 8–17 years 9–12 years (Mean/SD) dementia cases 9.9 (1.6) 9.8 (1.6) 10.3 (1.2) Average time to death (years) from each wave (mean/SD) 2.6 (1.5) 1.4 (0.7) 0.3 (0.2) aMMSE was not administered at wave 2, so wave 3 was considered as time 2 and wave 4 was considered as time 3 in these analyses. bSample of individuals who died during the study period in Newcastle 85+, representing those included in the current analyses. cDementia cases at baseline (N = 66 in Newcastle 85+ study) have been excluded from these analyses. aMMSE was not administered at wave 2, so wave 3 was considered as time 2 and wave 4 was considered as time 3 in these analyses. bSample of individuals who died during the study period in Newcastle 85+, representing those included in the current analyses. cDementia cases at baseline (N = 66 in Newcastle 85+ study) have been excluded from these analyses. Higher levels of SEP (medium and high) were not as- sociated with either the level of cognitive performance prior to death or with the rates of terminal decline com- pared with the low categories in these analyses. Although, education was positively associated with the estimated level of MMSE scores before death in the OCTO-Twin study (0.44 points per year of educa- tion, SE=0.14), it was not found to have an associa- tion with the level of performance prior to death in the Newcastle 85+ study. Furthermore, education did not appear to have a signiﬁcant impact on the rate of change in MMSE in either of the two cohorts investigated. Results Age at baseline was negatively associated with MMSE scores prior to the time of death in OCTO- Twin, but not in Newcastle 85+. For each year of age higher than the overall baseline age of 83years in OCTO-Twin, the MMSE score was estimated to be nal Journal of Geriatric Psychiatry Published by John Wiley & Sons Ltd. Int J Geriatr Psychiatry 2016; 31: 601–610 The Authors. International Journal of Geriatric Psychiatry Published by John Wiley & Sons Ltd. Int J Geriatr Psychia 606 D. Cadar et al. study MMSE in OCTO-Twin study MMSE in Newcastle 85+ study Coef. SE p-value Coef. Results SE p-value Fixed effects N = 552 N = 352a Intercept (Level of performance on MMSE prior to death) 22.44 0.68 <0.001 24.60 0.62 <0.001 Years to death from baseline 0.48 0.09 <0.001 0.10 0.23 0.67 Baseline age 0.49 0.10 <0.001 0.13 0.83 0.88 Education 0.44 0.14 0.009 0.19 0.23 0.44 Socio-economic position Medium 0.60 0.78 0.73 0.80 1.15 0.49 High 0.88 1.09 0.44 1.43 0.92 0.12 Female 0.85 0.16 0.41 0.48 0.79 0.55 Dementia cases 12.13 0.90 <0.001 10.68 1.48 <0.001 Linear slope (rate of decline) 1.60 0.21 <0.001 1.08 0.23 <0.001 Years to death from baseline 0.02 0.05 0.61 0.26 0.09 0.005 Baseline age 0.05 0.03 0.10 0.01 0.25 0.98 Education 0.03 0.05 0.61 0.01 0.05 0.86 Socioeconomic position Medium 0.01 0.21 0.78 0.21 0.25 0.40 High 0.04 0.24 0.98 0.20 0.25 0.42 Female 0.14 0.22 0.51 0.10 0.22 0.67 Dementia cases 2.31 0.25 <0.001 1.97 0.28 <0.001 Quadratic slope (acceleration) 0.12 0.02 <0.001 n/a n/a n/a Years to death from baseline 0.01 0.00 <0.001 — — — Baseline age 0.00 0.00 0.35 — — — Education 0.00 0.01 0.90 — — — Socioeconomic position Medium 0.00 0.01 0.90 — — — High 0.01 0.02 0.98 — — — Female 0.00 0.01 0.99 — — — Dementia cases 0.14 0.02 <0.001 — — — Random effects (variances) — — 95% CI — — 95% CI Level of performance 0.49 0.03 0.42–0.57 0.71 0.12 0.70–1.21 Linear rate of decline 5.53 0.23 5.09–5.92 5.03 0.39 5.70–7.23 Acceleration 0.94 0.02 0.89–0.96 — — — Residual 2.90 0.07 2.77–3.03 2.30 0.22 1.91–2.78 aThe samples included in these analyses represent the participants in each study with mortality data available during the study period, minimum 2 waves of MMSE and complete data on covariates, after excluding participants with dementia at baseline—reason for the reduction in the number of participants included relative to the full samples. aThe samples included in these analyses represent the participants in each study with mortality data available during the study period, minimum 2 waves of MMSE and complete data on covariates, after excluding participants with dementia at baseline—reason for the reduction in the number of participants included relative to the full samples. Discussion In the current study, we examined terminal change in MMSE, and the role of education and SEP on these trajectories, using a coordinated analytical approach in two European population-based samples of the oldest people from Sweden and the UK. The current investigation addressed how people differ in their ﬁnal years before death, presenting separate estimates for incident dementia and healthy individuals. ( ) In terms of cognitive instrument, MMSE is a screen- ing measure that can be used to systematically assess mental status and identify cognitive impairment, rather than a detailed and thorough measure of global cognition. However, MMSE relies mainly on verbal items (Xu et al., 2002) and lacks a detailed assessment of visuo-constructive abilities and executive functions (Kirby et al., 2001). Consequently, decline in the MMSE could represent a sensitivity threshold for the verbal task component in the presence of neurodegen- eration but could be particularly insensitive to frontal lobe, executive and social function disorders (Hodges, 2011). The maximum score of 30 should be attainable by anyone of average intelligence. However, many pa- tients with early dementia onset could score high on the MMSE, especially if they are younger, have higher education and advanced prior cognitive ability, resulting in a sensitivity reduction of the MMSE scores. y In OCTO-Twin, where it was possible to investigate polynomial change, the analyses revealed accelerated change prior to death. In Newcastle 85+, which had a shorter period of follow-up, we found a considerable linear decline. Education was signiﬁcantly associated with level of performance 6years before death in one of the two cohorts (OCTO-Twin), independent of the SEP and other common factors such as age at baseline, sex and time to death at the study entry, but was not associated with the rate of cognitive decline in either of these studies. Furthermore, SEP was not signiﬁcantly associated with either the level of performance on global cognition prior to death or with the rate of decline within each study. The current results are consistent with those reported by Wilson, describing terminal decline as a process which starts initially with a slower rate of change, followed by a more pronounced acceleration prior towards death (Wilson et al., 2007). Results aThe samples included in these analyses represent the participants in each study with mortality data available during the study period, minimum 2 waves of MMSE and complete data on covariates, after excluding participants with dementia at baseline—reason for the reduction in the number of participants included relative to the full samples. Figure 1 Model estimated mean curves for non-cases (blue lines) and incident dementia cases (red lines) for different values of education with the ad- ditional upper and lower bands of 3 years education (OCTO-Twin study, left panel and Newcastle 85+, right panel). Figure 1 Model estimated mean curves for non-cases (blue lines) and incident dementia cases (red lines) for different values of education with the ad- ditional upper and lower bands of 3 years education (OCTO-Twin study, left panel and Newcastle 85+, right panel). lower by 0.49 (SE=0.10) than the estimated mean score of 24.60 (SE=0.62). Baseline age was not associ- ated with rate of decline in MMSE in either of these lower by 0.49 (SE=0.10) than the estimated mean score of 24.60 (SE=0.62). Baseline age was not associ- ated with rate of decline in MMSE in either of these studies. Sex was not associated with either the inter- cept or the rate of decline in the MMSE in the current examination. studies. Sex was not associated with either the inter- cept or the rate of decline in the MMSE in the current examination. ational Journal of Geriatric Psychiatry Published by John Wiley & Sons Ltd. Int J Geriatr Psychiatry 2016; 31: 601–61 y & Sons Ltd. Int J Geriatr Psychiatry 2016; 31: 601–610 A cross-cohort analysis on education and terminal decline 607 maintain stable or only slightly decline, with a more severe deterioration indicating underlying neurobio- logical compromise. Periods characterised by slow- but-steady cognitive decline may be linked to other abrupt preterminal causes of death (e.g. neoplasms and stroke), which are also recognised to weaken cognitive function and exacerbate cognitive decline (Haj-Hassan et al., 2014, Lange et al., 2014). These more likely reﬂect the relatively acute nature of vascu- lar precursors or more speciﬁc biological conditions (e.g. organ failure, cancer, dementia, diabetes and respiratory conditions) associated with abrupt effects on cognition and mortality (Hassing et al., 2002, Anstey et al., 2006). Results Moreover, age is a crucial factor that has been shown to modify the association between the distinctive metabolic risk factors and cog- nitive decline (Siervo et al., 2014). Sensitivity analyses examining the rate of terminal decline within a follow-up period of only three waves in OCTO-Twin (to assure similarity to the Newcastle 85+study) revealed a similar rate of linear change as in Newcastle 85+ (see Supporting Information). The pattern of associations with education, SEP and other covariates remained the same. ational Journal of Geriatric Psychiatry Published by John Wiley & Sons Ltd. Int J Geriatr Psychiatry 2016; 31: 601–61 # 2015 The Authors. International Journal of Geriatric Psychiatry Published by John Wiley & Sons Ltd. Int J Geriatr Psychiatry 2016; 31: 601–610 # 2015 The Authors. International Journal of Geriatric Psychiatry Published by John Wiley & Sons Ltd. Int J G Discussion However, the differences across two studies analysed here, in terms of associations between education and level of cognitive performance, as an individual approaches the time to death, re- emphasise the inconsistency of ﬁndings in the literature (Muniz-Terrera et al., 2011). In the OCTO-Twin, higher education was predictive of higher global cognitive performance (almost half an MMSE point per year of ed- ucation) at six years from death, while in the Newcastle 85+, education was not a signiﬁcant predictor. To sum up, the process of terminal decline is a highly complex phenomenon for which we need to take into consideration multiple underlying and inter- vening factors. The current analyses highlighted an accelerated change in MMSE prior to death in one of the two cohorts investigated (OCTO-Twin), where the follow-up period was relatively longer, and a linear decline in the Newcastle 85+ study. More years of education were associated with higher MMSE scores prior to death, but not an attenuation of the rate of change, as reported by other studies. For example, Laukka and colleagues reported that those with more education experienced slower decline in test of Block Design in the proximity of death (Laukka et al., 2006), whilst others who investigated change in MMSE, information, block design and other cognitive domains reported an association of education with performance but not consistent associations with rate The various time metrics used in previous studies (e.g. chronological age, time in study or distance to death) are likely to contribute to the difference in ﬁndings related to the acceleration in cognitive decline prior to death. Another plausible mechanism is that in the absence of neuropathogenic cognitive decline, most people D. Cadar et al. 608 of decline (Muniz-Terrera et al., 2009) (Batterham et al., 2011) (Piccinin et al., 2011). of decline (Muniz-Terrera et al., 2009) (Batterham et al., 2011) (Piccinin et al., 2011). of decline (Muniz-Terrera et al., 2009) (Batterham et al., 2011) (Piccinin et al., 2011). in the oldest people, relying too much on verbal tasks and memory and not enough on other cognitive func- tions (e.g. speed and executive function), which are known to demonstrate age-related change. Further- more, we need to consider the probability of ‘healthy survival’ in longitudinal studies of oldest people and the substantial dropouts in population samples with older people. However, the methodology employed (random effects models) compensated for the missing data aspect. Conclusions Our results suggest that decline and acceleration of this decline were observable in these studies prior to death. Education was positively associated with MMSE scores in proximity to death, independent of other markers of cognitive reserve such as SEP, in one of the cohorts investigated, but did not appear to be pro- tective against faster rates of terminal decline in the oldest people. As such, our coordinated approach analysis revealed no consistent protection for the cog- nitive reserve hypothesis (as applied to MMSE), which stipulates a protective role for those with higher edu- cation in terms of slower terminal decline. g y To our knowledge, this is one of the few analyses of terminal decline in the oldest people population, where we were able to separately estimate the expected trajec- tory of those free of dementia from those diagnosed with dementia during the study period. The results highlight how cognitive decline differs across studies prior to death in two different countries from Europe and that educa- tion does not appear to have a neuroprotective role against faster rates of terminal decline, despite a good representation of education range in both cohorts inves- tigated. Lastly, this cross-cohort investigation provides valuable information regarding the rates of terminal de- cline in healthy individuals and in those who developed dementia during the study period in two different oldest people non-institutionalised populations. • Education protects cognitive performance prior to death, independent of socio-economic position, baseline age and sex in a twin population from Sweden, but not in an older population from the UK. • Education protects cognitive performance prior to death, independent of socio-economic position, baseline age and sex in a twin population from Sweden, but not in an older population from the UK. We are also aware of some limitations. The termi- nal decline phenomenon refers usually to an increase in rate of cognitive decline prior to death in individ- uals free of dementia. However, the British cohort had a shorter period of follow-up compared with the Swedish cohort, and therefore, we were not able to eval- uate whether a change in the rate of decline occurred in both studies. In addition, MMSE is a relatively crude instrument for detecting changes in cognitive function • Education was not moderating the terminal drop, offering only partial support to cognitive reserve hypothesis. Strengths and limitations A key strength of our study is the use of a coordinated analysis approach, which enables a comparison of results across cohorts. We addressed the same research question, using data from the same cognitive test ad- ministered in comparable population-based samples of 80+years at baseline, with the same methodological analysis plan, including a similar choice of covariates. Naturally, this type of coordinated analysis approach can provide a far-reaching foundation by facilitating efﬁcient analysis of cross-cohort analyses that could help maximise the comparability of results and permit evaluation of study differences (Hofer and Piccinin, 2009, Piccinin and Hofer, 2008). As a novel feature of this investigation, we examined cognitive change within both cohorts in relation to number of years to death, a potentially more sensitive and process-based parameterisation of time in this age-group compared with time in study or chronological age which are usually assessed (Sliwinski and Mogle, 2008). However, when moving to a different time metric, the inclusion of time to death from study entry and the baseline age play a similar role by helping to represent selection effects re- lated to age and mortality (Piccinin et al., 2011). Further investigations with more measurement occasions and longer follow-ups are necessary to bet- ter understand the transition from the subtle cognitive changes accompanying age to those of neurological substance. We also need more information about the underlying neurobiological mechanisms that tend to produce accelerated cognitive change prior to death. Discussion We also faced differences between the two investigated cohorts. The time period examined for the terminal decline was slightly different, with the Swedish cohort having a longer study period to describe an accelerated rate of change, in contrast to the relative shorter period of follow-up in the British cohort. # 2015 The Authors. International Journal of Geriatric Psychiatry Published by John Wiley & Sons Ltd. Int J G Key points References Journal of Clinical and Experimental Neuropsychology 25(5): 614–624 available from: PM:12815499. Collerton J, Barrass K, Bond J, et al. 2007. 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https://openalex.org/W2765594214	https://www.pure.ed.ac.uk/ws/files/48650458/riskbased_strategies_for_surveillance_of_tuberculosis_infection_in_cattle_for_lowrisk_areas_in_england_and_scotland.pdf	English	null	Risk-based strategies for surveillance of tuberculosis infection in cattle for low-risk areas in England and Scotland	Epidemiology and infection	2,017	cc-by	8,954	Edinburgh Research Explorer General rights C i h f h General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Digital Object Identifier (DOI): 10.1017/S0950268817001935 Link: Link to publication record in Edinburgh Research Explorer Risk‐based strategies for surveillance of bovine Tuberculosis infection in cattle for low risk areas in England and Scotland Citation for published version: Salvador, LCM, Deason, M, Enright, J, Bessell, P & Kao, R 2018, 'Risk‐based strategies for surveillance of bovine Tuberculosis infection in cattle for low risk areas in England and Scotland', Epidemiology & Infection, vol. 146, no. 1, pp. 107-118. https://doi.org/10.1017/S0950268817001935 Citation for published version: Salvador, LCM, Deason, M, Enright, J, Bessell, P & Kao, R 2018, 'Risk‐based strategies for surveillance of bovine Tuberculosis infection in cattle for low risk areas in England and Scotland', Epidemiology & Infection, vol. 146, no. 1, pp. 107-118. https://doi.org/10.1017/S0950268817001935 Citation for published version: Salvador, LCM, Deason, M, Enright, J, Bessell, P & Kao, R 2018, 'Risk‐based strategies for surveillance of bovine Tuberculosis infection in cattle for low risk areas in England and Scotland', Epidemiology & Infection, vol. 146, no. 1, pp. 107-118. https://doi.org/10.1017/S0950268817001935 Digital Object Identifier (DOI): 10.1017/S0950268817001935 Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record Published In: Epidemiology & Infection 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 Epidemiol. Infect., Page 1 of 12. © Cambridge University Press 2017 doi:10.1017/S0950268817001935 SUMMARY Disease surveillance can be made more effective by either improving disease detection, providing cost savings, or doing both. Currently, cattle herds in low-risk areas (LRAs) for bovine tuberculosis (bTB) in England are tested once every 4 years. In Scotland, the default herd testing frequency is also 4 years, but a risk-based system exempts some herds from testing altogether. To extend this approach to other areas, a bespoke understanding of at-risk herds and how risk-based surveillance can affect bTB detection is required. Here, we use a generalized linear mixed model to inform a Bayesian probabilistic model of freedom from infection and explore risk-based surveillance strategies in LRAs and Scotland. Our analyses show that in both areas the primary herd-level risk factors for bTB infection are the size of the herd and purchasing cattle from high- risk areas of Great Britain and/or Ireland. A risk-based approach can improve the current surveillance system by both increasing detection (9% and 7% fewer latent infections), and reducing testing burden (6% and 26% fewer animal tests) in LRAs and Scotland, respectively. Testing at-risk herds more frequently can also improve the level of detection by identifying more infected cases and reducing the hidden burden of the disease, and reduce surveillance effort by exempting low-risk herds from testing. Key words: Bovine tuberculosis, risk factors, risk-based surveillance, routine-herd-testing, slaughterhouse. L. C. M. SALVADOR1, M. DEASON1, J. ENRIGHT3, P. R. BESSELL2 AND R. R. KAO1* L. C. M. SALVADOR1, M. DEASON1, J. ENRIGHT3, P. R. BESSELL2 AND R. R. KAO1* L. C. M. SALVADOR1, M. DEASON1, J. ENRIGHT3, P. R. BESSELL2 AND R. R. KAO1* 1Boyd Orr Centre for Population and Ecosystem Health, Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK 2The Roslin Institute, The University of Edinburgh, Easter Bush, Midlothian, UK 3Computing Science and Mathematics, Faculty of Natural Sciences, University of Stirling, Stirling, UK Received 22 December 2016; Final revision 9 June 2017; Accepted 3 August 2017 * Author for correspondence R. R. Kao, Royal (Dick) School of Veterinary Science, Easter Bush Campus, Midlothian EH25 9RG, UK. (Email: rkao@exseed.ed.ac.uk) https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268817001935 Downloaded from https://www.cambridge.org/core. Edinburgh College of Art, on 11 Dec 2017 at 15:29:41, subject to the Cambridge Core terms of use, available at This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommon permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. INTRODUCTION Additional targeted testing is also performed in herds contiguous to herds with Ofﬁcially Tuberculosis Free Status Withdrawn (OTFW) due to fully conﬁrmed infection (contiguous and radial tests), in herds that have recently regained Ofﬁcially TB Free Status after suffering an episode of bTB (6- and 12-monthly check tests), herds with back traced reactors or cases detected through slaugh- ter surveillance (immediate check tests), herds in annual testing areas that send animals to other farms (pre-movement tests) and herds in LRAs that receive animals from annual testing areas (post-movement tests) or from Ireland (post-Irish imports). In Scotland, all eligible herds were tested every 4 years under the RHT policy until 31 December of 2011; since then, a risk-based surveillance system has been adopted exempting from RHT herds identiﬁed as low risk and with high chance of detection via abat- toir inspection, offering a cost reduction by requiring fewer herd and animal tests, and fewer false positives [23]. We hypothesise that, similar to Scotland, the cur- rent surveillance system in LRAs could be enhanced by a more targeted, risk-based approach that either reduces testing of cattle (thereby saving cost and effort), or enhances identiﬁcation of infection (thereby reducing onward risk), or does both. Based upon this, the objectives of this work are to (1) identify risk factors associated with breakdowns in these areas; (2) use a mixed logistic regression statistical model to compute the herd probability of freedom of infec- tion; and (3) use a risk-based surveillance model to evaluate alternative surveillance strategies. Traditionally, the bTB surveillance in GB regime allowed for different frequencies of routine herd testing (1 year RHT: HRAs, 2–3 year RHT: intermediate risk areas, 4 year RHT: LRAs), which was based upon the perceived risk of bTB in a given parish or the neigh- bouring parishes. In recent years more geographically streamlined designations have been introduced. All herds in Wales were ofﬁcially placed on annual RHT in 2010. INTRODUCTION Mycobacterium bovis and it affects an increasing number of cattle herds in GB, with an especially high incidence in the South West of England and in Wales (high-risk areas or HRAs) [4, 7]. While factors such as herd size, being a dairy farm, and some farm- ing practices have been identiﬁed as herd-level risks [3, 8–13], the presence of a wildlife reservoir, the Eurasian badger (Meles meles), makes the control of the disease particularly difﬁcult and controversial [2, 11, 14–16]. In contrast, wildlife reservoirs do not appear to make a meaningful contribution to the Bovine tuberculosis (bTB) in cattle is the most eco- nomically important disease of livestock in Great Britain (GB), with substantial impact on animal health and welfare, farmers’ livelihoods and their well-being [1–6]. A zoonosis, bTB is caused by L. C. M. Salvador and others 2 spread of bTB in cattle into low-risk areas (LRAs) such as the north and east of England, or into Scotland [4, 17]. While large outbreaks in cattle do occur here [18] and the risk of establishment in wildlife must be considered, in these areas control efforts can focus on infection in cattle, and particularly on imports from HRAs and from Ireland (both the Republic of Ireland and Northern Ireland), where bTB prevalence is also high [3, 12, 18–20]. The preva- lence of bTB in LRAs and in Scotland has been stable since 2006 and it remains at low levels (∼0·1 in LRAs and <0·01 in Scotland) [17]. While other supplemen- tary tests are used [21, 22], bTB cases are mostly iden- tiﬁed using the Single Intradermal Comparative Cervical test (SICCT or colloquially, the ‘skin test’) or by carcass inspection at the slaughterhouse. A posi- tive reaction is deﬁned as a difference of >4 mm in skin swelling when comparing responses with anti- genic derivatives from Mycobacterium avium and M. bovis, 72 h post injection. An animal with a posi- tive test (reactor) is slaughtered and movement restric- tions are imposed on the farm. A reactor is then conﬁrmed by identiﬁcation of at least one tuberculous lesion post-mortem or if M. bovis is successfully cul- tured from tissue samples. England. This change in the surveillance testing regime may come into effect during 2017. INTRODUCTION In England, a new bTB surveillance regime has been in place since 1 January 2013, whereby most herds in counties of the so-called low-risk area (LRA) are tested once every 4 years, whereas herds in the HRA of the West of England and a ‘transitional’ zone of intermediate bTB incidence known as the Edge Area (currently encompassing Hampshire, Berkshire, Buckinghamshire, Northamp- tonshire, Leicestershire, Nottinghamshire and parts of East Sussex, Oxfordshire, Warwickshire, Derbyshire and Cheshire) are tested annually (see online Supplementary Fig. S1). In order to speed up the detec- tion of infected herds in increasing bTB incidence parts of the Edge Area, a 6-monthly testing regime has been established since January 2015. Furthermore, at the end of 2016, Defra consulted on proposals to replace annual testing with 6 monthly herd testing in other high bTB incidence parts of the Edge Area of https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268817001935 Downloaded from https://www.cambridge.org/core. Edinburgh College of Art, on 11 Dec 2017 at 15:29:41, subject to the Cambridge Core terms of use, available at Surveillance scenarios A number of baseline and risk-based surveillance options were explored, considering both the likelihood of becoming infected and detected by SICCT and at the slaughterhouse. The baseline scenarios include slaughterhouse surveillance without live testing of herds, which represents a minimal model (the lowest amount of surveillance that a herd could be under) or, in addition, either 1-year (the maximal model that corresponds to the most prevalent surveillance approach in HRAs under the current testing regime), 2-year or 4-year RHT (the models that correspond to RHT surveillance every 2 or 4 years, respectively). Source data Analyses were performed using data between 2007 and 2013 from active holdings (determined by the unique County-Parish-Holding or CPH number, with all cattle associated with that holding assumed here to belong to one ‘herd’) in LRAs and in Scotland. Data on the historical SICCT test interval were used to identify herds in LRAs in England (on 4 yearly testing throughout the period considered), since the number of these herds has decreased over time (online Supplementary Fig. S1). Data on holdings, on historic bTB test results, on incidence and on animal life histories are taken, Risk-based strategies for surveillance of tuberculosis infection. 3 respectively, from the Defra animal health informa- tion system (SAM) provided by the Animal & Plant Health Agency (APHA) and from the British Cattle Movement System (BCMS) Cattle Tracing System Database (CTS). Data extraction were done as described in online Supplementary Section S2.1, which resulted in analyses on 13 327 herds in LRA and 10 145 herds in Scotland. respectively, from the Defra animal health informa- tion system (SAM) provided by the Animal & Plant Health Agency (APHA) and from the British Cattle Movement System (BCMS) Cattle Tracing System Database (CTS). Data extraction were done as described in online Supplementary Section S2.1, which resulted in analyses on 13 327 herds in LRA and 10 145 herds in Scotland. for each herd for every year between 2008 and 2013: herd size, herd type, herd-level prevalence of infection, number of batches from HRAs received in the previ- ous year, number of Irish imports received in the previous year and number of batches sent to slaugh- terhouse in the previous year. Source data Using the same study period as above, per herd data were extracted from the SAM and CTS databases and derived from the risk factor analysis (Section ‘Underlying risk factors for bTB breakdowns’). Batches of cattle in the previous year from holdings to slaughterhouse were extracted from CTS. Holdings that did not send any animals to the slaugh- terhouse were not considered, resulting in a total of 12 942 herds from 13 327 low-risk English herds and 9776 herds from 10 145 Scottish herds used in the ana- lysis in the section ‘Underlying risk factors for bTB breakdowns’. The following variables were calculated Statistical analysis Multivariate logistic mixed models were formulated to assess bTB candidate risk factors in herds in Scotland and in England (with 4-year testing) during 2008 and 2013. Both year and county were included as random effects and annual mean herd size, herd type (according to online Supplementary Table S1), Irish imports and high-risk movements were considered as ﬁxed effects (see online Supplementary Section S2.2 for more details). Models were evaluated using the AIC model score according to online Supplementary Section S2.3. Only the predictors that were signiﬁcant at P < 0·05 were included in the ﬁnal model. Interactions and associations between variables were also explored. The signiﬁcant ﬁxed effects are presented as odds ratios (Odds) with 95% conﬁdence intervals. The ﬁtted values extracted from the most signiﬁcant model correspond to the probability of the herd becoming infected in each year of the study (pintro). The risk-based surveillance scenarios were modelled considering the risk factors identiﬁed in the section ‘Underlying risk factors for bTB breakdowns’ together with the proportion of the herd’s total stock that is sent annually to slaughter as elements of infec- tion risk and detection. All modelled risk-based scen- arios also include routine slaughter surveillance (baseline). Table 2 provides a summary of the surveil- lance components of each scenario. ‘Scenarios 1–4’ test more often herds that import ‘high-risk’ animals and send a low proportion of the total herd to slaugh- ter every year; ‘scenarios 5–6’ test more often herds that import ‘high-risk’ animals, send a low proportion of animals to slaughter, and that have a large number of animals; ‘scenarios 7–8’ test more often herds that only have a large number of animals, and, the ‘Scottish scenario’ represents the surveillance that has been implemented in Scotland since 1 January 2012, in which herds that have a large number of ani- mals, import ‘high-risk’ animals and send a low num- ber of animals to slaughter are not exempt from testing. A detailed description of the elements of risk of infection, of the model assumptions and of the risk- based scenarios point-score system can be found in online Supplementary Section S3. The data were prepared using a combination of UNIX bash scripting and AWK commands for data extraction, and R [24] for data processing and management. The multivariate logistic mixed models and the model selec- tion were developed in the R lme4 [25, 26] and LMERConvenienceFunctions [27] packages. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268817001935 Downloaded from https://www.cambridge.org/core. Edinburgh College of Art, on 11 Dec 2017 at 15:29:41, subject to the Cambridge Core terms of use, available at Model evaluation The model was implemented in the R statistical envir- onment and run for 100 simulations using data between 2008 and 2013, with 2008 used as a ’burn-in’ period. The start of the routine herd testing cycle (e.g. every 1-, 2-, 4-years) was generated randomly for each simulation, to minimise cyclical effects. The risk-based scenarios were evaluated from 2009 to 2013 by com- paring the following variables to the equivalent ﬁtted values from the current surveillance scenarios in each area (LRA: 4 year-RHT, Scotland: Scottish scenario; the national totals for each term are given by summing the values for all herds). If the absolute difference between two values is <5%, then these values are considered similar. Derivation of each term for each holding at a time t is described below: sesystem = 1 −(1 −seherd)(1 −se part), in which seherd is the sensitivity of the SICCT imple- mented as a herd test, and separt is the part herd sensi- tivity for slaughterhouse surveillance. The herd sensitivity for a whole herd test is calculated as seherd = 1 −(1 −seSICCT)d, seherd = 1 −(1 −seSICCT)d, Where seSICCT is the animal-level sensitivity of the screening test and d is the number of infected animals in the herd deﬁned as: (a) The annual expected number of latent infections. Probability of being latently infected for each farm at time t: (1 −p(free)t). d = N × pstar. The value d is the product of a number drawn from the distribution of pstar and the annual average num- ber of animals in the herd N. The sensitivity for a part herd test for the proportion of the herd that is sent to slaughter is: (b) The total ﬁtted number of detected infections. Probability of a detected infection at time: (priort- −p(free)t). (c) The annual number of herds tested. (d) The annual number of cattle tested. se part = 1 −1 n × seslh N d , se part = 1 −1 n × seslh N d , (e) The annual expected number of false positives. The parameter spherd gives the probability of a given herd being a false positive. Therefore, sum- ming up spherd for all herds for a given year gives the expected number of false positives. where n is the number of animals sent to slaughter per annum. Model description To evaluate the likelihood of herd-level freedom from infection (herd probability of undetected infection) 17 at 15:29:41, subject to the Cambridge Core terms of use, available a L. C. M. Salvador and others 4 The probability of freedom (the posterior) at time t is given by: with bTB during a speciﬁed time period (t) the model requires the following parameters: p( free)t = 1 −priort (1 −priort) + priort × (1 −sesystem) , (a) The probability of the herd becoming infected at time t (p(intro)t) (from the section ‘Underlying risk factors for bTB breakdowns’); where priort is the prior probability that the herd is infected at time t. The prior for t + 1 is: where priort is the prior probability that the herd is infected at time t. The prior for t + 1 is: (b) Herd size at time t; (b) Herd size at time t; (b) Herd size at time t; (c) The efﬁcacy of the surveillance system implemen- ted on the farm (RHT and slaughterhouse testing) (d) The herd-level prevalence of infection pstar. (c) The efﬁcacy of the surveillance system implemen- ted on the farm (RHT and slaughterhouse testing) priort+1 = (1 −p( free)t) + p(intro)t −(1 −p( free)t)∗p(intro)t. priort+1 = (1 −p( free)t) + p(intro)t −(1 −p( free)t)∗p(intro)t. (d) The herd-level prevalence of infection pstar. The model only considers explicitly RHT and slaughterhouse surveillance. The efﬁcacy of the sur- veillance system is evaluated by calculating the herd-level test system sensitivity sesystem, which includes the RHT and part slaughterhouse testing: Model evaluation The false positive (unconﬁrmed breakdown) detec- tion rate of the whole herd test spherd is deﬁned as: A scenario is considered to have improved detection if it has similar or higher number of detections and lower number of latent infections. spherd = 1 −spn animal, spherd = 1 −spn animal, where spn animal with n = N is the speciﬁcity for whole herd tests. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268817001935 Downloaded from https://www.cambridge.org/core. Edinburgh College of Art, on 11 Dec 2017 at 15:29:41, subject to the Cambridge Core terms of use, available at Underlying risk factors for bTB breakdowns in LRAs and Scotland The models created for the risk-factor analysis have a substantial predictive value, as indicated by the area under the curve (LRA England, AUC = 0·8685; Scotland, AUC = 0·8533; online Supplementary Fig. S2). The signiﬁcance of the variables used to determine the risk factors in LRAs and in Scotland was estimated for the study period 2008–2013. Of the variables considered in the analysis, herd size, receiving batches from HRAs, and Irish imports were associated with bTB incidents in both LRAs and Scotland (Fig. 1, online Supplementary Table S2). The larger the herd, the higher is the association with bTB infection (Fig. 1 and online Supplementary Fig. S3 and Table S2). This associ- ation also applies across different herd types (online Supplementary Fig. S4), with the exception of dairy herds where there is a large overlap of the two distributions, and of store herds (young beef or mixed- breed animals reared and sold for ﬁnishing on fatten- ing farms) in Scotland, which did not suffer any breakdowns (online Supplementary Fig. S4-B). Fig. 1. The results of a mixed logistic regression model that computes the herd risk of infection and determines the risk factors associated with bovine Tuberculosis breakdowns in low-risk areas in England (a) and in Scotland (b) between 2008 and 2013. Odds ratios and 95% conﬁdence intervals express the contribution of each one of the signiﬁcant risk factors. schemes, especially in Scotland where some of the curves are overlapping. As expected, the high probabil- ity with slaughterhouse surveillance alone (grey line, Fig. 2a,c) decreases with increased RHT. Finishing herds have a lower probability of undetected infection (higher probability of freedom from infection) in both LRAs and in Scotland (online Supplementary Fig. S6). In LRAs, they are followed by dairy, by both beef and suckler, and by store, while for the period of the study, Scotland did not register any breakdowns in store herds (online Supplementary Fig. S4-B), making beef and suckler (herd of cattle composed of dams and their young calves up to the point of weaning) the riskiest herds, followed by dairy. There is a linear relationship between the average number of animals sent to slaughter per year and the average herd size with dis- tinct clustering of ﬁnishing and dairy herds both in LRAs and in Scotland (online Supplementary Fig. S7). Underlying risk factors for bTB breakdowns in LRAs and Scotland In LRAs, ﬁnishing herds are typically smal- ler than dairy herds (with the exception of some out- liers), however, they have more per capita movements to slaughter than dairy ones, followed by store, beef and suckler (online Supplementary Fig. S7-a). In Scotland, the trend is the same as in LRAs, with the exception that suckler herds have Receiving batches from HRAs and/or importing animals from Ireland in the previous year are also associated with bTB incidents in both areas (Fig. 1, online Supplementary Table S2). This outcome is con- sistent with an independent analysis of movements as risks (online Supplementary Section S1, Fig. S5). Risk-based strategies for surveillance of tuberculosis infection. 5 Fig. 1. The results of a mixed logistic regression model that computes the herd risk of infection and determines the risk factors associated with bovine Tuberculosis breakdowns in low-risk areas in England (a) and in Scotland (b) between 2008 and 2013. Odds ratios and 95% conﬁdence intervals express the contribution of each one of the signiﬁcant risk factors. to the English herds (see the section ‘Underlying risk factors for bTB breakdowns’). Should LRA herds with the same predicted probabilities under the Scottish model tend to have higher breakdown rates (or vice versa) this would imply an inherently different epidemiological risk between these areas, for example different demographics of the respective cattle popula- tions, presence of a wildlife reservoir or any other risk factor not captured by these models. Comparison of bTB risk between LRAs and Scotland To compare the risk of bTB infection between LRAs and Scotland, we have applied the predictor of the best-ﬁtted statistical model in LRAs to Scottish herds, and the predictor of the best-ﬁtted model in Scotland In this analysis, the values used for the test sensitiv- ities and speciﬁcities follow the meta-analysis of bTB diagnostic test performance in Great Britain per- formed by Downs et al. [28]. 17 at 15:29:41, subject to the Cambridge Core terms of use, available a https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268817001935 Downloaded from https://www.cambridge.org/core. Edinburgh College of Art, on 11 Dec 2017 at 15:29:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268817001935 Downloaded from https://www.cambridge.org/core. Edinburgh College of Art, on 11 Dec 2017 at 15:29:41, subject to the Cambridge Core terms of use, available at BTB surveillance in LRAs and in Scotland Probability of undetected infection In general, the probability of undetected infection per herd in LRAs is higher than in Scotland for all the pre- sented scenarios (Fig. 2). While the probabilities under different baseline scenarios are distinctly different, these differences are substantially reduced for the risk-based 6 6 L. C. M. Salvador and others Fig. 2. The cumulative distribution plot of the probability of each herd harbouring undetected infection at the end of each model time step for all eligible herds in LRA England (a) and in Scotland (b) between 2009 and 2013 from the four baseline surveillance scenarios. Subplots C and D show the cumulative distribution plot of the probability of each herd harbouring undetected infection at the end of each model time step for all eligible herds in LRA England (c) and in Scotland (d) during 2009–2013 from nine risk-based surveillance scenarios. The axes were truncated for clarity. Fig. 2. The cumulative distribution plot of the probability of each herd harbouring undetected infection at the end of each model time step for all eligible herds in LRA England (a) and in Scotland (b) between 2009 and 2013 from the four baseline surveillance scenarios. Subplots C and D show the cumulative distribution plot of the probability of each herd harbouring undetected infection at the end of each model time step for all eligible herds in LRA England (c) and in Scotland (d) during 2009–2013 from nine risk-based surveillance scenarios. The axes were truncated for clarity. more per capita movements to slaughter than beef and dairy (online Supplementary Fig. S7-b). latent and detected infections), testing burden (num- bers of animals and herds tested annually) and the number of SICCT false positive herd results gener- ated. The values of these variables were estimated for each scenario for both areas, were compared with the current surveillance regimes in each area (LRAs, ‘4 year RHT’; Scotland, ‘Scottish scenario’) and are presented in Figure 3 and in Table 1. Baseline scenarios perform as expected, with the not- able feature that increased frequency of RHTs (Table 1) greatly reduce the number of latent infec- tions with RHT frequency (4 and 15 compared with 32 in LRAs; 1 and 5 compared with 30 in Scotland), but with a minimal increase in the number of detected infections, while generating substantially more false positives (2 and 4 times more than the current systems). BTB surveillance in LRAs and in Scotland Probability of undetected infection When herd-level risk of infection is incorporated into surveillance scenarios, the cumulative herd level probabilities of harbouring undetected infection (calcu- lated by the cumulative distribution function (CDF)) are very similar across the different risk-based scenarios (Fig. 2), reﬂecting the importance of slaughterhouse surveillance for many herds (the one factor that is con- stant across scenarios). In LRAs, for higher values of proportion of herds tested, scenario 1 is the one that shows the lowest probability of undetected infection per herd, followed by scenarios 7 and 8. In Scotland, there are no major differences between the different scenarios, with the exception of scenarios 5, 6 and Scottish scenarios, which present slightly higher prob- abilities of harbouring undetected infection per herd. Results of the risk-based surveillance model (all ﬁgures per annum) are presented in Figure 3 and Tables 1 and 2. These can be divided into three differ- ent categories, all of which reduce the burden of testing: https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268817001935 Downloaded from https://www.cambridge.org/core. Edinburgh College of Art, on 11 Dec 2017 at 15:29:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268817001935 Downloaded from https://www.cambridge.org/core. Edinburgh College of Art, on 11 Dec 2017 at 15:29:41, subject to the Cambridge Core terms of use, available at Risk-based strategies for surveillance of tuberculosis infection. 7 g. 3. Risk-based surveillance scenarios comparison between LRA England and Scotland. Each panel represents the rveillance variables used in the probability of freedom model and the x- and y-axes represent the rankings of each i E h l h diff i d h i l l h h i Fig. 3. Risk-based surveillance scenarios comparison between LRA England and Scotland. Each panel represents the surveillance variables used in the probability of freedom model and the x- and y-axes represent the rankings of each scenario. Each colour represents the different scenarios and the triangle overlap represents that more than one scenario had the same ranking related to the variable in study. The 4-year scenario was also included in the analysis for comparison with the risk-based ones. Surveillance scenarios ranking The different surveillance scenarios were ranked according to their levels of detection (numbers of i.org/10.1017/S0950268817001935 e. Edinburgh College of Art, on 11 Dec 2017 at 15:29:41, subject to the Cambridge Core terms of use, available at Risk-based strategies for surveillance of tuberculosis infection. (1) Scenarios that improve detection (1) Scenarios that improve detection number of animals tested (290 579 compared with 391 613), and 24% on the number of false positives (56 compared with 72) in LRAs when compared with the current 4-year RHT regime. In Scotland scenario 6 reduces the herd testing by 17% (1328 compared with 1602), the animal testing by 20% (244 088 compared with 306 499), and the number of false positives by 24% (31 compared with 41) when compared with the Scottish scenario. (1) Scenarios that improve detection In LRAs, scenario 1 decreases latent infections by 9% (29 compared with 32) for a similar level of detected infections, it tests 5% fewer herds annually (3065 compared with 3235), 6% fewer animals (369 673 compared with 391 613), and generates 6% fewer false positives (68 compared with 72), when compared with the current 4-Year RHT regime. In Scotland, scenario 2 reduces latent infections by 7% (12 compared with 14) for a similar number of detected cases (within 5%), tests 26% fewer animals (304 968 compared with 306 499 for a slight higher number of herds tested) and generates 10% fewer false positives (45 compared with 41) than the current surveillance regime (Scottish scenario). (3) (3) Scenarios that substantially reduce the burden of testing but lower the levels of detection. (3) Scenarios that substantially reduce the burden of testing but lower the levels of detection. The scenario that offers savings on the burden of testing but at the expense of reduced detection of infected herds in LRAs is scenario 3. This scen- ario would reduce the testing burden by testing 44% fewer animals (220 621 compared with 391 613), 36% fewer herds (2074 compared with 3235) and generate 38% fewer false positives (45 compared with 72) while increasing the num- ber of latent infections by 9% (35 compared with (2) Scenarios that reproduce similar levels of detection. For the same level of detection, scenario 2 offers a saving of 22% on the number of herds tested annually (2531 compared with 3235), 26% on the (2) Scenarios that reproduce similar levels of detection. For the same level of detection, scenario 2 offers a saving of 22% on the number of herds tested annually (2531 compared with 3235), 26% on the L. C. M. Salvador and others 8 Table 1. DISCUSSION The development of our proposed risk-based surveil- lance strategy has two major aims. The ﬁrst is to iden- tify inefﬁciencies: in the case of bTB, this amounts to identifying where slaughterhouse surveillance works well and therefore RHT can most safely be discarded. The second is to identify those herds most likely to present an infection risk: both those most likely to become infected and (more difﬁcult to deﬁne) those more likely to present risks to others. For bTB in the absence of a wildlife reservoir, larger herds and those that purchase cattle from HRAs are more likely to become infected, while herds that sell on many cat- tle to other premises, present the greatest infection risk. As our statistical models do not capture this onward transmission related risks, and as some of these risks are captured indirectly by herd characteris- tics, intelligent decision-making is sometimes required to balance off these issues. For example, ﬁnishing herds bring in many cattle and therefore are a high risk of becoming infected, however they also send many cattle to slaughter and do not sell on many cat- tle to other premises, and therefore present a low risk to others. The development of our proposed risk-based surveil- lance strategy has two major aims. The ﬁrst is to iden- tify inefﬁciencies: in the case of bTB, this amounts to identifying where slaughterhouse surveillance works well and therefore RHT can most safely be discarded. The remaining scenarios would increase the burden of testing with at best minimal detection beneﬁts, and therefore would not contribute to an increased per- formance of the surveillance system and are not con- sidered further here. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268817001935 Downloaded from https://www.cambridge.org/core. Edinburgh College of Art, on 11 Dec 2017 at 15:29:41, subject to the Cambridge Core terms of use, available at (1) Scenarios that improve detection Summary of the bovine TB surveillance analysis results for low-risk areas in England and for Scotland between 2009 and 2013 Surveillance scenario Interval (years) Herds tested Animals tested Detected infections Latent infections False positives LRA Scotland LRA Scotland LRA Scotland LRA Scotland LRA Scotland Baseline scenarios Slaughterhouse n.a. 0 0 0 0 120 33 80 30 0 0 4 Year 4 3235 2444 391 613 412 293 144 45 32 12 72 58 2 Year 2 6471 4888 783 226 824 587 142 46 15 5 143 115 1 Year 1 12 942 9776 1 566 452 1 649 175 136 45 4 1 286 231 Risk-based surveillance Scenario 1 0/2/4 3065 2090 369 673 349 453 147 45 29 11 68 49 Scenario 2 0/4 2531 1930 290 579 304 968 143 45 33 12 56 45 Scenario 3 0/4 2074 1629 220 621 227 379 142 44 35 13 45 37 Scenario 4 0/1/2/4 2827 2262 448 135 454 829 143 46 33 11 68 57 Scenario 5 0/1/2/4 1803 1483 264 090 308 083 139 43 39 14 41 36 Scenario 6 0/1/2/4 1682 1328 213 163 244 088 138 42 40 14 37 31 Scenario 7 1/4 3878 3428 735 237 1 352 799 145 46 30 3 270 207 Scenario 8 1/4 3486 2871 570 664 736 476 144 45 31 11 80 72 Scottish scenario 0/4 2166 1602 292 074 306 499 139 43 37 14 51 41 The ‘4 year’ baseline scenario is the one currently implemented in LRAs and the ‘current’ risk-based scenario is the one that has been implemented in Scotland since January 2012. Table 1. Summary of the bovine TB surveillance analysis results for low-risk areas in England and for Scotland between 2009 and 2013 Table 1. Summary of the bovine TB surveillance analysis results for low-risk areas in England and for Scotland between 2009 and 2013 The ‘4 year’ baseline scenario is the one currently implemented in LRAs and the ‘current’ risk-based scenario is the one that has been implemented in Scotland since January 2012. 32) for the same level of detection. This scenario would also miss 3 out of 13 breakdowns identiﬁed by RHT (Table 2). Comparison of bTB risk between LRAs and Scotland The herd probability of infection given by the English and Scottish predictors is presented in Figure 4. The estimated herd probability of bTB infection is lower in Scotland than in LRAs (μ(Scot) = 0·00080, σ2(Scot) = 2·5e-06; μ(LRA) = 0·0020, σ2(LRA) = 4·1e-05; online Supplementary Fig. S8) and if Scottish herds were exposed to the same risk as herds in LRAs the average risk per herd would increase to 0·0013 (σ2 = 7·2 × 10−6, online Supplemen- tary Fig. S9). However, if LRA English herds were exposed to the same risk as herds in Scotland, the pre- dicted risk of infection would be similar to Scotland (μ = 0·00080, σ2 = 2·3 × 10−6) suggesting that there is an inherent geographical difference between the two areas, with the risk of bTB infection higher in LRAs. Despite some broad similarities (the same risk fac- tors and some similarities in the cattle industry), d cenarios for low-risk areas in England and Scotland during 2009–2013 Points Testing interval No. Comparison of bTB risk between LRAs and Scotland RHT column represents the breakdowns that were detected by routine herd testing risks compared across LRAs and Scotland have some subtle differences, notably the mean risk of infection of bTB per herd (LRAs have higher probability of risk of infection per herd than Scotland), and differ- ences in per capita movements to slaughter (in LRAs, beef herds have more per capita movements to slaughter than suckler and dairy herds, while in Scotland suckler herds have more per capita move- ments to slaughter than beef and dairy). Store herds have the lowest probability of freedom from infection with slaughterhouse surveillance only, while in Scotland they are the least risky after ﬁnishing herds. England and Scotland have proceeded along differ- ent surveillance paths until now, mainly as a result of differences in the underlying legislative and policy environments. As the EU granted ofﬁcial bTB free (OTF) status to Scotland in September 2009, this offered the opportunity to do away with routine test- ing of low-risk herds (while maintaining very similar detection rates), and so a risk-based surveillance strat- egy based on our previous work [23] was adopted. The evidence presented here suggests some reﬁnements that could offer a higher reduction on the testing bur- den while maintaining the current level of bTB detec- tion in Scotland (scenarios 2 and 6). By contrast, in the LRA of England the process to secure OTF status has already begun, but has not yet been achieved. Therefore, any reduction of testing at this stage would present a greater risk and could jeopardise this process. However, it is something that policy- makers should consider if the LRA is recognised as OTF in due course. On the other hand, the present study shows that there is little to be gained from increased routine herd testing frequencies in the LRA. Whether or not changes in surveillance in LRAs are currently merited, nonetheless it is import- ant to understand the extent to which the areas consid- ered low risk in England may differ from Scotland, as this may be an indicator of the feasibility of achieve- ment of OTF status in LRAs. A good prediction of the risk of infection in both LRAs and in Scotland is determined by the size of the herd, by movements from HRAs, and by whether herds receive animals from Ireland. Comparison of bTB risk between LRAs and Scotland herds (%) bTB cases RHT LRA Scotland LRA Scotland LRA Scotland Having >20 animals and receiving ‘high-risk’ animals more than once in the last 4 years and/or slaughtering >25% of stock and receiving ‘high-risk’ animals more than once in the last 4 years and/or slaughtering >40% of stock 1 1 point = 4 year testing 8665 (67) 6410 (65·6) 54 27 1 2 0 points = no testing 4277 (33) 3366 (34·4) 92 18 17 11 The testing interval column represents the time frame of the bTB testing, which depends on the level of risk based on a score point system (0 = no testing, 1 = 4 year testing, 2 = 2 year testing, 3 = annual testing). The No. herds column corresponds to the number of herds (and percentage of the total number of herds) that fell into each testing interval category. bTB is the number of conﬁrmed breakdowns for the eligible herds between 2009 and 2013. RHT column represents the breakdowns that were detected b ro tine herd testing ( ) Criteria Points Testing interval No. herds (%) bTB cases RHT LRA Scotland LRA Scotland LRA Scotland Having >20 animals and receiving ‘high-risk’ animals more than once in the last 4 years and/or slaughtering >25% of stock and receiving ‘high-risk’ animals more than once in the last 4 years and/or slaughtering >40% of stock 1 1 point = 4 year testing 8665 (67) 6410 (65·6) 54 27 1 2 0 points = no testing 4277 (33) 3366 (34·4) 92 18 17 11 The testing interval column represents the time frame of the bTB testing, which depends on the level of risk based on a score point system (0 = no testing, 1 = 4 year testing, 2 = 2 year testing, 3 = annual testing). The No. herds column corresponds to the number of herds (and percentage of the total number of herds) that fell into each testing interval category. bTB is the number of conﬁrmed breakdowns for the eligible herds between 2009 and 2013. Comparison of bTB risk between LRAs and Scotland herds (%) bTB cases RHT LRA Scotland LRA Scotland LRA Scotland +1 0 points = no testing 2632 (20·3) 2015 (20·6) 93 18 2 2 ing +1 1 point = 4 year testing 8359 (64·6) 7159 (73·2) 34 22 10 9 2 points = 2 year testing 1951 (15·1) 602 (6·2) 19 5 6 2 ’ 1 0 points = no testing 2815 (21·8) 2053 (21) 94 19 2 2 1 point = 4 year testing 10 127 (78·2) 7723 (79) 52 26 16 11 k’ 1 0 points = no testing 4643 (35·9) 3259 (33·3) 106 26 4 3 1 point = 4 year testing 8299 (64·1) 6517 (66·7) 40 19 14 10 −1 −1 or 0 points = no testing 4524 (35) 3152 (35) 27 12 4 3 +1 1 point = 4 year testing 6166 (47·6) 4585 (46·9) 101 22 8 4 +1 2 points = 2 year testing 1932 (14·9) 1845 (18·9) 14 10 6 4 +1 3 points = 1 year testing 320 (2·5) 194 (2) 4 1 0 2 −1 −1 or 0 points = no testing 6828 (52·8) 4504 (46·1) 72 25 8 4 +1 1 point = 4 year testing 5076 (39·2) 4679 (47·9) 67 15 9 6 +1 2 points = 2 year testing 1007 (7·8) 559 (5·7) 7 5 1 3 +1 3 points = 1 year testing 31 (0·2) 34 (0·3) 0 0 0 0 −1 −1 or 0 points = no testing 7160 (55·3) 4878 (49·9) 89 31 11 6 +1 1 point = 4 year testing 4859 (37·5) 4499 (46) 50 10 6 5 +1 2 points = 2 year testing 910 (7) 390 (4) 7 4 1 2 +1 3 points = 1 year testing 13 (0·1) 9 (0·1) 0 0 0 0 1 1 point = 4 year testing 857 (6·6) 1312 (13·4) 58 14 3 4 3 3 points = 1 year testing 12 085 (93·4) 8464 (86·6) 88 31 15 9 1 1 point = 4 year testing 12 607 (97·4) 9206 (94·2) 110 37 17 11 3 3 points = 1 year testing 335 (2·6) 570 (5·8) 36 8 1 2 10 L. C. M. Salvador and others Criteria Points Testing interval No. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268817001935 Downloaded from https://www.cambridge.org/core. Edinburgh College of Art, on 11 Dec 2017 at 15:29:41, subject to the Cambridge Core terms of use, available at Comparison of bTB risk between LRAs and Scotland Larger herds have a greater likelihood of infection in both areas, conﬁ- rming previous results in an earlier time period [4, 29]. The continued signiﬁcance of the high-risk movements despite pre-movement testing suggests that greater test sensitivity could accrue substantial beneﬁts (possibly even if there is a cost of reduced test speciﬁcity). The risk of bTB breakdowns can also be due to local effects https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268817001935 Downloaded from https://www.cambridge.org/core. Edinburgh College of Art, on 11 Dec 2017 at 15:29:41, subject to the Cambridge Core terms of use, available at Risk-based strategies for surveillance of tuberculosis infection. 11 Fig. 4. Herd probability of infection given by the English (LRA) and Scottish predictors. The different colours represent the location of each herd (orange: LRA England, light blue: Scotland). If herds in Scotland were exposed to the same level of risk as herds in LRA England, their probability of getting infected would be higher. From the other hand, if herds in LRA England were exposed to the same level of risk as herds in Scotland, their probability of getting infected would be lower. implementation and the differential impact on the various sectors of the industry, our analyses show that the current schemes in LRAs and in Scotland are effective and a more frequent testing regime (1 or 2 years interval) will provide higher freedom from disease (originate fewer latent infections), but will not improve the current number of detected break- downs and will substantially increase the burden asso- ciated with testing. However, surveillance in LRAs can be improved if herds that are at higher risk are tar- geted. It is not expected that these factors affect the performance of the model, however. As the frame- work used is scalable and adaptable, it can be extended with other surveillance scenarios, as well as other policy measures currently implemented in both LRAs and Scotland, such as pre-movement and post- movement testing. Fig. 4. Herd probability of infection given by the English (LRA) and Scottish predictors. The different colours represent the location of each herd (orange: LRA England, light blue: Scotland). If herds in Scotland were exposed to the same level of risk as herds in LRA England, their probability of getting infected would be higher. From the other hand, if herds in LRA England were exposed to the same level of risk as herds in Scotland, their probability of getting infected would be lower. SUPPLEMENTARY MATERIAL The supplementary material for this article can be found at https://doi.org/10.1017/S0950268817001935 CONCLUSION This paper has demonstrated that the determinants of bTB infections in LRAs of England and in Scotland are very similar to each other, but that the magnitude of risk in LRAs is markedly higher than in Scotland, suggesting some underlying risks not captured in this analysis. Risk-based surveillance is an effective and efﬁcient method that reduces the cost by testing fewer animals whilst improving/maintaining the level of disease detection. A full evaluation of the best strat- egy would entail a cost-beneﬁt analysis considering the impacts of all changes in testing rates, impact on herd restrictions, and risk of onward transmission, and more subtly, changes in surveillance that may have unintended consequences for farmer behaviour. While such analyses lie outside the scope of this paper, the results presented here, specifying the purely epidemiological beneﬁts of risk-based surveillance, are the essential groundwork for such a broader task. such as the history of bTB cases in the herd [4, 19] and poor farm biosecurity [30]. These factors have not been considered in the analysis, but it is likely that they also play a role and be responsible for the persistence and spread of the disease. That herds in LRAs have substan- tially higher risk of infection than in Scotland even under the same risk model is notable, suggesting that there are important epidemiological factors not captured in our models; this merits further study beyond the scope of this paper. In this work, a model of freedom from infection was used to evaluate different strategies for surveil- lance for bTB in LRAs and Scotland. By exploiting statistical analyses deﬁning herd level probability of freedom from disease layered by signiﬁcant risk factors, testing frequencies can be reﬁned to test highly probable infections more frequently, and conversely potentially reducing testing of low-risk herds. The strategies modelled here focused on signiﬁcant indica- tors of risk, penalizing herds with large sizes that receive ‘risky’ imports (movements from HRAs and Ireland). Herds that send a small proportion of their stock to slaughterhouse are also penalised, as this makes slaughterhouse detection less likely. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268817001935 Downloaded from https://www.cambridge.org/core. Edinburgh College of Art, on 11 Dec 2017 at 15:29:41, subject to the Cambridge Core terms of use, available at REFERENCES 19. Green DM, et al. Estimates for local and movement- based transmission of bovine tuberculosis in British cat- tle. 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https://openalex.org/W2064060001	https://literator.org.za/index.php/literator/article/download/816/986	English	null	The representation of ideology: Orwell’s (re)reading of ‘Boys’ Weeklies’	Literator	1,990	cc-by	5,976	1. This article is based on a chapter from the present writer’s MA dissertation entitled Artful Propaganda: A Study of George Orwell’s Technique in Selected Essays (UNISA, 1987). Material used with permission. ISSN 0258-2279 LITERATOR 11 No. 3 November 1990 ISSN 0258-2279 LITERATOR 11 No. 3 November 1990 ISSN 0258-2279 ISSN 0258-2279 November 1990 Abstract The aim of this paper is to unmask the underlying assumptions of Orwell’s reading strategy in order to illustrate that texts written from a conscious and specific ideological perspective is much more vulnerable to ideological “unmasking” than texts which do not have such a specific ideological bias. A number of key aspects with regard to commitment, ideology and representation are identified and used as criteria for a critical reading of “Boys’ Weeklies”, one of Orwell’s most famous essays. 2. Theses • Commitment is the representation of a writer’s implicit or explicit ideology and manifests itself in literary or critical texts in which the writer either pleads openly for a particular point of view or ideology of a political, social or religious nature, or subtly tries to manipulate his readers into accepting his particular ideology or point of view with the ultimate aim of affecting a change in his reader’s attitude and approach (Bachrach, 1980: 372; my translation - AMdeL). • The concept ideology is used in a wide variety of contexts and can therefore often be misunderstood if it is not clearly defined within each context. McCormick and Waller (1987: 195-196) emphasize that it should not merely be seen as a set of alien or wrong ideas, but should be seen as pointing to “(t)hose common values, practices, ideas, and assumptions of a particular society that, in fact, hold it together: the deeply ingrained, sometimes only partly conscious, habits, beliefs, lifestyles of a particular time and place.” The major function of an ideology is to define and limit the cultural practices of society by structuring their experience of reality in a way which would suggest that the existing order of things is permanent, natural, universally acknowledged and true. As such it provides coherence and a lo^cal pattern to a society’s social, political and cultural expression and practices. Language is a writer’s only medium by means of which he can effect a change of heart in his readers. It is therefore imperative for the committed writer to accept the arbitrary nature of the various codes embedded in a linguistic system and therefore also the premise that language can engage meaningfully with reality by inventing, representing, cheating, playing, enchanting and animating (Nuttall, 1984: 192-193). A committed writer invariably invents a new reality which has a very definite and recognizable link with reality as perceived by the reader, but at the same time perverts the language to emphasize his interpretation of reality, or to point out why somebody else’s interpretation of reality is invalid or not as valid or truthful as his. 1. Introduction Frederic Jameson, in the preface to The Political Unconscious: Narrative as a Socially Symbolic Act (1986), states that the interpretation of texts is not an isolated act, but takes place within a Homeric battlefield, on which a host of interpretative options are either openly or implicitly in conflict and that only “another, stronger interpretation can overthrow and practically refute an interpretation already in place” (1986; 13). The aim of this article is to take to the Homeric battlefield in a somewhat unconventional and undercover way by unmasking the underlying ideology of Orwell’s interpretative strategies and in so doing argue that a text which has been written from an explicit ideological point of view is even more vulnerable to ideological “unmasking” than texts which have been written without any explicit bias. 108 The method followed is as follows: a number of theses regarding commitment, ideology and language as representative medium are posited, and then used as criteria against which Orwell’s approach and style in a key essay in his critical oeuvre are measured after his basic ideological stance has been delineated. 23 McCormick and Waller (1987: 194) define literary and general ideology as follows: “The term ideology ... refers to the shared though very diverse beliefs, assumptions, habits and practices of a particular society. Some of these are specifically literar>' matters, such as whether the author is seen as a unique genius or as a spokesperson for society, what literary genres and conventions are most highly valued, whether women’s writing is valued as highly as men’s, and so forth. We refer to such aspects as literary ideology. We refer to all other, non-literary, matters as general ideology". 2. Theses While it is readily acknowledged that all literary works attempt to influence their readers in some way or another, it should be emphasized that this is the committed writer’s primary aim, and care should be taken not to be misled by the writer’s “perversion of words” (Orwell, 1980b: 20), but to look for the subtleties and nuances when studying the ways and means through which a committed writer tries to effect this change. Language is a writer’s only medium by means of which he can effect a change of heart in his readers. It is therefore imperative for the committed writer to accept the arbitrary nature of the various codes embedded in a linguistic system and therefore also the premise that language can engage meaningfully with reality by inventing, representing, cheating, playing, enchanting and animating (Nuttall, 1984: 192-193). A committed writer invariably invents a new reality which has a very definite and recognizable link with reality as perceived by the reader, but at the same time perverts the language to emphasize his interpretation of reality, or to point out why somebody else’s interpretation of reality is invalid or not as valid or truthful as his. While it is readily acknowledged that all literary works attempt to influence their readers in some way or another, it should be emphasized that this is the committed writer’s primary aim, and care should be taken not to be misled by the writer’s “perversion of words” (Orwell, 1980b: 20), but to look for the subtleties and nuances when studying the ways and means through which a committed writer tries to effect this change. 109 The key question which logically falls within the ambit of this article, is; how does a writer’s ideology effect or influence his own literary and critical texts, as well as his interpretation of other texts? McCormick and Waller see this influence as analogous to “a powerful force hovering over us as we write or read a text ... [always reminding] us of what is correct, commonsensical, or ‘natural’. It tries ... toguide the readings of a text.. (1987: 197; my italics - AMdeL). 4. Orwell never outlined his ideology as such. However, the most coherent representation of his ideas and beliefs was outlined in The Road to Wigan Pier (1980). His ideology is based on two key concepts, justice and common decency. 5. Orwell’s reminiscences about his schooldays were published in a very negative essay, “Such, Such Were the Joys” (1982b: 379-422) and evoked severe criticism from his old school friends. 2. Theses One could therefore argue that a committed writer’s ideology, embedded implicitly or explicitly in his work, will attempt to guide or manipulate a reading of a text much more strongly than would, say, that of a text dealing not so much with societal issues but with very personal and intense emotions. These guiding principles will therefore either be strengthened or opposed by the society’s literary ideology,^ as this shows how a society’s general ideology^ is articulated through its literary and cultural practises. These guiding principles will furthermore paradoxically expose the writer’s own ideology as these will differ markedly from the society’s literary ideology. The greatest effect is achieved when a writer succeeds in camouflaging his approach so that the disparity between a reader’s ideology and the writer’s ideology seems insignificant and not at all relevant. Adorno (1%2: 303-304) emphasizes this aspect strongly when he distinguishes between commitment and tendency: In esthetic theory, ‘commitment’ should be distinguished from ‘tendency’. Committed art in the proper sense is not intended to generate ameliorative measures, legislative arts or practical institutions - like earlier propagandist (tendency) plays against syphilis, duels, abortion or borstals - but to work at the level of fundamental attitudes... But what gives commitment its esthetic advantage over tendentiousness also renders the content to which the artist commits himself inherently ambiguous. Before investigating the underlying ideology represented in Orwell’s essay “Boys’ Weeklies” (1982a: 505-531), it will first be necessary to refresh one’s memory with regard to the context in which it originated, as well as Orwell’s ideology as self-appointed spokesman of the working-class, his experiences in the Spanish Civil War, his personal experience of the English educational system, and, finally, his views on the relationship between writer and society. 110 6. Years later Orwell expressed his guilt feelings about his parents’ poverty through Gordon Comstock, the protagonist of Keep the Aspidistra Flying: 3. Orwell’s ideology^ Orwell’s personal ideology must first be defined if one is to see the subtle, and sometimes not so subtle, representation of this ideology in his reading of “Boys’ Weeklies”. This ideology was influenced by many things, three of the most important being his stay at Eton, the machinations of the British class system in general and the Spanish Civil War. Orwell’s experience of the English educational system had a lasting effect on his view of the English class system, and later became a full-scale attack on the public-school ethos.* As a schoolboy coming from “a lower-upper middle-class family” (Crick, 1980: 58), he experienced both the subtle and blatant nuances of class prejudice. His parents could only manage to send him to St Cyprians, a second-rate preparatory school, where, unknown to Orwell, he was kept at half-fees.* He eventually overcame his problems at St Cyprians and won a scholarship to Eton, where he experienced upper-class snobbery at first hand. T.R. Fyvel accurately articulates the effect Eton had on the development of Orwell’s outlook when he says that “Eton also gave him a sharp idea of how the English class system worked and who was truly of the English upper-class and who was not” (1982: 48). Orwell’s awareness of his parents’ lack of money and his experience at Eton changed his attitude from feelings of guilt - which caused him many hours of anguish and shame at St Cyprians - to a determined stubbornness. To excel at Eton meant to conform to the norms and expectations 5. Orwell’s reminiscences about his schooldays were published in a very negative essay, “Such, Such Were the Joys” (1982b: 379-422) and evoked severe criticism from his old school friends. 6. Years later Orwell expressed his guilt feelings about his parents’ poverty through Gordon Comstock, the protagonist of Keep the Aspidistra Flying: Even at the third-rate schools to which Gordon was sent nearly all the boys were richer than himself. They soon found out his poverty, of course, and gave him hell because of it. Probably the greatest cruelty one can inflict on a child is to send it to a school among children richer than itself. A child conscious of poverty will suffer snobbish agonies such as a grown-up person can scarcely imagine (1981: 46). 7. Cyril Connolly once summarized Orwell’s stubbornness at Eton as follows; “I was a stage rebel. Orwell was a real one” (1%1: 178). 8. POiJM: Partido Obrero de Unification Marxista (Worker’s Party of Marxist Unification). 10. Orwell often repeated this idea, two of the most prominent reiterations being: “... no book is genuinely free from political bias” (1982a: 26) and “But every writer, especially every novelist, has a ‘message’, whether he likes it or not, and the minutest details of his work are influenced by it. All art is propaganda” (1982a: 492). 9. These essays are “Why I Joined the Independent Labour Party” [1938] (1982a: 373- 375); “Why I Write” [1946] (1982a: 23-30); “The Prevention of Literature” [1946] (1980b: 81-95); “Politics vs. Literature: An Examination of Gulliver's Travels [1946] (1980b: 241-261) and “Writers and Leviathan” [1948] (1980b: 463-470). 3. Orwell’s ideology^ Ill of the upper-class and he firmly decided against this7 His career at Eton was an unglamorous one; no prizes, no university entrance, and no position in the Civil Service. Orwell’s experience at Eton, poignantly emphasized by the following statement - “The English class system is so subtle an instrument of discomfort that its victims can suffer pains quite imperceptible to those on the receiving end of a sharper divisiveness. Orwell was a connoisseur of social shame both upwards and downwards: a poor boy at Eton, an Etonian among the poor” (Hope, 1971:10) - formed the basis of his hatred of everything which had to do with the English class system. While his ideology found its most coherent articulation in The Road to Wigan Pier (1980), it was given impetus by his experiences as a policeman in Burma and as an unemployed writer-to-be, “slumming” in Paris and London during the thirties. Orwell, as spokesman for the working-class, fought against everything upper-class and stated his intentions quite clearly: For perhaps ten years past [1928-38] I have had some grasp of the real nature of capitalist society. I have seen British imperialism at work in Burma and I have seen something of the effects of poverty and unemployment in Britain. In so far as I have struggled against the system, it has been mainly by writing books which I hoped would influence the reading public. I shall continue to do that... (1982a: 374) His experiences during the Spanish Civil War and the insights he gained proved to be a major formative influence. His first-hand contact with Communist propaganda made a deep impression on his developing ideas. This is emphasized by Stansky and Abrahams: “At the heart of his socialism ... was a belief in honour and decency. And the way in which the POUM® was being misrepresented by its enemies seemed to him indecent and dishonourable: the politics of lying, the malignant distortions of language” (1981: 229). 4. Orwell’s “Poetics of Commitment” 4. Orwell’s “Poetics of Commitment” Orwell wrote a great deal about the relationship between a writer and his 112 society. 3. Orwell’s ideology^ Gerard Manley Hopkins) is really, if one looks closely, making a desperate attempt to use them straightforwardly. Whereas a writer who seems to have no tricks whatever... [is] making an especially subtle flank attack upon positions that are impregnable from the front. (1980a: 19-20; my italics - AMdeL) When one bears all this in mind, the representation of Orwell’s ideology becomes much more transparent in his rereading and unmasking of the subtleties and falsehoods of language and upper-class ideology in such apparently innocent texts as “Boys’ Weeklies”. 3. Orwell’s ideology^ If one looks at the six key essays® regarding this relationship one can almost posit an Orwellian “poetics of commitment” which will delimit the parameters within which he operated and help one to grasp the subtleties of his approach as well as the pitfalls which even a writer as subtle and cunning as Orwell cannot escape. Each writer, says Orwell, has a desire not to become involved in politics, but is inevitably drawn into making his allegiances public because of the increasing attack on freedom of speech. “All issues are political issues, and politics itself is a mass of lies, evasions, folly, hatred and schizophrenia” (Orwell, 1980b; 167). Because a writer [in the 20th century] lives in a tumultuous age, his spiritual hinterland will be one from which he cannot escape, one which forces a writer to become a committed writer. The age determines that there is no such thing as “non-political literature” (Orwell, 1980b: 88). Eagleton (1986: 59) underscores Orwell’s view when he states that in cases where authorial ideologies are in conflict with a dominant general ideology, “their modes of ideological disinheritance from that contemporary historical moment are determined, in the last instance, by the nature of that moment itself’. Given the situation that all issues are political issues, a modern writer should use his work to guide his readers in a particular direction. In stating four universal motives which guide all writers, Orwell states emphatically that one of these motives is “... a desire to push the world in a certain direction, to alter other people’s idea of the kind of society that they should strive after” (1982a: 26). The committed writer should strive to transmit his message which is inherent in every text and from which no text can escape clearly and unequivocally. If a committed writer has set himself these aims, how then should he go about changing the readers’ attitudes? Orwell says that a writer should not consciously tell lies, but should report events truthfully, “or as truthfully as is consistent with the ignorance, lies and self-deception from which every observer necessarily 113 suffers” (1980b: 83). Comhiitted writing is, says Orwell: Largely a perversion of words, and I would even say that the less obvious the perversion is, the more thoroughly it has been done. For a writer who seems to twist words out of their meanings (e.g. 11. Orwell’s hatred of the class system is nowhere as poignantly articulated as in the following passage in The Road to Wigan Pier 5. Orwell’s (re)reading of “Boys’ Weeklies” 114 generating of “a complete fantasy life” (Hunter, 1984: 112) and for bemg “fantastically unlike life at a real public-school” (Orwell, 1982a: 507) as no mention is made of the beatings and humiliation suffered by the boys. On the other hand the glamour and paraphernalia of the public-school, the “lock-up, roll-call, house matches, fagging, speeches [and] cosy teas round the study fire” (1982a: 511) are exploited fully and presented in such a way that life at a public school becomes an ideal for the readers. Upper-class names, manners and moral abound: Talbot, Manners and Lowther are frequently used and readers are often reminded of the presence of titled boys: “Gussy is the honourable Arthur A. D’Arcy, son of Lord Eastwood ... Jack Blake is heir to ‘broad acres’ ... Hurree Jamset Ram Singh (nicknamed Inky) is the Nabob of Bhanipur ... [and] Vernon-Smith’s father is a millionaire” (1982a: 511). Their speech mannerisms are typical of upper-class speech, albeit in a caricatured form: “Bai Jove! This is a go, deah boy!... I have been thrown into quite a fluttah! Oogh! The wuffians! The fearful outsidahs!” (1982a: 508). Orwell sees the underlying morality as similar to that of the Boy Scout Movement.'^ The “good” boys are good in the “cleanliving Englishman tradition - they keep in hard training, wash behind their ears, never hit below the belt” (1982a: 509), while the “bad” boys, on the other hand, regularly visit pubs, smoke and place bets. Orwell’s ridicule of upper-class ideology is palpable. As stated earlier, it is interesting to note that Orwell’s “re-reading” of “Boys’ Weeklies” provoked quite a caustic retort from Frank Richards, the author of many of the stories in the Gem and Magnet. If one studies Richard’s rebuttal closely, his own personal ideology is clearly represented in a way which exposes the points at which Orwell’s underlying ideology differs from that of Richards. Thus by contrasting the two writers’ views, the differences, strengths and weaknesses of each writer’s ideology are exposed. In answering to Orwell’s charges of cleanliness and snobbishness, Richards implicitly confirms the fact that the weeklies are produced from within a particular ideological framework. His sarcastic retort - “Now, although Mr Orwell may not suspect is, the word ‘aristocrat’ has not wholly lost its original Greek meaning. 12. A Boy Scout had to “promise that on his honour he would do his best to do his duty to God and his country (or sovereign), to help other people at all times and to obey the scout law, itself a code of chivalrous behaviour easily understood by the boy” (Corbett, 1971: 41). 5. Orwell’s (re)reading of “Boys’ Weeklies” The main theme of the weekly stories Orwell selects for scrutiny is the public- school life at the imaginary schools of Greyfriars and St Jim’s, which are depicted as “ancient and fashionable foundations of the type of Eton or Winchester” (1982a: 507). Orwell raises a number of objections to this theme: the harsh realities of life that had to be endured as a result of the British class system'* allow no places for fantasy, not even in a boys’ paper. He objects to the 11. Orwell’s hatred of the class system is nowhere as poignantly articulated as in the following passage in The Road to Wigan Pier The train bore me away, through the monstrous scenery of slagheaps, chimneys, piled scrap-iron, foul canals, paths of cindery mud criss-crossed by the prints of clogs. This was March, but the weather had been horribly cold and everywhere there were mounds of blackened snow. As we moved slowly through the outskirts of the town we passed row after row of little grey slum houses turning at right angles to the embankment. At the back of one of the houses a young woman was kneeling on the stones, poking a stick up the leaden waste-pipe which ran from the sink inside and which I suppose was blocked. I had time to see everything about her - her sacking apron, her clumsy clogs, her arms reddened by the cold. She looked up as the train passed, and I was almost near enough to catch her eye. She had a round pale face, the usual exhausted face of the slum girl who is twenty-five and looks forty, thanks to miscarriages and drudgery; and it wore, for the second in which I saw it, the most desolate, hopeless expression I have ever seen. It struck me then that we are mistaken when we say that ‘it isn’t the same for them as it would be for us’, and that the people bred in the slums can imagine nothing but the slums. For what I saw in her face was not the ignorant suffering of an animal. She knew well enough what was happening to her - understood as well as I did how dreadful a destiny it was to be kneeling there in the bitter cold, on the slimy stones of a slum backyard, poking a stick up a foul drain-pipe (1980: 16-17). 5. Orwell’s (re)reading of “Boys’ Weeklies” It is an actual fact that, in this country at least, nobleman are generally better fellows than commoners” (1982a: 535) - not only hints at Orwell’s own background, but also reveals the typical upper-class morality which Orwell has set out to expose. Richards, however, seems to realize the implications of such a stance a bit 115 further in his essay and weakens his argument by stating that “they [the workers] are not only the backbone of the nation; they are the nation”; all other classes being merely trimmings” (1982a: 536). This back-pedalling, if not downright contradiction, is an attempt prompted less by honest conviction than by hard headed expediency, to allay working class readers’ annoyance and only confirms Orwell’s stand that these weeklies are propagandist. Orwell’s criticism of the manners and morality shows that these are represented in the characters’ attitudes in ways that would influence readers to blithely and unquestioningly accept the political and social status quo. As such the weeklies are themselves instruments in the hands of their upper-class owners and the educational system. The obvious attempt to emphasize the difference in education merely serves to focus even more sharply on the crucial issue of wealth. The characters are portrayed in a way which, says Orwell, reinforces the position of the upper-class. The characters are the typical upper-class public school characters: there are the born leaders, the boy-assistants, the studious lads, the eccentrics and - of particular importance to Orwell’s argument - the scholarship boy: “Then there is the scholarshipboy, an important figure in this class of story because he makes it possible for the boys from the very poor homes to project themselves into the public school atmosphere” (1982a: 514). The autobiographical strain of this adds a certain poignancy to Orwell’s argument. He maintains further that while most of the characters are from the upper-class, the working class characters are presented only in an unfavourable light: “The working-classes only enter into the Gem and Magnet as comics or semi villains ... [they] appear as prize-fighters, acrobats, cowboys, professional footballers and Foreign Legionnaires - in other words, as adventurers. There is no facing of the facts about working-class life, or indeed, about working life of every description” (1982a: 526). Thus the unattainable ideal serves to impress their deprived status even upon their already conditioned minds. 5. Orwell’s (re)reading of “Boys’ Weeklies” Another accusation levelled at the weeklies is that, despite the fact that some of the characters are ostensibly working class, they are all living at several pounds a week above their income. And needless to say, that is just the impression that is intended... Not only is a five-to-six pound a week standard of life set up as the ideal, it is tacitly assumed that that is how the working class people really do live. The major faas are simply not faced. It is admitted, for instance, that people lose their jobs; but then the dark clouds roll away and they get better jobs instead. No mention is made of the dole, no mention of tradeunionism. No suggestion anywhere that there can be anything wrong with the system as a system. (Orwell, 1982a: 527) 116 It seems then as if Orwell is suggesting that the working class characters and their lifestyles are presented in ways which undermine the morale of the working class and make them feel inferior, their presentation being based on the assumption that “inferior” people will not challenge the validity of a system dominated by “superior” people. Richard’s (see Orwell, 1982a: 531-540) answers to these accusations serve as a clear indication of how Orwell’s ideology leads him to use the very methods of propaganda which he abhors. Richards states firmly that a misrepresentation of the working class would not only be bad manners, but bad business as well, as circulation figures depend on working class readers. Orwell seems to (conveniently?) lose sight of the aims of a boys’ paper’, namely entertainment and escape. Richards’s rhetorical “are these [strikes, slumps and unemployment] really subjects for young people to meditate upon?” (Orwell, 1982a: 537) underlines the fact that Orwell deliberately seems to “forget” what boys papers set out to do, or is deliberately distorting the real issues. However, Orwell’s analysis does not end with the weeklies only. He also turns his attention to the. readers, whom he divides into three significant groups: boys who attend public schools and read Gem and Magnet up to twelve years of age; boys at private schools who cannot afford to go to public schools and read the weeklies for much longer, lingering on the impossible dream of a public school education; and working class boys who work in offices, factories and mines and are enthralled by the glamour of the public schools. 5. Orwell’s (re)reading of “Boys’ Weeklies” Orwell argues that the weeklies are aimed at precisely the third group, many of whom will never read anything else other than newspapers, the assumption being that the readers with some subordinate job are led to identify with people in positions of command and that therefore they will never come to question “the system”. Certain political and social implications presented in these papers strike Orwell with a particular force, namely that nothing ever changes and that all foreigners arc funny. The ironical summary of the pre-1914 political attitude - “the King is on his throne and the pound is worth a pound” - conveying a lack of political evolution, forms a corollary to Orwell’s argument that the status quo will never be questioned. What is again significant is that there is no facing of realities, thus deluding their readers as to the nature of the adult world: “The outlook inculcated by all these papers is that of a rather stupid member of the Navy League in the year 1910” (Orwell, 1982a: 528). Hitler, the Nazis and the threat of Communism are only mentioned in occasional remarks: “The clock was stopped at 1910. Britannia rules the waves, and no-one has heard of slumps, 117 booms, unemployment, dictatorships, purges or concentration camps” (1982a; 525). booms, unemployment, dictatorships, purges or concentration camps” (1982a; 525). Richards’s ignorance with regard to dangers of totalitarianism is a point in Orwell’s favour. Note, however, that Orwell is pleading for the same things that are being done by the weeklies, namely indoctrination, albeit in a different form as he seems to be pleading that a more realistic approach in the weeklies will prepare (indoctrinate?) the youthful readers for the events to come. The attitude to foreigners derives from English group insularity and xenophobia’^ fostering the concomitant belief that everybody outside this group is necessarily a clown. The lack of social progress depicted in the weeklies runs parallel to the lack of political development. Any improvement in the work situation of the working class chiu^acters are depicted as resulting from the employers’ benevolence and not from honest, hard work. 13. Orwell’s critique of English xenophobia is developed further in “The English People” (1982b: 15-56). 6. Concluding remarks A study of Orwell’s accusations and an analysis of Richards’s replies clearly expose the ideological parameters of each. It also informs a central concern of Orwell’s, namely the use and abuse of language as a propaganda medium. Orwell’s Spanish experiences brought him into contact with Communist propaganda methods. In “Spilling the Spanish Beans” (Orwell, 1982a: 301-309) he mentions that Communist propaganda depends on “terrifying the people with the (quite real) horrors of Fascism” (1982a; 306). His criticism of the boys’ weeklies seems to imply that the reverse is also quite true, that is that the upper-class owners of the weeklies aim to foster an attitude of subordmation and dependence among working class readers by convincing them that the class system is not so unfair as is generally believed and that the relative welfare of the workers is the result of the upper class’ benevolent concern. As in Spain, where there was a deliberate conspiracy to prevent the real situation from being understood (1982a: 308), so a similar situation seems to exist in England: while the Communists in Spain instilled fear into the people and deliberately misinformed them, the British upper-class seems to prefer the far more subtle methods of “persuasion” through subtle emotional blackmail. Yet Orwell himself is not free from distortion, from “the powerful force hovering over us as we write or read a text” (McCormick and Waller, 1987: 197). In his essay “Boys’ Weeklies” Orwell uses methods of distortion to bring the weeklies into disrepute. He criticizes the weeklies for indoctrinating their readers to accept the class system and all its ramifications, ignoring obvious facts and emphasizing only that which will strengthen his argument. He criticizes the weeklies for their plagiarism (which in fact is shown to be Orwell’s lack of knowledge about Alice and Chaucer), being outdated and snobbish. Yet by championing the cause of the working class through ridiculing the things which the upper class hold dear - a love of titles, cleanliness of mind and patriotism - Orwell is paradoxically also being “snobbish” and class conscious, by only allowing the working class breathing space. “Boys’ Weeklies” is one of the best examples of how Orwell’s ideology encroaches on his approach and technique. While not denying the sharp focus and perspective of his observations and his skilful use of rhetorical devices, he grossly overstates his case. The prose is “like a window pane” (1982a: 30). 5. Orwell’s (re)reading of “Boys’ Weeklies” The clearest evidence of the representation of Orwell’s ideology can be found in his artistic perversion of w or^, his ability to manipulate words and phrases in such a confident and assertive way that the reader is almost certain to believe him without questioning the truth of validity of his statements. He criticizes the style and idiom of the stories, pointing out their tautologous nature. The following few examples will suffice to prove the point; note how Orwell tries to persuade his readers by leaning heavily on loaded adjectives, rhetorical questions and bland assertions: So far as / know, there are extremely few stories in foreign languages (1982a: 511; my italics - AMdeL). So far as / know, there are extremely few stories in foreign languages (1982a: 511; my italics - AMdeL). All I can say from my own observations is this... (1982a: 512; my italics - AMdeL). Needless to say, these stories are fantastically unlike life at a real public school (1982a: 509; my italics - AMdeL). Needless to say, these stories are fantastically unlike life at a real public school (1982a: 509; my italics - AMdeL). Needless to say, these stories are fantastically unlike life at a real public school (1982a: 509; my italics - AMdeL). This kind of thing is a perfectly deliberate incitement to wealth fantasy (1982a: 511; my italics - AMdeL). The reason, obviously, is that in England education is a matter of status (1982a: 511; my italics - AMdeL). 13. Orwell’s critique of English xenophobia is developed further in “The English People” (1982b: 15-56). 118 These examples serve as illustrations of the practical manifestations of Orwell’s ideological assumptions. By retracing his steps through his argument, as it were, one is able to identify and unmask the words and concepts which embody his ideological presuppositions. 6. Concluding remarks Orwell’s spectacles, however, do not seem to be quite as spotless. 119 Bibliography Adomo. T.W. 1962. Commitment (In Arato, A. and Gebhardt, E. (eds). 1978. The Essential Frankfurt School Reader. Oxford: Blackwell). f ) Arato, A. and Gebhardt, E. (eds). 1978. The Essential Frankfurt School Reader. Oxford; Blackwell. Bachrach, A.G.H. et. al. 1980. 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Nuttall, A.D. 1984. A New Mimesis: Shakespeare a,td the Representation of Reality. London: Methuen. Orwell, G. 1981. Keep the Aspidistra Flying. Harmondsworth: Penguin. Orwell, G. 1980. The Road to Wigan Pier. Harmondsworth: Penguin. Orwell, Sonia and Angus, L (eds). 1980a. The Collected Essays, Journalism and Letters of George Orwell. Vol. 3. Harmondsworth: Penguin. f g g Orwell, Sonia and Angus, L (eds). 1980b. The Collected Essays, Journalism and Letters of George Orwell. Vol. 4. Harmondsworth: Penguin. of George Orwell. Vol. 4. Harmondsworth: Penguin. f g g Orwell, Sonia and Angus, L (eds). 1982a. The Collected Essays, Journalism and Letters of George Orwell. Vol. 1. Harmondsworth; Penguin. Orwell, Sonia and Angus, L (eds). 1982a. The Collecte Orwell, Sonia and Angus, L (eds). 1982a. The Collecte of George Orwell. Vol. 1. Harmondsworth; Penguin. of George Orwell. Vol. 1. Harmondsworth; Penguin. f g g Onvell, Sonia and Angus, L (eds). 1982b. 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https://openalex.org/W4249003678	https://bmcnephrol.biomedcentral.com/track/pdf/10.1186/s12882-020-02190-0	English	null	Efficacy and safety of the long-acting C5 inhibitor ravulizumab in patients with atypical hemolytic uremic syndrome triggered by pregnancy: a subgroup analysis	Research Square (Research Square)	2,020	cc-by	5,921	Open Access Efficacy and safety of the long-acting C5 inhibitor ravulizumab in patients with atypical hemolytic uremic syndrome triggered by pregnancy: a subgroup analysis Anja Gäckler1, Ulf Schönermarck2, Vladimir Dobronravov3, Gaetano La Manna4, Andrew Denker5, Peng Liu5, Maria Vinogradova6, Sung-Soo Yoon7 and Manuel Praga8 © The Author(s). 2021, corrected publication 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/ licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: Atypical hemolytic uremic syndrome (aHUS) triggered by pregnancy is a rare disease caused by dysregulation of the alternative complement pathway that occurs in approximately 1 in 25,000 pregnancies. The 311 phase 3 trial (NCT02949128) showed that ravulizumab, a long-acting C5 inhibitor obtained through selective modifications to eculizumab, is efficacious in inhibiting complement-mediated thrombotic microangiopathy (TMA) in patients with aHUS. In this analysis, we report outcomes in a subgroup of patients from the 311 study who developed TMA postpartum. Methods: This was a phase 3, multicenter trial evaluating efficacy and safety of ravulizumab in adults (≥18 years of age) with aHUS naïve to complement inhibitor treatment. The primary endpoint was complete TMA response (simultaneous platelet count normalization [≥150 × 109/L], lactate dehydrogenase normalization [≤246 U/L] and 25% improvement in serum creatinine) through the 183-day initial evaluation period. Additional efficacy endpoints included time to complete TMA response, hematologic normalization, and dialysis requirement status. Results: Eight patients presenting with TMA postpartum (median age of 37.7 [range; 22.1–45.2] years) were diagnosed with aHUS and received ≥1 dose of ravulizumab. Five patients (63%) were on dialysis at baseline. Complete TMA response was achieved in 7/8 patients (87.5%) in a median time of 31.5 days. Hematologic normalization was observed in all patients. All patients on dialysis at baseline discontinued dialysis within 21 days after treatment with ravulizumab. All patients showed continued improvements in the estimated glomerular filtration rate from baseline to Day 183. Three possible treatment-related adverse events were observed in 2 patients (arthralgia and nasopharyngitis [both non-severe]; urinary tract infection). No deaths or meningococcal infections occurred. (Continued on next page) Correspondence: Anja.Gaeckler@uk-essen.de 1Department of Nephrology, University Hospital Essen, University Duisburg-Essen, Essen, Germany Full list of author information is available at the end of the article Gäckler et al. BMC Nephrology (2021) 22:5 https://doi.org/10.1186/s12882-020-02190-0 Gäckler et al. BMC Nephrology (2021) 22:5 https://doi.org/10.1186/s12882-020-02190-0 (Continued from previous page) (Continued from previous page) (Continued from previous page) Conclusions: Treatment with ravulizumab provided immediate and complete C5 inhibition, resulting in rapid clinical and laboratory improvements and complete TMA response through 183 days in patients with aHUS triggered by pregnancy. The safety profile observed in this subset of patients analysed is consistent with the 311 study investigating ravulizumab in patients with aHUS naïve to complement treatment. Trial registration: Clinical trial identifier: NCT02949128. Keywords: Ravulizumab, Thrombotic microangiopathy, Pregnancy microangiopathies, Atypical hemolytic uremic syndrome blocks terminal complement activation at C5 [11, 12], has significantly improved the clinical outcomes in pa- tients with aHUS. The efficacy and safety profile of ecu- lizumab has been demonstrated across 4 prospective clinical trials [13–17], registry and non-trial patient data [8, 18], including studies on patients with aHUS trig- gered by pregnancy [8, 19–21]. Ravulizumab (Alexion Pharmaceuticals, Inc., Boston, MA, USA) is a new long- acting monoclonal antibody obtained through selective modifications to eculizumab, allowing extended main- tenance dosing from every 2 to every 8 weeks [22]. Ravu- lizumab was recently approved for the treatment of adults and children with aHUS in the United States [23]. The phase 3 ALXN1210-aHUS-311 clinical trial (hereon referred to as ‘311’) demonstrated the efficacy and safety profile in a cohort of 56 adults with aHUS naïve to complement inhibitor therapy treated for acute TMA [24]. The purpose of this analysis is to report both clinical characteristics and outcomes in a subset of 8 pa- tients with aHUS from the 311 study who presented postpartum and received ravulizumab. This is the largest cohort of patients from a clinical trial to evaluate the ef- ficacy and safety of C5 inhibitors in postpartum aHUS. Background blocks terminal complement activation at C5 [11, 12], has significantly improved the clinical outcomes in pa- tients with aHUS. The efficacy and safety profile of ecu- lizumab has been demonstrated across 4 prospective clinical trials [13–17], registry and non-trial patient data [8, 18], including studies on patients with aHUS trig- gered by pregnancy [8, 19–21]. Ravulizumab (Alexion Pharmaceuticals, Inc., Boston, MA, USA) is a new long- acting monoclonal antibody obtained through selective modifications to eculizumab, allowing extended main- tenance dosing from every 2 to every 8 weeks [22]. Ravu- lizumab was recently approved for the treatment of adults and children with aHUS in the United States [23]. Atypical hemolytic uremic syndrome (aHUS) is a rare, life-threatening disease caused by dysregulation of the alternative complement pathway, presenting as throm- botic microangiopathy (TMA; hemolytic anemia, thrombocytopenia, and organ injury – usually the kid- ney) [1, 2]. Over the past few years, there is increasing consensus that, in the majority of patients, aHUS may involve both genetic predisposition (e.g., pathogenic vari- ants, autoantibodies or at-risk polymorphisms in com- plement genes) and a triggering condition in order for the clinical event of a TMA to occur [3–5]. However, a genetic predisposition is not always identified and is not required for diagnosis. Pregnancy is a known triggering condition for the manifestation of the disease. aHUS triggered by preg- nancy is a rare and under-recognized complement- mediated TMA [5], occurring in approximately 1 in 25,000 pregnancies [6] and in 4% of diagnosed cases of aHUS [3]. The onset of aHUS triggered by pregnancy is more common in the postpartum period (79%) [7], but can also occur in the peripartum period. Diagnosis is often difficult because aHUS shares similar clinical fea- tures to other syndromes triggered by pregnancy, includ- ing ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) deficiency-associated thrombotic thrombocytopenic pur- pura (TTP), pre-eclampsia, and HELLP (hemolysis, ele- vated liver enzymes and low platelets) syndrome [1]. © The Author(s). 2021, corrected publication 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/ licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. (2021) 22:5 Gäckler et al. BMC Nephrology (2021) 22:5 Page 2 of 9 Gäckler et al. BMC Nephrology Trial oversight and study design Safety and tolerability of ravulizumab were evaluated by clinical and laboratory assessment and frequency of adverse events (AEs) and serious AEs (SAE). Full details on the study methodology have been previously described in detail in the primary analysis [24]. Patients must have received meningococcal vaccin- ation according to local and national guidelines at the time of commencing therapy and were also required to receive antibiotic prophylaxis from the time of first dose of ravulizumab until at least 2 weeks after vaccination. Trial oversight and study design The 311 clinical trial (NCT02949128) is a phase 3, single arm, multicenter study designed to evaluate the efficacy and safety of ravulizumab administered by intravenous (IV) infusion to adults with aHUS naïve to complement inhibitor treatment. Patients from the trial included in this analysis were ≥18 years of age and weighed ≥40 kg with active TMA (thrombocytopenia, evidence of hemolysis and kidney dysfunction) at postpartum. All patients included in this analysis had evidence of TMA lasting for ≥3 days. Patients could meet the platelet and lactate dehydrogenase (LDH) criteria (<150 × 109 and ≥1.5 x upper limit of normal, respectively) based on results from local laboratories, but the serum creatinine criteria (≥upper limit of normal) must have been con- firmed by the central laboratory at baseline. aHUS triggered by pregnancy is considered a medical emergency, requiring hospitalization and prompt initi- ation of appropriate treatment for optimal maternal out- comes. Overall, the disease course in aHUS triggered by pregnancy and aHUS not related to pregnancy is similar, with some studies reporting that two-thirds of patients require dialysis at onset, and more than half reach end- stage renal disease (ESRD) within 1 month of onset [8], despite treatment with corticosteroids or plasma ex- change [9]. Generally, outcomes for patients with aHUS triggered by pregnancy are poor without terminal com- plement inhibitor treatment [10]. The study consisted of an initial evaluation period of 183 days. Ravulizumab was administered via IV loading Eculizumab (Alexion Pharmaceuticals, Inc., Boston, MA, USA), a humanized monoclonal antibody that Page 3 of 9 Page 3 of 9 Gäckler et al. BMC Nephrology Gäckler et al. BMC Nephrology (2021) 22:5 Page 3 of 9 dose of 2400 mg, 2700 mg or 3000 mg in patients weigh- ing ≥40–< 60 kg, ≥60–<100 kg, or ≥100 kg, respectively, on Day 1. Maintenance doses of 3000 mg, 3300 mg, 3600 mg, respectively, were administered on Day 15 and then every 8 weeks thereafter. Baseline was defined as the period of screening up to before the point of the first study drug infusion, including Day 1 (study design de- scribed previously in detail [24, 25]). dialysis requirement status. Exploratory genetic analysis by whole exome sequencing was conducted on patients in the original study. Additional genetic analyses per- formed at the centers treating the individual patients in this analysis were also included. Patient characteristics Patients with ADAMTS13 deficiency (activity <5%); Shiga toxin-producing Escherichia coli-HUS; hematopoietic stem cell transplantation in the 6 months prior to screening and history of malignancy within 5 years of screening were excluded. Patients who had received complement inhibitors, immunosuppressive therapies (except for kidney transplant regimens), steroids, patients who received tranexamic acid within 7 days, and patients on chronic dialysis were also excluded. Plasma exchange/infusion (PE/PI) was allowed up to, but not after, the first dose of ravulizumab, but patients were excluded if therapy exceeded 28 days. Eight postpartum patients with a median age of 37.7 (range; 22.1–45.2) years met the inclusion criteria, were enrolled and received ≥1 dose of ravulizumab. None of the patients reported breastfeeding during the study. Baseline demographics and disease characteristics are shown in Table 1. At baseline, and prior to any ravulizumab dose, all pa- tients presented with acute, severe medical emergency associated with the pregnancy or delivery. All patients had complicated deliveries and most also had antenatal complications. Pre-eclampsia and hypertension were re- ported in 2 patients each, and renal failure and gesta- tional diabetes in 1 patient each. Two patients suffered placental abruption, resulting in antenatal fetal death in 1 of these cases. Five patients underwent emergency cesarean section with complications occurring in 4 cases; hemorrhage in 2, secondary hysterectomy in 2 (1 with hemorrhage) and fetal death in 1 case. Additional data on the events prior to TMA are detailed in Supplemen- tary Table 1. The protocol was approved by the Institutional Review Board or Independent Ethics Committee at each partici- pating center, and the study was conducted in accord- ance with the Declaration of Helsinki and the Council for International Organizations of Medical Sciences International Ethical Guidelines. Written informed con- sent was obtained from all individual participants or legal guardians, as applicable. All patients completed the 183-day initial evaluation period with no study or drug discontinuations. Efficacy and safety endpoints Full details of the efficacy and safety endpoints can be found in the primary analysis publication [24]. The pri- mary efficacy endpoint was complete TMA response through an initial evaluation period of 183 days. The cri- teria for complete TMA response were platelet count normalization (≥150 × 109/L), LDH normalization (≤246 U/L) and ≥25% improvement in serum creatinine from baseline met concurrently and at two separate assess- ments ≥28 days apart, and any measurement in between. When a patient was on dialysis at baseline, the baseline value used for serum creatinine response assessment was the first value at 6 or more days post-dialysis. Patients were considered as being on dialysis at baseline if dialy- sis occurred within 5 days prior to ravulizumab initiation. Safety Adverse events post-treatment initiation were observed in all 8 patients included in the analysis (Table 2). The most common adverse events reported in at least 2 pa- tients were headache and pyrexia (occurring in 3 pa- tients each). Other adverse events reported in 2 patients Data not available aBaseline values may be after PE/PI in some patients. beGFR in patients on dialysis was set to 10 mL/min/1.73 m2, and eGFR was calculated using the Modification of Diet in Renal Disease formula. Data displayed as n (%) unless otherwise stated ADAMTS13 a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13, aHUS atypical hemolytic uremic syndrome, CFB complement factor B, CFH complement factor H, eGFR estimated glomerular filtration rate, HGB hemoglobin, ICU intensive care unit, LDH lactate dehydrogenase, PE plasma exchange, PI plasma infusion, TMA thrombotic microangiopathy Secondary endpoints each included constipation, urinary tract infection, naso- pharyngitis, alopecia, hypertension, arthralgia, increased alanine aminotransferase, and increased aspartate ami- notransferase. Three possible treatment-related adverse events (as determined by the investigator) were noted in 2 patients (arthralgia and nasopharyngitis [both non- severe]; urinary tract infection). Both patients recovered from these events. One serious adverse event was re- ported. This event was a routine renal biopsy unrelated to treatment with ravulizumab. No deaths or meningo- coccal infections occurred. The median (95% CI) time to complete TMA response was 31.5 (9.0, 46.0) days (Fig. 1). Hematologic normalization, platelet count and LDH normalization were observed in all patients (100%). Platelet count, LDH and eGFR all improved rapidly (Fig. 2, Fig. 3, Fig. 4). All patients on dialysis at baseline were able to discontinue dialysis within 21 days of commencing treat- ment with ravulizumab. Primary endpoint h During the initial evaluation period, 7 of 8 patients (87.5%) met the primary endpoint of complete TMA response (Fig. 1). The patient that did not achieve complete TMA response had a rapid response to ravulizumab treatment, with normalization of both platelets and LDH on Day 8. She had a dialysis ses- sion 5 days before first dose, and baseline creatinine was the value obtained on Day 8 (≥6 days after last dialysis session as defined by protocol). On Day 8, this patient had already shown improved serum cre- atinine levels to 51 μmol/L, which is well within the normal range, and an additional improvement of 25% in serum creatinine from this value was not reached, which would have been the requirement to meet complete TMA response criteria. Secondary objectives of the study included time to complete TMA response; change in hematologic vari- ables (platelets, LDH, and hemoglobin); change in esti- mated glomerular filtration rate (eGFR) values; and Page 4 of 9 Gäckler et al. BMC Nephrology (2021) 22:5 Gäckler et al. BMC Nephrology Secondary endpoints each included constipation urinary tract infection naso Table 1 Baseline demographics and disease characteristics Variable Overall (N = 8) Median (min, max) age at first infusion, years 37.7 (22.1, 45.2) Age at time of first infusion (years) category 18 to <30 years 2 (25.0) 30 to <40 years 3 (37.5) 40 to <50 years 3 (37.5) Race Asian 1 (12.5) White 7 (87.5) ADAMTS13 activity >5% 8 (100) Extrarenal signs or symptoms of aHUS prior to first infusion of drug 6 (75) Baseline laboratory values, median (min, max) a Platelet count, ×109/L 119 (36, 473) LDH, U/L 576 (280, 876) Serum creatinine, μmol/L 408 (51, 758) HGB, g/L 72.8 (63, 105.5) eGFR, mL/min/1.73 m2(b) 10.0 (10, 18) Median (min, max) time from delivery to first dose, days 11 (5, 19) ICU care required 7 (87.5) Median (min, max) stay in ICU, days 9 (2, 21) Received PE/PI related to this TMA prior to first infusion of drug 6 (75) On dialysis within 5 days of first dose 5 (62.5) Median (min, max) time on dialysis prior to first dose, days 5 (4, 8) Patients with ≥1 identified pathogenic variant or autoantibody 2 (25) CFB pathogenic variant 1 (12.5) Anti-CFH antibodies 1 (12.5) None identified 5 (65.5) Data not available 1 (12.5) aBaseline values may be after PE/PI in some patients. Primary endpoint h beGFR in patients on dialysis was set to 10 mL/min/1.73 m2, and eGFR was calculated using the Modification of Diet in Renal Disease formula. Data displayed as n (%) unless otherwise stated ADAMTS13 a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13, aHUS atypical hemolytic uremic syndrome, CFB complement factor B, CFH complement factor H, eGFR estimated glomerular filtration rate, HGB hemoglobin, ICU intensive care unit, LDH lactate dehydrogenase, PE plasma exchange, PI plasma infusion, TMA thrombotic microangiopathy Age at time of first infusion (years) category CFB pathogenic variant Anti-CFH antibodies None identified Discussion This is the largest cohort of patients from a prospective interventional study to evaluate the efficacy and safety of C5 inhibitors in postpartum aHUS. All patients Gäckler et al. BMC Nephrology (2021) 22:5 Gäckler et al. BMC Nephrology Page 5 of 9 Fig. 1 Kaplan–Meier graph depicting time to complete TMA response. BL baseline, TMA thrombotic microangiopathy patient could not achieve a 25% improvement in serum creatinine levels required to fulfil the primary endpoint. No safety concerns were identified in this study. presented in a severe condition; 62.5% of patients re- quired dialysis at baseline and 87.5% of patients required ICU level care, similar to other reports detailing patients with aHUS triggered by pregnancy [7]. All patients responded rapidly to treatment with ravulizumab, with a median time to complete TMA response of 31.5 days. Although one patient did not achieve the primary end- point of TMA response, this patient had a rapid re- sponse to ravulizumab treatment, with normalization of both platelets and LDH by Day 8. Serum creatinine levels were within normal range on Day 8, thus the The data obtained in this subset analysis show that a higher proportion of patients presenting postpartum re- solved TMA with ravulizumab treatment compared to the full cohort of patients with aHUS in the 311 study [24]. Patients in this analysis received ravulizumab treat- ment soon after delivery (median 11 [range, 5–19] days), whereas the time to treatment in the overall 311 study was broader, with patients receiving their first dose of Fig. 2 Observed platelet count value over time. Data are shown as mean (error bars, 95% confidence interval). BL baseline Gäckler et al. BMC Nephrology (2021) 22:5 Page 6 of 9 Gäckler et al. BMC Nephrology Fig. 3 Observed lactate dehydrogenase values over time. Data are shown as mean (error bars, 95% confidence interval). BL baseline treatment within 7 days of disease manifestation than in patients initiating treatment after 7 days [26]. Although, by the criteria of the study, 1 patient did not achieve a complete TMA response with ravulizumab, this patient responded to treatment and improved in all clinical pa- rameters, including serum creatinine levels, normalization of platelets and LDH by Day 8, and complete recovery of renal function at last follow-up. ravulizumab as early as at the onset of symptoms or as late as 215 months after the first symptom of aHUS, highlighting the importance of early treatment. Supplementary Information Th li i i l Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12882-020-02190-0. The online version contains supplementary material available at https://doi. org/10.1186/s12882-020-02190-0. Additional file 1: Supplementary Table S1. Patients clinical profile. Additional file 2. The PLS (Patient Lay Summary). Additional file 1: Supplementary Table S1. Patients clinical profile. Additional file 2. The PLS (Patient Lay Summary). The proportion of treatment-related AEs in the full 311 cohort was similar to that of the subgroup of pa- tients in this analysis (34.5 and 37.5%, respectively). The current subgroup did not show a preponderance of any type of safety signal. In the full 311 cohort the most common AEs were headache, diarrhea, and vomiting [24], while headache and pyrexia were the most com- mon in this subgroup. No patients died or contracted a meningococcal infection. Abbreviations ADAMTS13: A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; AEs: Adverse events; aHUS: Atypical hemolytic uremic syndrome; BL: Baseline; CFB: Complement factor B; CFH: Complement factor H; CI: Confidence interval; eGFR: Estimated glomerular filtration rate; Esrd: End-stage renal disease; Hellp: Hemolysis, elevated liver enzymes and low platelets; HGB: Hemoglobin; ICU: Intensive care unit; IV: Intravenous; LDH: Lactate dehydrogenase; PE: Plasma exchange; PI: Plasma infusion; SAE: Serious adverse event; TEAE: Treatment-emergent adverse event; TESAE: Treatment-emergent serious adverse event; TMA: Thrombotic microangiopathy; TTP: Thrombocytopenic purpura This study has limitations that must be noted. aHUS is an ultra-rare disease with an estimated prevalence of 4.9 per million and annual incidence rate between 0.23–1.9 per million [27]. As postpartum patients account for only around 4% of diagnosed cases of aHUS [3], there is not a large enough pool of complement inhibitor-naïve Table 2 Summary of adverse events Table 2 Summary of adverse events Table 2 Summary of adverse events Overall (N = 8) AE Categories n (%) Events Any AE 8 (100.0) 71 Any SAE 1 (12.5) 1 Fatal TEAEs 0 (0.0) 0 TEAEs or TESAEs resulting in drug discontinuation 0 (0.0) 0 TEAEs or TESAEs resulting in study discontinuation 0 (0.0) 0 Meningococcal infections 0 (0.0) 0 Treatment-related AEs (all considered possibly-related) 2 (25.0) 3 AE adverse event, SAE serious adverse event, TEAE treatment-emergent adverse event, TESAE treatment-emergent serious adverse event Regarding genetic analysis, we found no association between the identification of a pathogenic complement abnormality and response to ravulizumab (1 patient had a complement factor B (CFB) variant and 1 had anti- complement factor H (CFH) antibodies). The subgroup of patients analyzed here had complicated deliveries with significant bleeding, which could have triggered the syn- drome, as suggested in other studies [8]. One patient with a severe predisposition (CFB pathogenic variant) had a normal delivery, whereas three patients with no identified pathogenic variants had severe bleeding com- plications, hypertension or pre-eclampsia, and conse- quently more severe clinical presentations. Five patients underwent emergency deliveries, 4 of which were by cesarean sections (a fifth patient also had a previously planned cesarean with complications postoperatively). Based on these observations in this subgroup and with only 2 patients testing positive for pathogenic variants or autoantibodies in our study, we hypothesize that patients with a severe genetic predisposition do not necessarily need a severe trigger to develop aHUS, whereas patients with a more severe trigger might not need an identified pathogenic variant in order to develop the disease. Nevertheless, all patients in this analysis had a severe presentation, regardless of genetic background and responded well to ravulizumab treatment. patients presenting postpartum to conduct a placebo- controlled trial. Currently this sub-analysis is the largest postpartum dataset utilized in a prospective study of pa- tients with aHUS; no comparator or control group was utilized for the full 311 cohort, meaning that conclusions drawn from this subanalysis must be interpreted with caution. Further studies, if possible, with larger sample sizes, are required to fully confirm these findings. Conclusion In this first prospective interventional trial assessing the efficacy and safety of the long-acting C5 inhibitor ravuli- zumab, TMA caused by aHUS was rapidly resolved in the subset of postpartum patients, with continued im- provement over time and an acceptable safety profile. The results from this subset analysis suggest that ravuli- zumab is effective with a favorable safety profile in women presenting with aHUS postpartum. Discussion More- over, the median time to complete TMA response in this subgroup of patients was shorter than the full cohort in the 311 study (31.5 days vs 86 days). Previous clinical trial data have demonstrated that renal outcomes are better in patients initiating complement inhibitor Fig. 4 Observed eGFR values over time. Data are shown as mean (error bars, 95% confidence interval). BL baseline, eGFR estimated glomerular filtration rate ig. 4 Observed eGFR values over time. Data are shown as mean (error bars, 95% confidence interval). BL baseline, eGFR esti tration rate Page 7 of 9 Gäckler et al. BMC Nephrology (2021) 22:5 Ethics approval and consent to participate The protocol was approved by the Institutional Review Board or Independent Ethics Committee at each participating center, and the study was conducted in accordance with the Declaration of Helsinki and the Council for International Organizations of Medical Sciences International Ethical Guidelines. Written informed consent was obtained from all individual participants or legal guardians, as applicable. 10. Vinogradova M, Kirsanova T, Shmakov R: Thrombotic microangiopathies associated with pregnancy: diagnostic and therapeutic challenges. Blood. 2018;132(Suppl 1):4810. 11. EMA: EU/3/09/653. Available at: https://www.ema.europa.eu/en/medicines/ human/orphan-designations/eu309653 [Accessed Jan, 2019]. 2011. 11. EMA: EU/3/09/653. Available at: https://www.ema.europa.eu/en/medicines/ human/orphan-designations/eu309653 [Accessed Jan, 2019]. 2011. 12. FDA: Eculizumab (Soliris). Available at: http://wayback.archive-it.org/7993/201 70113081126/http://www.fda.gov/AboutFDA/CentersOffices/ 12. FDA: Eculizumab (Soliris). Available at: http://wayback.archive-it.org/7993/201 70113081126/http://www.fda.gov/AboutFDA/CentersOffices/ OfficeofMedicalProductsandTobacco/CDER/ucm273089.htm [Accessed Jan, 2019]. 2011. References 1. Scully M. Thrombotic thrombocytopenic Purpura and atypical hemolytic uremic syndrome Microangiopathy in pregnancy. Semin Thromb Hemost. 2016;42(7):774–9. 2. Fakhouri F, Zuber J, Fremeaux-Bacchi V, Loirat C. Haemolytic uraemic syndrome. Lancet. 2017;390(10095):681–96. 2. Fakhouri F, Zuber J, Fremeaux-Bacchi V, Loirat C. Haemolytic uraemic syndrome. Lancet. 2017;390(10095):681–96. Authors’ contributions AG, US, VD, GLM, AD, PL, MV, S-SY, and MP discussed and agreed the con- tent. The first draft of this manuscript was developed by the medical writer under guidance from AG, US, VD, GLM, AD, PL, MV, S-SY, and MP. AG, US, VD, GLM, AD, PL, MV, S-SY, and MP reviewed and provided comprehensive feed- back of all manuscript drafts and approved the final version for journal submission. Availability of data and materials Alexion will consider requests for disclosure of clinical study participant-level data provided that participant privacy is assured through methods like data de-identification, pseudonymization, or anonymization (as required by applic- able law), and if such disclosure was included in the relevant study informed consent form or similar documentation. Qualified academic investigators may request participant-level clinical data and supporting documents (statis- tical analysis plan and protocol) pertaining to Alexion-sponsored studies. Fur- ther details regarding data availability and instructions for requesting information are available in the Alexion Clinical Trials Disclosure and Trans- parency Policy at http://alexion.com/research-development. Link to Data Request Form (https://alexion.com/contact-alexion/medical- information). 6. Dashe JS, Ramin SM, Cunningham FG. The long-term consequences of thrombotic microangiopathy (thrombotic thrombocytopenic purpura and hemolytic uremic syndrome) in pregnancy. Obstet Gynecol. 1998;91(5 Pt 1): 662–8. 7. Fakhouri F, Roumenina L, Provot F, Sallee M, Caillard S, Couzi L, Essig M, Ribes D, Dragon-Durey MA, Bridoux F, et al. Pregnancy-associated hemolytic uremic syndrome revisited in the era of complement gene mutations. J Am Soc Nephrol. 2010;21(5):859–67. 8. Bruel A, Kavanagh D, Noris M, Delmas Y, Wong EKS, Bresin E, Provot F, Brocklebank V, Mele C, Remuzzi G, et al. Hemolytic uremic syndrome in pregnancy and postpartum. Clin J Am Soc Nephrol. 2017;12(8):1237–47. Link to Data Request Form (https://alexion.com/contact-alexion/medical- information). 9. Gupta M, Govindappagari S, Burwick RM. Pregnancy-associated atypical hemolytic uremic syndrome: a systematic review. Obstet Gynecol. 2020; 135(1):46–58. Funding This study was funded by Alexion Pharmaceuticals, Inc. Boston, MA, US. Alexion Pharmaceuticals, Inc. (the sponsor) was responsible for the study design, funded the medical writing assistance, and provided a formal review of the publication. Authors retain control and final authority of publication content and decisions, including the choice of journal. Open Access funding enabled and organized by Projekt DEAL. 4. Noris M, Caprioli J, Bresin E, Mossali C, Pianetti G, Gamba S, Daina E, Fenili C, Castelletti F, Sorosina A, et al. Relative role of genetic complement abnormalities in sporadic and familial aHUS and their impact on clinical phenotype. Clin J Am Soc Nephrol. 2010;5(10):1844–59. 4. Noris M, Caprioli J, Bresin E, Mossali C, Pianetti G, Gamba S, Daina E, Fenili C, Castelletti F, Sorosina A, et al. Relative role of genetic complement abnormalities in sporadic and familial aHUS and their impact on clinical phenotype. Clin J Am Soc Nephrol. 2010;5(10):1844–59. 5. Fakhouri F, Vercel C, Fremeaux-Bacchi V. Obstetric nephrology: AKI and thrombotic microangiopathies in pregnancy. Clin J Am Soc Nephrol. 2012; 7(12):2100–6. 5. Fakhouri F, Vercel C, Fremeaux-Bacchi V. Obstetric nephrology: AKI and thrombotic microangiopathies in pregnancy. Clin J Am Soc Nephrol. 2012; 7(12):2100–6. Acknowledgements The authors would like to thank all study sites, investigators and patients who took part in this study. The authors would also like to thank: Rui-Ru Ji (Alexion Pharmaceuticals Inc.), who conducted the genetic analysis for this study; and Dr. Åsa Lommelé, PhD and Dr. Peter Chen, PhD (Alexion Pharmaceuticals Inc.) for their critical review of the manuscript. Page 8 of 9 Page 8 of 9 Page 8 of 9 Gäckler et al. BMC Nephrology (2021) 22:5 Gäckler et al. BMC Nephrology (2021) 22:5 Gäckler et al. BMC Nephrology Medical writing support, funded by Alexion Pharmaceuticals Inc., Boston, MA, was provided by Leia Silvagnoli-Compston, MSc, and Ciaran Wright, PhD of Bioscript, Macclesfield, UK. Gynecology, Moscow, Russia. 7Seoul National University Hospital, Seoul, Republic of Korea. 8Instituto de Investigación Hospital Universitario 12 de Octubre i+12, Madrid, Spain. Received: 14 April 2020 Accepted: 26 November 2020 Received: 14 April 2020 Accepted: 26 November 2020 Competing interests 13. Fakhouri F, Hourmant M, Campistol JM, Cataland SR, Espinosa M, Gaber AO, Menne J, Minetti EE, Provot F, Rondeau E, et al. Terminal complement inhibitor Eculizumab in adult patients with atypical hemolytic uremic syndrome: a single-arm, open-label trial. Am J Kidney Dis. 2016;68(1):84–93. Anja Gäckler: Honoraria and lecture fees from Alexion, Ablynx/Sanofi and Novartis. Ulf Schönermarck: study fees, travel support and consultancy fees from Alexion, and study fees and consultancy fees from Ablynx. Ulf Schönermarck: study fees, travel support and consultancy fees from Alexion, and study fees and consultancy fees from Ablynx. 14. Greenbaum LA, Fila M, Ardissino G, Al-Akash SI, Evans J, Henning P, Lieberman KV, Maringhini S, Pape L, Rees L, et al. Eculizumab is a safe and effective treatment in pediatric patients with atypical hemolytic uremic syndrome. Kidney Int. 2016;89(3):701–11. Vladimir Dobronravov: no disclosures. Gaetano La Manna: no disclosures. Andrew Denker: employee of Alexion at the time of the study and owns stock in the company. 15. Legendre CM, Licht C, Muus P, Greenbaum LA, Babu S, Bedrosian C, Bingham C, Cohen DJ, Delmas Y, Douglas K, et al. Terminal complement inhibitor eculizumab in atypical hemolytic-uremic syndrome. N Engl J Med. 2013;368(23):2169–81. Peng Liu: employee and shareholder of Alexion. Maria Vinogradova: no disclosures. Sung-Soo Yoon: no disclosures. Manuel Praga: personal fees from Otsuka, grants and personal fees from Alexion, personal fees from Retrophin. Manuel Praga: personal fees from Otsuka, grants and personal fees from Alexion, personal fees from Retrophin. 16. Licht C, Greenbaum LA, Muus P, Babu S, Bedrosian CL, Cohen DJ, Delmas Y, Douglas K, Furman RR, Gaber OA, et al. Efficacy and safety of eculizumab in atypical hemolytic uremic syndrome from 2-year extensions of phase 2 studies. Kidney Int. 2015;87(5):1061–73. Authors’ information Not included. 3. Schönermarck U, Ries W, Schröppel B, Pape L, Dunaj-Kazmierowska M, Burs 3. Schönermarck U, Ries W, Schröppel B, Pape L, Dunaj-Kazmierowska M, Burst V, Mitzner S, Basara N, Starck M, Schmidbauer D, et al. Relative incidence of thrombotic thrombocytopenic purpura and haemolytic uraemic syndrome in clinically suspected cases of thrombotic microangiopathy. Clin Kidney J. 2019;sfz066. https://doi.org/10.1093/ckj/sfz1066. Author details 1 1Department of Nephrology, University Hospital Essen, University Duisburg-Essen, Essen, Germany. 2Medizinische Klinik IV, LMU Klinikum, LMU, Munich, Germany. 3Pavlov University, Research Institute of Nephrology, St. Petersburg, Russia. 4Department of Experimental Diagnostic and Specialty Medicine (DIMES), Nephrology, Dialysis and Renal Transplant Unit, St. Orsola Hospital, University of Bologna, Bologna, Italy. 5Alexion Pharmaceuticals, Inc., Boston, USA. 6National Medical Research Centre for Obstetrics and 1Department of Nephrology, University Hospital Essen, University Duisburg-Essen, Essen, Germany. 2Medizinische Klinik IV, LMU Klinikum, LMU, Munich, Germany. 3Pavlov University, Research Institute of Nephrology, St. Petersburg, Russia. 4Department of Experimental Diagnostic and Specialty Medicine (DIMES), Nephrology, Dialysis and Renal Transplant Unit, St. Orsola Hospital, University of Bologna, Bologna, Italy. 5Alexion Pharmaceuticals, Inc., Boston, USA. 6National Medical Research Centre for Obstetrics and 17. Vilalta R, Al-Akash S, Davin J, Diaz J, Gruppo R, Hernandez J, Jungraithmayr T, Langman C, Lapeyraque A, Macher M, et al. Eculizumab therapy for pediatric patients with atypical hemolytic uremic syndrome: efficacy and safety outcomes of a retrospective study. Haematologica. 2012;97:479. 18. Zuber J, Fakhouri F, Roumenina LT, Loirat C, Fremeaux-Bacchi V. French study group for a HCG: use of eculizumab for atypical haemolytic uraemic syndrome and C3 glomerulopathies. Nat Rev Nephrol. 2012;8(11):643–57. Page 9 of 9 Gäckler et al. BMC Nephrology (2021) 22:5 Gäckler et al. BMC Nephrology Gäckler et al. BMC Nephrology (2021) 22:5 19. Antonio F, Saad JR, Wyble A, Luis D. Pacheco: pregnancy-associated atypical hemolytic uremic syndrome. Am J Perinatol. 2016;6:125–8. 20. Demir E, Yazici H, Ozluk Y, Kilicaslan I, Turkmen A. Pregnant woman with atypical hemolytic uremic syndrome delivered a healthy newborn under eculizumab treatment. Case Rep Nephrol Dial. 2016;6(3):143–8. 21. Gately R, San A, Kurtkoti J, Parnham A. Life-threatening pregnancy- associated atypical haemolytic uraemic syndrome and its response to eculizumab. Nephrology (Carlton). 2017;22(Suppl 1):32–5. 22. Sheridan D, Yu ZX, Zhang Y, Patel R, Sun F, Lasaro MA, Bouchard K, Andrien B, Marozsan A, Wang Y, et al. Design and preclinical characterization of ALXN1210: a novel anti-C5 antibody with extended duration of action. PLoS One. 2018;13(4):e0195909. 23. FDA: Ravulizumab (ULTOMIRIS). Available at: https://alexion.com/ Documents/Ultomiris_USPI.pdf [accessed Nov 2019]. 2019. 24. Rondeau E, Scully M, Ariceta G, Barbour T, Cataland S, Heyne N, Miyakawa Y, Ortiz S, Swenson E, Vallee M, et al. The long-acting C5 inhibitor, Ravulizumab, is effective and safe in adult patients with atypical hemolytic uremic syndrome naïve to complement inhibitor treatment. Kidney Int. 2020;97(6):1287–96. 25. Author details 1 Fremeaux-Bacchi V, Miller EC, Liszewski MK, Strain L, Blouin J, Brown AL, Moghal N, Kaplan BS, Weiss RA, Lhotta K, et al. Mutations in complement C3 predispose to development of atypical hemolytic uremic syndrome. Blood. 2008;112(13):4948–52. 26. Vande Walle J, Delmas Y, Ardissino G, Wang J, Kincaid JF, Haller H. Improved renal recovery in patients with atypical hemolytic uremic syndrome following rapid initiation of eculizumab treatment. J Nephrol. 2017;30(1): 127–34. 27. Yan K, Desai K, Gullapalli L, Druyts E, Balijepalli C. Epidemiology of atypical hemolytic uremic syndrome: a systematic literature review. Clin Epidemiol. 2020;12:295–305. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W3115469089	https://anuariomusical.revistas.csic.es/index.php/anuariomusical/article/download/358/387	es		“La Voz del Pueblo, una Canción, una Frase Notable, una Rima, lograron sobrevivir”. El nacimiento de la Estética Musical y el momento musicológico global	Anuario musical	2,020	cc-by	8,086	ANUARIO MUSICAL, N.º 75 enero-diciembre 2020, 11-22 ISSN: 0211-3538 https://doi.org/10.3989/anuariomusical.2020.75.02 ‘THE VOICE OF THE PEOPLE, A SONG, A NOTABLE PHRASE, A RHYME, MANAGED TO SURVIVE’.1 THE BIRTH OF MUSICAL AESTHETICS AND THE GLOBAL MUSICOLOGICAL MOMENT “LA VOZ DEL PUEBLO, UNA CANCIÓN, UNA FRASE NOTABLE, UNA RIMA, LOGRARON SOBREVIVIR”. EL NACIMIENTO DE LA ESTÉTICA MUSICAL Y EL MOMENTO MUSICOLÓGICO GLOBAL Philip V. Bohlman University of Chicago boh6@uchicago.edu ORCID ID: 0000-0002-0216-4055 Resumen En esta historia intelectual de la aparición de la estética musical global, trato del nacimiento de la estética como un campo filosófico moderno y la expansión de la historiografía musical hasta incluir el mundo entero. Estos momentos musicológicos globales son muy evidentes en los primeros escritos de Johann Gottfried Herder (17441803), quien canalizaría y mantendría esa confluencia a lo largo de su vasta obra publicada. El título de este artículo procede de Herder, específicamente de los volúmenes que llamó Volkslieder ( Canciones populares, 1778/79), en los que conecta sus escritos fundacionales sobre estética, Kritische Wälder (Selvas críticas), con el primer concepto integral de estética musical global. La historia de la recepción de Herder es en sí un hilo conductor crítico en la historia intelectual de la estética musical que conecta el siglo XVIII con el XXI. Sigo este hilo conductor hasta el sur de Asia en el siglo XVIII a la búsque-da de una moderna estética musical hindú fundacional, particularmen-te en el género clásico kriti, del sur de la India, y el vasto dominio estético de la teoría de los rāga, que también dio un giro hacia lo moderno a finales del siglo XVIII. El artículo concluye volviendo al presente con ejemplos de principios estéticos de Herder aplicados a interpretaciones musicales en la actual crisis migratoria global. Es esa historia intelectual, con ejemplos del pasado y del presente, de la Ilus-tración en Europa y en el sur de Asia, especialmente sus dimensiones Abstract In this intellectual history of the emergence of modern musical aesthetics globally, I concern myself with the birth of aesthetics as a modern philosophical field and the expansion of music historiography to include the entire world. These global musicological moments are abundantly evident in the earliest writings of Johann Gottfried Herder (1744-1803), who would channel and sustain their confluence throughout his vast body of publications. The title of the essay comes from Herder, specifically from the seminal volumes he called Volkslieder (Folk Songs, 1778/79), in which he connects his foundational writings on aesthetics, the Kritische Wälder (Critical Forests) to the first comprehensive concept of global music aesthetics. The history of Herder reception is itself a critical thread in the intellectual history of musical aesthetics that links the eighteenth to the twenty-first century. I follow this critical thread to eight eenth-century South Asia in search of a foundational modern Indian musical aesthetics, particularly in the South Indian classical genre, kriti, and the vast aesthetic domain of rāga theory, which also took a turn toward the modern at the end of the eighteenth century. The essay concludes by returning to the present with examples of Her der’s aesthetic principles in musical performance in today’s global migration crisis. It is that intellectual history, with the cases from past and present, enlightenments in Europe and South Asia, espe- The title of this essay comes from the “Introduction” to Johann Gottfried Herder, Volkslieder, vol. 1, part II (Leipzig: Weygandsche Buchhandlung, 1778/1779). It is translated in Johann Gottfried Herder and Philip V. Bohlman, Song Loves the Masses: Herder on Music and Nationalism (Berkeley: University of California Press, 2017), p. 54. An earlier version of this essay was delivered as a keynote address at the conference, “Twenty-First-Century Challenges to the History of Eighteenth-Century Aesthetics”, Turin, 11-12 June 2018. © 2020 CSIC. The online edition of this journal is distributed under the terms of Creative Commons Attribution 4.0 Internaional (CC BY 4.0) License. ANUARIO MUSICAL, N.º 75, enero-diciembre 2020, 11-22. ISSN: 0211-3538 https://doi.org/10.3989/anuariomusical.2020.75.02 1 Cómo citar este artículo/Citation: Bohlman, Philip V. “‘The Voice of the People, a Song, a Notable Phrase, a Rhyme, Managed to Survive’. The Birth of Musical Aesthetics and the Global Musicological Moment”. Anuario Musical, 75 (2020), pp. 11-22. doi: https://doi.org/10.3989/anuariomusical.2020.75.02 12 Philip V. Bohlman globales, la que da forma a la teoría de momentos musicológicos globales que constituye el núcleo de este artículo. cially their global dimensions, that shapes the theory of global musicological moments at the heart of this essay. Palabras clave Ilustración, momento musicológico global, Johann Gottfried Herder, kriti, teoría de los rāga, estética musical, Volkslieder, “Zaid and Zaida”. Keywords Enlightenment, global musicological moment, Johann Gott fried Herder, kriti, rāga theory, musical aesthetics, Volkslieder, “Zaid and Zaida”. AVANT PROPOS. HERDER’S JOURNEY AND THE BIRTH OF MUSICAL AESTHETICS In 1773, Johann Gottfried Herder (1744-1803) proclaimed a new way to understand the very being of music, proposing a radical new way to experience it as at once everyday, intimate, and sublime. Herder would claim a new place for music in human experience through the 1770s and beyond, over the course of his lifetime as one of the great philosophers, theologians, anthropologists, polyglots, and polymaths of the Enlightenment — and as the thinker who coined the term, Volkslied, folk song.3 Herder not only coined the term, but he also gathered folk songs, as an ethnographer no less than as a philologist, published them in anthologies, set debates about their meaning in motion, and profoundly rerouted the history of music. This was a moment of song, indeed, a moment after which the art and aesthetics of music would never be the same. The song that Herder called forth as ontological fact, first in 1773, had limitless potential, for he truly be- lieved, as did many who heeded his call, that song could be sensitive to “the entire being of life.”4 Herder’s Enlightenment moment of folk song in the 1770s also produced his first engaged attempt to implement a new musical aesthetics, which he had developed systematically and theoretically during the late 1760s, when he entered actively into the debates about the new philosophical attention to what, since first used by Alexander Gottlieb Baumgarten in his Meditationes philosophicae, had been called aesthetics.5 Herder embraced the potential of aesthetics fully, proclaiming in his earliest writings from the mid1760s that “not poetry, but aesthetics should be the field of the Germans.”6 Herder’s earliest aesthetic writings, particularly the four volumes of the Kritische Wälder (Critical Forests), would become a prolegomenon to what we now would understand as a full-blown field of musical aesthetics. In the fourth Critical Forest of 1769 —which was not published in Herder’s lifetime— he sought to understand music as the art-form that contained the “properties of the delightful that penetrated most deeply into the soul and moved it most powerfully.”7 To forge a theory that would lead to an understanding why this was so would be no less than a “gateway to a new aesthetics.”8 It was also in 1769, the year in which the Critical Forests appeared, that Herder would embark on a different journey, fully through the “gateway” he had imagined as metaphor in the prolegomenon. In May 1769, Herder, already a twenty-five-year-old theologian, anthropologist, philosopher, and music scholar, embarked on a sea 2 Translated in Herder and Bohlman, Song Loves the Mass es, p. 70. 3 The term “folk songs” appears for the first time in two related forms, “Lieder des Volks” and “Volkslieder,” in Johann Gottfried Herder, “Auszug aus dem Briefwechsel über Ossian und die Lieder alter Völker,” in Von deutscher Art und Kunst: Einige fliegende Blätter, ed. J. G. Herder (Hamburg: Bode, 1973), translated in Herder and Bohlman, Song Loves the Mass es, pp. 140-67. In 1778, “folk songs” appeared as the title of Herder, Volkslieder, the first anthology of folk songs from throughout the world. 4 Herder, “Auszug aus einem Briefwechsel”, translated in Herder and Bohlman, Song Loves the Masses, p. 165. 5 Alexander Gottlieb Baumgarten, Meditationes philosophicae de nunullis ad poema pertinentibus (Halle: I. H. Grunerti, 1735). 6 Johann Gottfried Herder, “Versuch über das Sein”, in Jo hann Gottfried Herder Werke, ed. Ulrich Gaier (Frankfurt am Main: Deutscher Klassiker Verlag, 1985 [1764]), vol. 1, pp. 9-21. 7 Cited in Johann Gottfried Herder, Selected Writings on Aesthetics, ed. and trans. by Gregory Moore (Princeton, NJ: Princeton University Press, 2006), p. 207. 8 Herder, Selected Writings on Aesthetics, p. 207. I began early on to collect what would become a history of lyrical song, and I disdained nothing that would not serve this end. … Whoever speaks about folk songs also comes to understand her or his own time and all that is part of it, even after ceasing to speak about folk songs. —J. G. Herder, Volkslieder, part 2, 1779.2 ANUARIO MUSICAL, N.º 75, enero-diciembre 2020, 11-22. ISSN: 0211-3538 https://doi.org/10.3989/anuariomusical.2020.75.02 ‘The Voice of the People, a Song, a Notable Phrase, a Rhyme, Managed to Survive’… journey that would take him far from his Baltic home in Riga, Latvia, and across the great seas of human experience before he would find his way to Germany, where he would produce a lifetime of thought that, among other influences, would transform the understanding of what music in the world was and could be, in other words, a full-blown theory of musical aesthetics. Herder kept a journal, perhaps better understood as elaborate field notes, on his sea journey, in which he intimately traced the transformation of personal encounter to the global experience of a common humanity.9 In his notes, which like the contemporaneous fourth volume of Critical Forests he himself never published, he made the challenge he was embracing as a scholar indebted to an aesthetics with explicit global dimensions. What had earlier been tentative theory now introduced ethnography as encounter and experience to an aesthetics that would stretch beyond Europe and beyond the eighteenth century: For this purpose I wish to collect data about the history of every historical moment, each evoking a picture of its own use, function, custom, burdens, and pleasures. Accordingly, I shall assemble everything I can, leading up to the present day, in order to put it to good use.10 It is the spirit of “moving beyond,” made possible by the journey immanent in musical aesthetics, that I follow through this essay. It is this spirit that formed at the confluence of historiography and anthropology in Her der’s writings in the late Enlightenment, laying the foundations for a musical aesthetics that was at once modern and global. It is the aesthetic capacity to move beyond, moreover, that connects three different histories that begin in the eighteenth century and that have extensive resonance in the twenty-first century, connected as they are by the global and globalization (Table 1). 1) The birth and history of musical aesthetics 2) The birth of global history 3) The birth and history of world music Table 1. Three Histories Converging in the Eighteenth Century. Johann Gottfried Herder, Journal meiner Reise im Jahr 1769 (Stuttgart: Reclam, 1997 [1769]). 10 Herder, Journal meiner Reise, cited in Herder and Bohlman, Song Loves the Masses, p. 266. 9 13 Critical to my understanding of the paradigm shift brought about by all three is the crucial role of folk song, experienced and given aesthetic dimensions by Herder. My approach to the moments of folk song I locate historically throughout this essay is broadly ontological, because I search for the moment —actually a history that returns to folk song across many moments— as a chronotope (time and place) in which the ontology of folk song —its very being— acquires renewed meaning and identity. Whereas my focus here is on folk song, I also let folk song represent the much larger concept of music, in its many aesthetic and ontological forms, across genres, geographies, and histories. The moments of music about which I reflect are notable because they are never static, hence music’s ontological power is unleashed through history.11 The narrative threads that interconnect in the historical longue durée of musical aesthetics I trace through this essay often begin with Herder and run through his substantial body of work on music.12 Of considerable importance is that these narrative threads return to Herder and to history, whether it be in the eighteenth or the twenty-first century. Herder’s ontological moment in the Volks lieder thus resurfaces in the moment of intimacy we find in Herder’s Lieder der Liebe (Songs of Love), his reflections on the biblical Song of Songs, and also in his translation of the great epic of Spain, Cantar de mio Cid.13 To expand the aesthetic chronotope I move through early and modern moments beyond Europe, especially to India, not only because these moments are the focus of my research, but because it was Herder who was the most important Enlightenment German scholar to call attention to music in early Sanskrit writings, and then to translate several critical texts, for example the Bhagavad Gita.14 11 See also Philip V. Bohlman, “Ontologies of Music”, in Re thinking Music, ed. Nicholas Cook and Mark Everist (Oxford: Oxford University Press, 1999), pp. 17-34. 12 Nine of his most influential writings on music are translated in Herder and Bohlman, Song Loves the Masses. 13 Cf. Johann Gottfried Herder, Lieder der Liebe: Die äl testen und schönsten aus Morgenlande, nebst vier und vierzig alten Minneliedern (Leipzig: Weygandsche Buchhandlung, 1778), and idem, Der Cid—Geschichte des Don Ruy Diaz, Grafen von Bivar, nach spanischen Romanzen, in Johann Gott fried Herder Werke, ed. Ulrich Gaier (Frankfurt am Main: Deutscher Klassiker Verlag, 1990), vol. 3, pp. 545-693. 14 Richard H. Davis, The Bhagavad Gita: A Biography (Princeton, NJ: Princeton University Press, 2015), pp. 84-87. ANUARIO MUSICAL, N.º 75, enero-diciembre 2020, 11-22. ISSN: 0211-3538 https://doi.org/10.3989/anuariomusical.2020.75.02 14 Philip V. Bohlman Before moving more expansively to extend the historical and geographic landscape of folk song’s ontology, I should like to turn briefly to the questions that so preoccupied Herder and the Enlightenment when folk song came into being. I do this consciously and deliberately because I believe that Herder’s aesthetic of song and music has been foundational for what music scholars —internationally and across disciplinary boundaries— undertake as a common field of musical aesthetics. No work poses these questions more sweepingly than the first major collection of folk songs, which appeared as two volumes, each containing six compact folios, called first Volkslied er, and in later editions, Stimmen der Völker in Liedern (Voices of the People in Songs).15 In the 1778/79 Volks lieder, folk songs acquire new functions, both different and distinctive. On one hand, folk songs retain Herder’s goal of illustrating the historical qualities of song, with emphasis on the old and the ancient. Rather than establishing these historical qualities more specifically for German and Scottish repertories as he had in 1773, Herder looks beyond the historical and linguistic borders of Northern and Western Europe to establish age and oral transmission as qualities of folk song in cultures throughout the world.16 THE GLOBAL MUSICOLOGICAL MOMENT. AN AESTHETIC MATRIX IN THE EIGHTEENTH CENTURY AND BEYOND Herder’s moment of global encounter in 1769 quickly and sweepingly left its impact on music, for among the data he collected were the songs of peoples throughout the world. Within four years, in 1773, he created a new word to describe these songs: Volkslieder, or “folk songs”.17 As he gathered the songs, he began publishing them in collections in 1774, leading finally to an anthology of 194 songs published in 1778 and 1779, called simply Volkslieder. The influence of Herder’s “folk songs” was enormous, indeed, a paradigm shift in musical aesthetics with global proportions. Herder’s writings on folk songs and other forms and genres of music represent what I call in recent work a “global musicological moment”.18 I invite music scholars Herder, Volkslieder. See Herder, Volkslieder and “Aus einem Briefwechsel”. 17 Herder, “Aus einem Briefwechsel”. 18 See, e.g., Philip V. Bohlman, “’All This Requires but a Moment of Open Revelation’: Johann Gottfried Herder, Robert 15 16 ANUARIO MUSICAL, N.º 75, enero-diciembre 2020, 11-22. ISSN: 0211-3538 https://doi.org/10.3989/anuariomusical.2020.75.02 to think about the temporality of such moments as having two dimensions. First, there is the moment itself, when a change of revolutionary proportions takes place because of the transformation of musical object (a song, for example) to subjectivity (the ways in which songs shape human society throughout the world).19 Second, global musicological moments change history, both as it is understood in the past and as it unfolds in the future. Herder’s global musicological moment was especially significant because it represents a moment in which the first concept of world music was invented and given aesthetic dimensions. His volumes begin with songs from northern Europe and the Baltics (e.g., the Estonian “Jörru, Jörru”) and conclude with songs from Madagascar and Peru (e.g., “To His Child”).20 He wrote major works on biblical song (e.g., the Song of Songs) and medieval Iberian epics (Cantar de mio Cid). Herder’s global musicological moment, nonetheless, was not isolated in the history of musical aesthetics. There were those who came before and those who would come thereafter, and it is this intellectual history of musical aesthetics that is my larger concern in this essay. Among the works in which concepts of world music emerge during shorter or longer moments prior to Her der’s Enlightenment world, I should like to mention three here, though there are many other candidates. Of these three moments, the earliest is the one that forms around the Indian treatise on music, theater, and dance from roughly the third century of the Common Era, the Nāṭyaśāstra.21 The search for music’s universality became an historical leitmotif in medieval Islamic works on the global reach of history, for example, Ibn Khaldūn’s (1332-1406) fourteenth-century Muqaddimah, an “Introduction” to the history of the universe, with abundant references to music in culture.22 During the sixteenth and Lachmann, and the Global Ethnomusicological Moment’”, in Remixing Music Studies: Essays in Honour of Nicholas Cook, ed. Ananay Aguilar, Ross Cole, Matthew Pritchard, and Eric Clarke (London: Routledge, 2021), pp. 131-147. 19 I use the concepts of scientific revolution and paradigm shift as in the classic Thomas S. Kuhn, The Structure of Scien tific Revolutions (Chicago: University of Chicago Press, 1962). 20 See Herder and Bohlman, Song Loves the Masses, pp. 63-64 and 104. 21 Nāṭyaśāstra, The Nāṭyaśāstra, trans. by Adya Rangacharya (New Delhi: Munshiram Manoharlal, 1984). 22 Ibn Khaldûn The Muqadimmah: An Introduction to History, 3 vols., trans. by Franz Rosenthal (New York: Pantheon, 1958). ‘The Voice of the People, a Song, a Notable Phrase, a Rhyme, Managed to Survive’… seventeenth centuries, the European Age of Encounter would also produce the potential for global musicological moments, the best known of which is probably Athana sius Kircher’s 1650 compendium of musical objects from the world, Musurgia universalis (see Figure 1).23 15 J. Ellis and Guido Adler who would integrate various social and physical sciences into modern musical scholarship, notably expanding the field to encompass growing musical and human diversity.24 Adler’s 1885 essay on the “Scope, Methods, and Aims of Musicology” was notable for the ways in which it was globally and disciplinarily inclusive, specifying exactly what music history, music theory, and ethnomusicology could achieve.25 It is the confluence of aesthetic ideas at these moments that is especially significant for the formation of ethnomusicological moments, and therefore I should like to give a bit more theoretical shape to the aesthetic matrix that yields such moments, particularly that in the eight eenth century. In the model I propose here, the global musicological moment occupies a central position among four other moments, which proceed chronologically in the following way (Table 2): 1) The Moment of Encounter 2) The Moment of Audibility 3) The Ontological Moment 4) The Moment of Revelation Table 2. Aesthetic Moments of the Global Musicological Moment. Figure 1. Frontispiece of Athanasius Kircher, Musurgia universalis (1650). In the centuries since Herder, there has been a proliferation of global musicological moments, and it is upon these that the modern fields of music scholarship have been built. A century after Herder, it would be Alexander 23 Athanasius Kircher, Musurgia universalis (Rome: Francisco Corbelletti 1650). None of these moments is static, but rather each is transformative as it expands the aesthetic capacity of musical thought to perceive and understand musical experience. The movement afforded by the four processes is one of expansion, a dynamic process of globalization. The universal qualities we witness in music are, for example, both internally implicit and expansively explicit. Song and melody, moving across these moments, might be globally comparable, even similar, not because of their sameness, but rather because of their differences. Music history, by its very nature, moves from the local to the global, represented in the aesthetic matrix below as processes that coalesce at its center (see Figure 2). See Alexander J. Ellis, “On the Musical Scales of Various Nations”, Journal of the Society of Acoustics, 33 (1885), pp. 485-527, and Harry Liebersohn, Music and the New Global Culture from the Great Exhibitions to the Jazz Age (Chicago: University of Chicago Press, 2019). 25 Guido Adler, “Umfang, Methode und Ziel der Musikwissenschaft”, Vierteljahresschrift für Musikwissenschaft, 1/1 (1885), pp. 5-20. 24 ANUARIO MUSICAL, N.º 75, enero-diciembre 2020, 11-22. ISSN: 0211-3538 https://doi.org/10.3989/anuariomusical.2020.75.02 16 Philip V. Bohlman the second millennium BCE.27 The aesthetics of the Vedic hymns emerges from an expansive range of metaphors, both musical and material, for example, in passages such as the “Creation of Sacrifice” (Table 3): Figure 2. The Global Musicological Moment. The Aesthetic Matrix of the Eighteenth Century and Beyond. THE EIGHTEENTH CENTURY AND THE GLOBALIZATION OF MUSICAL AESTHETICS Critical to the intellectual history of musical aesthetics that I examine here is its acquisition of global dimensions, the globalization that eventually leads us to the twenty-first century and beyond. The modern emergence of musical aesthetics in the Enlightenment shared by Baumgarten and Herder, therefore, also shaped the enlightenments that were emerging elsewhere in the world at the same time. Among the many cases for what I call global enlightenments, I should like to focus here on the rise of modern musical aesthetics in India, not in small measure because of the direct and indirect connections to Herder and his generation.26 I do so by first turning to one of the earliest sources to shape Indian aesthetics, the Rg Veda, an extensive anthology of sacred hymns, that entered written Sanskrit verses from oral tradition during Philip V. Bohlman, “Musical Thought in the Global Enlightenments”, in Studies on a Global History of Music: A Balzan Musicology Project, ed. Reinhard Strohm (London and New York: Routledge, 2018), pp. 61-80. 26 ANUARIO MUSICAL, N.º 75, enero-diciembre 2020, 11-22. ISSN: 0211-3538 https://doi.org/10.3989/anuariomusical.2020.75.02 1) The sacrifice that is spread out with threads on all sides, drawn tight with a hundred and one divine acts, is woven by these fathers as they come near: “Weave forward, weave backward,” they say as they sit by the loom that is stretched tight. 2) The Man stretches the warp and draws the weft; the Man has spread it out upon this dome of the sky. These are the pegs, that are fastened in place; they made the melodies into the shuttles for weaving. 3) What was the original model, and what was the copy, and what was the connection between them? What was the butter, and what the enclosing wood? What was the metre, what was the invocation, and the chant, when all the gods sacrificed the god? 4) The Gāyatrī metre was the yoke-mate of Agni; Savitṛ joined with the Uṣṇi metre, and with the Anuṣṭubh metre was Soma that reverberates with the chants. The Bṛhatī metre resonated in the voice of Bṛhaspati. 5) The Virāj metre was the privilege of Mitra and Varuṇa; the Triṣṭubh metre was part of the day of Indra. The Jagatī entered into all the gods. That was the model for the human sages. Table 3. From “The Creation of Sacrifice,” Rg Veda.28 Among the Vedic hymns that Johann Gottfried Herder translated, the “Creation of Sacrifice” was one of Herder’s favorites. The Vedic hymns provided him one of the most immediate points of entry to Brahmanic Hinduism, which he situated among the other world religions in which he searched for common meanings of sacred texts, expressed through intersecting aesthetic practices. Critically, the act of translation, as it did so often in Herder’s writings, took on historical meaning: Herder came to understand that he was translating from one enlightenment to another, which is to say, producing translation between an Indian and a European aesthetics. Translation between The standard English edition and translation is Wendy Doniger O’Flaherty, The Rig Veda (Harmondsworth: Penguin, 1981). 28 O’Flaherty, The Rig Veda, p. 31. 27 ‘The Voice of the People, a Song, a Notable Phrase, a Rhyme, Managed to Survive’… and among global enlightenments provided Herder a means to gain a sense of the music and of musical aesthetics, as expressed in language and text. Pranabendra Ghosh, making one of the strongest cases for Herder’s knowledge of Sanskrit, reflects on why translation was so important, indeed, as an act of aesthetic realization, thus “also a matter of faithfulness to form, which, to him, in addition to numerical and metrical form, is also sound, melody, rhythm and tone and, therefore, organic form”.29 It was at these eighteenth-century moments of the global aesthetic matrix that we also witness ontological change of remarkable proportions, a parallel aesthetic modernization in Europe and India, and beyond. We witness, for example, modern forms of classical music emerging at moments of encounter, contestation, and political change in the surfeit of global enlightenments. In South India at the same moment of enlightenment, the “trinity of saint composers” of South Indian music, Thyāgarāja (1767-1847), Muthuswāmi Dikṣitar (1775-1835), and Śyāmā Śāstri (1762-1827), created a canon of compositions that standardized the structure and improvisation of the genre, kriti, whose formal structures share much with the spread of sonata form in European music. Table 4 illustrates the three-part form that became canonic in the eighteenth century with Thyāgarāja’s kriti, “Maregulara”. Indian musical aesthetics formed from this moment of global enlightenment, not just in the centuries leading to the eighteenth century, but also in the global music history that leads to the present. Of particular interest for expanding the history of musical aesthetics are the ways in which melodic mode —rāga— became global and came to serve as the primary symbol of an aesthetic system that is both ancient and modern. By the end of the eighteenth century, as the British colonial Raj fully took hold of India, systematic musical thought —above all, the classification of rāga— had entered a domain of global exchange. William Jones, living in colonial Calcutta and conducting research at the Asiatic Society, published one of the most influential texts on rāga, “On the Modes of the Hindoos,” in 1784.30 It would be the Bengali music 29 Pranabendra Nath Ghosh, Johann Gottfried Herder’s Im age of India (Santiniketan: Visva-Bharati Research Publications, 1990), p. 268. 30 William Jones, “On the Modes of the Hindoos”, in Hindu Music from Various Authors, ed. S. M. Tagore (Varanasi: Chowkhamba Sanskrit Series, 1964), pp. 88-112. 17 Pallavi Marugelara o Rhaghava Why this screen separating me from you? O Rhagava! Anupallavi Marugela caracara rupa paratpara Surya sudhakara locana Why this screen? Between me and the Lord of all forms, Who has the sun and moon as eyes! Charanam Anni nivanucu antaranga muna Tinnaga vetagi telusukontinayya Ninne gani madinenna jalanorula Nannu brovavayya tyagaraja nuta After inner soul searching, I now see You are in everything! I can think of none but You! Please protect me, Thyāgarāja! Main theme reprised Marugelara o Rhagava Why this screen separating me from you? O Rhagava! Table 4. Thyāgarāja, “Marugelara”. Lyrics in Telugu; the melodic mode, or rāga, is jayantasri; the meter, or tāla, is aditāla. scholar and collector, Sourindro Mohun Tagore, who would republish Jones in India, but as a parallel historical material with his own extensive works on the Uni versal History of Music (Tagore 1896/1963),31 and detailed organological works, which accompanied, among others, the actual collections of musical instruments that he sent to the Berlin Phonogram Archive in which Curt Sachs and Erich von Hornbostel would later work and propose their standard classification system for musical instruments, based on Indian models. I make this historical observation to illustrate that, when we write about exchange from the eighteenth century onward, it moved in both directions, following paths prescribed by a global aesthetics. 31 Sourindro Mohun Tagore, Universal History of Music, Compiled from Divers Sources (Varanasi: Chowkhamba Sanskrit Series, 1963). Originally published in 1896. ANUARIO MUSICAL, N.º 75, enero-diciembre 2020, 11-22. ISSN: 0211-3538 https://doi.org/10.3989/anuariomusical.2020.75.02 18 Philip V. Bohlman The importance of rāga in that emerging global aesthetics should not be underestimated, and for that reason I make a brief excursion to Indian musical aesthetics that potentially allows us to reroute the intellectual history of music scholarship more generally. I turn, in fact, to one rāga, the many manifestations of which came to enter a new global musical aesthetics in the eighteenth century. I speak here of the rāga, or rāgini, Todi, for whom an aesthetic language coalesced in what we can understand as the Indian enlightenment. The aesthetic language used to express the ontological meanings of rāga encompasses a remarkable range of the arts, among them the religious narratives and visual designs of the tradition of miniature painting known as rāgamāla (see Figure 3). thus evoking the vastness of music’s ontologies. The goddess Todi’s many musical forms and avatars notwithstanding, there are several that are especially suggestive for the ways she becomes an avatar for Indian musical aesthetics. Clearly, the Todi entering a forest clearing in this rāgamāla from village West Bengal, a folk depiction I collected in Kolkata, is richly endowed with musical form and meaning. Rāgas and rāginis contain and express differences in gender, and rāgini Todi makes it very clear why this is so. The goddess/rāga Todi we see here traditionally carries a vīṇā over her left shoulder. The vīṇā is not just any musical instrument, but rather it is the instrument that is most closely connected to Sarasvatī, the Hindu goddess associated with learning and the arts, particularly music. In some interpretations, Sarasvatī’s body and the anthropomorphic body of the vīṇā are considered the same: the goddess is music, and music is the goddess.32 Stories always accompany rāgamāla paintings, and in counterpoint with the visual images they expand the universe of rāga’s representational meanings and the forms in which they appear. Characteristically, the stories that describe Todi revel in her beauty. The stories circulate widely as mythological tales, but they also appear frequently on the paintings of a rāga, such as the following story by Chaupayi on a rāgamāla for Todi. Chaupayi’s story: The Almighty has made a wondrous creature in Todi. He appears to have spared no charm and grace in this act. Holding the nectar in her hands, she stands in a garden, and the world around her is filled with deep love. Hearing the enchanting sounds of Todi, herds of deer lose their way. The beauty of Todi is so enchanting that eyes drop after a glance at her. Nearby is a pond of clear, sweet water, sacred as “Gangajal” [waters of the Ganges; sacred water]. Taking her to be his own, Sarang extends his hands and beckons her to his side.33 Todi is an especially eloquent storyteller. In her long history as a rāgini, she has assumed many forms and captivated many to admire the narratives that resonate with her songs. She invites many other rāgas to join her, to weave Figure 3. Rāgamāla Painting of Rāgini Todi. (Personal collection of Philip V. Bohlman) A rāga with primarily feminine attributes, hence a rāgini, Todi shapes herself as an avatar for melodic mode, ANUARIO MUSICAL, N.º 75, enero-diciembre 2020, 11-22. ISSN: 0211-3538 https://doi.org/10.3989/anuariomusical.2020.75.02 32 For a more extended analysis of this rāgamāla, see Philip V. Bohlman, “Foreword: Todi in the Forest of Song”, in Kirin Narayan, Everyday Creativity and Well-Being: Singing God desses in the Himalayan Foothills (Chicago: University of Chicago Press, 2016), pp. ix-xvi. 33 Cited and translated in Walter Kaufmann, The Ragas of North India (Bloomington: Indiana University Press, 1968). ‘The Voice of the People, a Song, a Notable Phrase, a Rhyme, Managed to Survive’… their stories into hers through melodic modulation. All accounts of Todi’s presence in Indian musical aesthetics acknowledge that she has a remarkable presence. As a rāg (the North Indian form of rāga), she owes the importance of that presence to her familial genealogy, for she is one of the noblest members of the family, or thāta, of rāgs to which she also gives her name, Todi thāta. The family of rāgs that bears her name has a particularly interesting lineage. We know of it as a distinct thāta since at least the fifteenth century, but Todi as a rāg would enjoy an extensive familiarity with another rāg of considerable presence, Bhairavi, until the eighteenth century, when Bhairavi —also a rāgini, representing Bhairavi worshiping Lord Bhairava on Mount Kailasha, often with cymbals in her hands— would go her own way in North Indian classical music but retain a scalar filial relationship in South Indian Karnatak classical music. The scales —the bare notes of the melodic mode that we extract from countless stories sung by the rāginis Todi and Bhairavi, North and South— are intriguing both for what they say and for what they do not say. Above all, what they say tells us a great deal about the changing relations of kinship, religion, and gender in the eighteenth century. Perhaps in its simplest form, we could represent Todi’s ascent as a scale with the following Indian (sargam) notation and its Western equivalent, locating the primary note of sa on middle C (Table 5).34 19 seduce new songs and stories into her world. No rāg has responded more fully to this invitation than Bilaskhani Todi. The naming of Bilaskhani Todi —tradition claims that Bilas Khan, the son of the great sixteenth-century musician and composer at the Mughal court, Tansen (ca. 1490s-1586), sang the rāg as an alternative to Bhairavi while mourning his deceased father— stresses its syncretism and hybridity. Muslim tradition enters the narratives of Hinduism; male and female forms of rāg sound the potential of their union. It is this aesthetic union, realized through the formation of the modern genealogy, or pa rampara, of the gharānā, that becomes the catalyst for a new musical aesthetics in eighteenth-century North India. THE RETURN OF EIGHTEENTH-CENTURY AESTHETICS TO THE TWENTY-FIRST CENTURY Todi makes it abundantly clear that the melodic and aesthetic sameness with rāg Bhairavi is the source of a particular attraction. Of particular interest, moreover, are rāgs that form in the narrative and musical spaces that conjoin Todi and Bhairavi. Rāgs that fill these spaces enact a type of ambiguity, which complicates the gendered roles of Todi and Bhairavi as rāginis. It is into such aesthetic spaces of ambiguity that Todi invites new rāgs, as if to As I draw toward the conclusion of this essay on global aesthetics, I return to the intellectual history of musical aesthetics as I began, through folk song, and I do so with a ballad, “Zaid and Zaida,” that Johann Gottfried Herder published in several variants at the beginning of his first folio of Volkslieder in 1778. Because ballads, as a narrative genre, with specific linguistic histories, were the first to capture Herder’s attention, it might not seem surprising that Herder should include the Spanish romance, or ballad, as five variants (songs 7-11) in his Folk Songs, including translations from Spanish, English, and what Herder calls “moorisch”, a standardized version of Maghrebi Arabic (see the variant that was published as song 8, “Zaid und Zaide”, in Figure 4). The history of “Zaid and Zaida,” however, is far more complex and global than first meets the eye. The ballad’s origins are in medieval al-Andalus, and like other early ballads from the Mediterranean Middle Ages, the narrative moves through many languages in oral and written tradition, in the case of “Zaid and Zaida” Arabic and multiple Iberian Romance languages, among them Castilian, Portuguese, and Ladino. Herder translates from a historical work by Ginés Pérez de Hita, published in 1647.35 Among other reasons he was attracted to the song was its etymological relation to the Cid epic. Zaid and Sa‘īd are 34 N. A. Jairazbhoy, The Rāgs of North Indian Music: Their Structure and Evolution (Middletown, CT: Wesleyan University Press, 1971), pp. 97-99. 35 Ginés Pérez de Hita, Historia de los vandos de los Ze gries, y Abencerrages, cavalleros moros de Granada, de las civiles guerras (Barcelona: S. de Cormellas, 1647). Rāg Todi sa—re (komal)—ga (komal)—ma (tivra)—pa— dha (komal)—ni (śuddha)—sa C—D♭—E♭—F♯—G—A♭—B—C Table 5. Rāg Todi’s Scale in Sargam and Western Notation. ANUARIO MUSICAL, N.º 75, enero-diciembre 2020, 11-22. ISSN: 0211-3538 https://doi.org/10.3989/anuariomusical.2020.75.02 20 Philip V. Bohlman Durch die Strasse seiner Dame Wandelt Zaid auf und nieder, Harrend, daß die Stunde komme, Endlich komme, sie zu sprechen. Through the streets on which his lady lived Paced Zaid back and forth, Waiting for the hour to come, Finally to come, so that he might speak with her. Und schon geht der Mohr verzweifelnd, Da es sich so lange zögert, Denket: nur von ihr ein Anblick Wird all meine Flammen kühlen. And, in doubt, the Moor was already thinking That it had been such a long delay, He thought: If I could but once glance upon her, All my flames would be quelled. Und da sieht er sie! Am Fenster Tritt hervor sie, wie die Sonne Aufgeht in dem Ungewitter, Wie der Mond im Dunkel aufgeht. And then he saw her there! She approached the window like the sun Rising after the storm, Like the moon rising in the night. Leise tritt ihr Zaid näher: Alla mit dir, schöne Mohrin! Ist es wahr, was meine Pagen, Deine Dienerinnen sagen? […] Warst du’s, die auf dieser Stelle Zu mir sprach, noch jenen Abend? Dein bin ich, dein bin ich ewig! Dein, o du mein Leben, Zaid!« Quietly Zaid drew closer to her: Allah be with you, lovely Moor! Is it true, what my pages, What your servants say? Was it not you, who on this very spot, Spoke to me on that very evening? I am yours, I am yours forever! Yours, oh, you, my life, Zaid!” Figure 4. “Zaid und Zaide / Zaid and Zaida”, Song 8 in J. G. Herder, Volkslieder (verses 1-4 and 14). both Arabic predecessors to the name “Cid.” “Zaid and Zaida,” moreover, is a captivity narrative, a genre that was widespread in Mediterranean song long before it entered the history of colonial encounter, the classic 137th Psalm, “By the Waters of Babylon”, being one of the earliest examples. Even this history attracted Herder, for he worked through and translated the 137th Psalm on several occasions. The history Herder might have attempted to realize by publishing variants of “Zaid and Zaida” in the 1770s, however, would unfold as a history of aesthetics that even he might have imagined. As a narrative ballad with a captivity narrative, the story of the lovers, “Zaid and Zaida”, also captured the attention of writers and musicians in Enlightenment Europe, among them Jean-Philippe Rameau and Wolfgang Amadeus Mozart, who began composing the Singspiel, Zaide, in 1780. Its arias and ensemble components were eventually gathered as K. 344, but there is no overture or third act, thus leaving the Singspiel incomplete. Zaide’s narrative remained open, and it surely influenced that best known of operas with a captivANUARIO MUSICAL, N.º 75, enero-diciembre 2020, 11-22. ISSN: 0211-3538 https://doi.org/10.3989/anuariomusical.2020.75.02 ity narrative, Mozart’s Entführung aus dem Serail, K. 384, which was premiered in 1782. It is the unfinished history of Zaide, passing through medieval, Renaissance, and early modern variants, but then entering the modern aesthetics of Herder’s Enlightenment, that extends its narrative arc to the twenty-first-century refugee crisis faced by so many from the Mediterranean, Africa, and Middle East in the contemporary migration crisis of the present, for it now becomes the source for new variant performances that extend the Andalusian, Arabic, Spanish, Herderian, Mozartean history to the present. We experience these performances by refugee and immigrant ensembles in Central Europe, for example the productions of Zaide, eine Flucht (Zaide, a Refugee’s Journey), which employ an aesthetic counterpoint that mixes Middle Eastern and European Enlightenment sounds. It is, in fact, Flüchtlinge, refugees seeking refuge in Europe, who weave their histories into new libretti and performances in Europe. They interweave improvised recitatives about daily dangers and the difficulties of crossing national borders that have been fenced ‘The Voice of the People, a Song, a Notable Phrase, a Rhyme, Managed to Survive’… into the arias and ensemble songs composed by Mozart for Zaide.36 In these, refugee and non-refugee, the aesthetic possibilities of past and present, the classical and the modern, the medieval al-Andalus and postcolonial migration crisis, converge in the global musicological moment we together witness in real time. I conclude this essay by reflecting on the aesthetic space in which the historical longue durée and the history of the present converge, and I do so by returning to its beginning, Johann Gottfried Herder and the birth of a new musical aesthetics in song. I listen for the global moment that is so presciently palpable in the final passage of Herder’s last major essay on song, the chapter called “Von Musik” (On Music) in the 1800 book that encompassed his aesthetic vision at the end of his life, Kalli gone. I began with Herder’s reflections on the possibility of a global aesthetics as he embarked on his journey of discovery in 1769, and I close with his own final return to the history of song through a summation of his thought on musical aesthetics. If one gathers, without any prejudice, the affects that melodies and songs invest in human feeling and afford to all collectivities of human society, from the family to the nation, music would rise as a narrative of miraculous tales from the lowest rung to which it has been assigned to realize its relation to the culture of humanity. “The concepts of music come from transitory impressions; either they disappear entirely, or if they are deliberately repeated from the strength of imagination, they are more burdensome than unpleasant”.37 … As we know so very well, it is music that we experience within ourselves that lifts us up from the earth at the very moment of death.38 Upon returning to song, upon returning to music at the end of his own life, Herder envisions a musical aesFor videos of performance extracts, see, Zaide: Eine Flucht; https://www.youtube.com/watch?v=8xPPndgffRs; most recent access on October 29, 2020. 37 Herder quotes here from his own essay on immortality, Johann Gottfried Herder, “Über die menschliche Unsterblichkeit”, in Johann Gottfried Herder Werke, ed. Hans Dietrich Irmscher (Frankfurt am Main: Deutscher Klassiker Verlag, 1998), vol. 8, pp. 203-219. 38 Johann Gottfried Herder, Kalligone, in Johann Gottfried Herder Werke, ed. Hans Dietrich Irmscher (Frankfurt am Main: Deutscher Klassiker Verlag, 1998), vol. 8, pp. 641-964; translated in Herder and Bohlman, Song Loves the Masses, p. 259. 36 21 thetics embodied by a global collective, Menge —of choral singing and communal dance— that expands considerably upon his earlier and more comfortable reliance on speech and the philosophy of language. “We cannot listen often enough to the intimate passages that move us. How they echo, and we wish for their return without satisfaction, until we (so we imagine) absorb these passages and they become part of our soul”.39 Herder’s return to history through music becomes more focused and more human, while at the same time the range of music’s aesthetic matrix becomes more expansive and more universal. Accordingly, he foresees the future, maybe even the twenty-first century, and wonders whether “the voice of the people, a song, a notable phrase, a rhyme, managed to survive”. Herder’s late reflections on the aesthetic moment of song in the final stage of his life opened upon a vision of transcendence. Herder hesitated not at all to call that moment, having arisen from the birth of musical aesthetics, also a moment of death. That moment contained humanity and the universe, life and death. Music and aesthetics, which live through our return to them, however transitory such return sometimes is, are endowed with remarkable force, the very power to realize life itself, and to do so in the moment in which music is transcendent, when music enters the aesthetic realm beyond itself. WORKS CITED Adler, Guido. “Umfang, Methode und Ziel der Musikwissenschaft”. Vierteljahresschrift für Musikwis senschaft 1/1 (1885), pp. 5-20. Baumgarten, Alexander Gottlieb. Meditationes philo sophicae de nunullis ad poema pertinentibus. Halle: I. H. Grunerti, 1735. Bohlman, Philip V. “Ontologies of Music”. In Rethinking Music, ed. Nicholas Cook and Mark Everist. Oxford: Oxford University Press, 1999, pp. 17-34. . “Foreword: Todi in the Forest of Song”. In Kirin Narayan, Everyday Creativity and Well-Being: Sing ing Goddesses in the Himalayan Foothills. Chicago: University of Chicago Press, 2016, pp. ix-xvi. . “Musical Thought in the Global Enlightenments”. In Studies on a Global History of Music: A Balzan 39 Herder, Kalligone; translated in Herder and Bohlman, Song Loves the Masses, p. 258. ANUARIO MUSICAL, N.º 75, enero-diciembre 2020, 11-22. ISSN: 0211-3538 https://doi.org/10.3989/anuariomusical.2020.75.02 22 Philip V. Bohlman Musicology Project, ed. Reinhard Strohm. London and New York: Routledge, 2018, pp. 61-80. . “’All This Requires but a Moment of Open Revelation’: Johann Gottfried Herder, Robert Lachmann, and the Global Ethnomusicological Moment’”. In Remixing Music Studies: Essays in Honour of Nich olas Cook, ed. Ananay Aguilar, Ross Cole, Matthew Pritchard, and Eric Clarke. London and New York: Routledge, 2020, pp. 131-147. Davis, Richard H. The Bhagavad Gita: A Biography. Princeton, NJ: Princeton University Press, 2015. Doniger O’Flaherty, Wendy. The Rig Veda. Harmonds worth: Penguin, 1981. Ellis, Alexander J. “On the Musical Scales of Various Nations”. Journal of the Society of Acoustics, 33 (1885), pp. 485-527. Ghosh, Pranabendra Nath. Johann Gottfried Herder’s Im age of India. Santiniketan: Visva-Bharati Research Publications, 1990. Herder, Johann Gottfried. “Versuch über das Sein”. In Jo hann Gottfried Herder Werke, vol. 1: Johann Gott fried Herder: Frühe Schriften, 1764-1772, ed. Ulrich Gaier. Frankfurt am Main: Deutscher Klassiker Verlag, 1985 [1764], pp. 9-21. . Journal meiner Reise im Jahr 1769. Stuttgart: Reclam, 1997 [1769]. . Von deutscher Art und Kunst. Hamburg: Bode, 1773. . “Auszug aus einem Briefwechsel über Oßian und die Lieder alter Völker”. In idem, Von deutscher Art und Kunst. Stuttgart: Reclam, 1966 [1773], pp. 5-62. . Volkslieder, 2 vols., in 6 folios. Leipzig: Wey gandsche Buchhandlung, 1778/1779. . Lieder der Liebe: Die ältesten und schönsten aus Morgenlande, nebst vier und vierzig alten Minne liedern. Leipzig: Weygandsche Buchhandlung, 1778. . Der Cid — Geschichte des Don Ruy Diaz, Grafen von Bivar, nach spanischen Romanzen. In Johann Gottfried Herder Werke, Vol. 3, ed. Ulrich Gaier. Frankfurt am Main: Deutscher Klassiker Verlag, 1990, pp. 545-693. . Kalligone. In Johann Gottfried Herder Werke. Vol. 8. Frankfurt am Main: Deutscher Klassiker Verlag, 1998 [1800], 641-964. ANUARIO MUSICAL, N.º 75, enero-diciembre 2020, 11-22. ISSN: 0211-3538 https://doi.org/10.3989/anuariomusical.2020.75.02 . “Über die menschliche Unsterblichkeit”. In Jo hann Gottfried Herder Werke. Vol. 8, ed. Hans Dietrich Irmscher. Frankfurt am Main: Deutscher Klassiker Verlag, 1998, pp. 203-219. . Selected Writings on Aesthetics, ed. and trans. by Gregory Moore. Princeton, NJ: Princeton University Press, 2006. , and Philip V. Bohlman. Song Loves the Masses: Herder on Music and Nationalism. Berkeley: University of California Press, 2016. Ibn Khaldûn. The Muqadimmah: An Introduction to His tory, 3 vols., trans. by Franz Rosenthal. New York: Pantheon, 1958. Jairazbhoy, N. A. The Rāgs of North Indian Music: Their Structure and Evolution. Middletown, CT: Wesleyan University Press, 1971. Jones, William. “On the Modes of the Hindoos”. In Hin du Music from Various Authors, ed. S. M. Tagore. Varanasi: Chowkhamba Sanskrit Series, 1964, pp. 88-112. Originally published in Asiatick Re searches, 1784. Kaufmann, Walter. The Ragas of North India. Bloomington: Indiana University Press, 1968. Kircher, Athanasius. Musurgia universalis. Rome: Francisco Corbelletti, 1650. Kuhn, Thomas S. The Structure of Scientific Revolutions. Chicago: University of Chicago Press, 1962. Liebersohn, Harry. Music and the New Global Culture from the Great Exhibitions to the Jazz Age. Chicago: University of Chicago Press, 2019. Nāṭyaśāstra. The Nāṭyaśāstra, trans. by Adya Rangacharya. New Delhi: Munshiram Manoharlal, 1984. Pérez de Hita, Ginés. Historia de los vandos de los Ze gries, y Abencerrages, cavalleros moros de Gra nada, de las civiles guerras. Barcelona: S. de Cormellas, 1647. Tagore, S. M. Universal History of Music, Compiled from Divers Sources. Varanasi: Chowkhamba Sanskrit Series, 1963. Originally published in 1896. Zaide: Eine Flucht: https://www.youtube.com/watch?v=8xPPndgffRs; most recent access on 29 October 2020. Recibido: 01.11.2020 Aceptado: 08.11.2020
https://openalex.org/W3116014880	https://revista.sabnet.org/ojs/index.php/sab/article/download/845/4	Portuguese	null	Acervos arqueológicos	Revista de Arqueologia	2,020	cc-by-sa	5,393	Maria Cristina Oliveira Bruno* Maria Cristina Oliveira Bruno* RESUMO RESUMO A abordagem sobre acervos arqueológicos, neste texto, está pautada pela problematização referente à longevidade dos acervos e suas implicações histórico-culturais para as sociedades que são responsáveis pelas suas constituições, manutenções e preservações, com projeção especial sobre algumas questões brasileiras na contemporaneidade e ainda no que corresponde às responsabilidades curatoriais. A R T I G O Submissão: 14/09/2020 Aprovação: 06/10/2020 Publicação: 28/12/2020 REVISTA DE ARQUEOLOGIAVolume 33 No. 3 Setembro–Dezembro 2020Edição Especial: Gestão de Acervos Arqueológicos Submissão: 14/09/2020 Aprovação: 06/10/2020 Publicação: 28/12/2020 REVISTA DE ARQUEOLOGIAVolume 33 No. 3 Setembro–Dezembro 2020Edição Especial: Gestão de Acervos Arqueológicos Acervos arqueológicos… \| Maria Cristina Oliveira Bruno Palavras-chave: acervo; longevidade; curadoria. Palavras-chave: acervo; longevidade; curadoria. * Museóloga, Professora Titular em Museologia do Museu de Arqueologia e Etnologia da Universidade de São Paulo, onde coordena o Programa de Pós-Graduação em Museologia. E-mail: mcobruno@uol.com.br. ORCID: https://orcid.org/0000-0003-2172-9071. DOI: https://doi.org/10.24885/sab.v33i3.845 DOI: https://doi.org/10.24885/sab.v33i3.845 DOI: https://doi.org/10.24885/sab.v33i3.845 A R T I C L E 1 A citação em epígrafe contou com atualização gramatical. 2 Paulo Duarte figura entre aqueles que lutaram para a aprovação da Lei 3924 de 26 de julho de 1961, de proteção dos sítios arqueológicos e matriz de uma longa lista de normativas e legislações direcionadas para os bens arqueológicos. Criou e dirigiu o Instituto de Pré-História da Universidade de São Paulo na década dos anos de 1960. 3 Observa-se, desde os anos da década de 1990, um crescimento exponencial de trabalhos acadêmicos voltados para a proteção do patrimônio arqueológico, como também: os desdobramentos dos eventos científicos registrados em documentos, criação de grupos de trabalhos e organização de movimentos coletivos. 4 Este tema, nos últimos anos, tem impulsionado a organização de dossiês em publicações da área, como por exemplo, o Dossiê “Musealização da Arqueologia e produção acadêmica: novos problemas, novos desafios”, organizado em duas partes: Parte 1. A produção Acadêmica em Musealização da Arqueologia no Brasil e Parte 2. Musealização da Arqueologia e o Legado Patrimonial no Brasil, publicado na Revista de Arqueologia 2013/2014 da Sociedade Arqueologia Brasileira. APRESENTAÇÃO E estamos esperando até hoje. Enquanto isso as jazidas pré-históricas continuam à mercê do vandalismo e do extermínio. O mesmo destino das matas e florestas, arrasadas para o fabrico do carvão; o mesmo destino dos animais selvagens, dizimados pelos caçadores mais selvagens do que eles; o mesmo destino do Brasil, cujo território vai sendo vendido aos pedaços a estrangeiros por brasileiros sem escrúpulos... Brasil da displicência, Brasil do abandono. Brasil de tantos messias que não sabem sociologia… 1 (PAULO DUARTE, 1970, p. 372)1 A abordagem sobre os dilemas que envolvem o tratamento de acervos arqueológicos no Brasil tem recebido atenção de muitos profissionais, estudantes e instituições de pesquisa e de preservação patrimonial nas últimas décadas. É possível sublinhar que Paulo Duarte2 tenha sido a primeira voz mais eloquente e obstinada a favor dos estudos e da proteção dos vestígios das sociedades que nos precederam. Hoje, é possível reconhecer que esta abordagem tem sido uma preocupação transgeracional 3 e tem impulsionado ações individuais e coletivas no que se refere à identificação das características e dos problemas e à proposição de soluções que levem em consideração não só a pesquisa, mas os seus necessários desdobramentos de tratamento patrimonial e divulgação museológica. g g Algumas dessas abordagens têm um escopo internacional e são provenientes de problemas e novas inquietações científicas globais mas outras, para além dos desafios gerais, encontram um cenário muito específico no Brasil, que tem sido verificado de forma muito detalhada por diversos(as) autores(as) em seus trabalhos acadêmicos e artigos científicos, como por exemplo: Bruno (1984, 1995, 2000, 2005, 2013/2014), Caldarelli e Bruno (1982); Moraes Wichers (2010, 2011, 2013/2014), Saladino (2013/2014), apenas para mencionar alguns(mas) autores(as) 4 . Esses mesmos profissionais e estudantes têm impulsionado movimentos coletivos – como a criação do próprio Grupo de Trabalho Acervos Arqueológicos da Sociedade de Arqueologia Brasileira (SAB) e da Rede de Museus e Acervos Arqueológicos e Etnológicos (REMMAE) – e debatido essas questões de forma sistemática e elaborado documentos de orientação e de reivindicação inerentes ao tratamento dos acervos arqueológicos entre nós. A partir dessas considerações iniciais e rememorando análises já divulgadas em textos precedentes, neste momento, gostaria de compartilhar e recolocar algumas reflexões sobre as relevâncias, problemas e desafios, mas, pontuando que esses desafios estão sustentados por características que evidenciam uma expressiva longevidade no tempo, o que afeta a valorização das relevâncias dos acervos pelas sociedades contemporâneas. Acervos arqueológicos… \| Maria Cristina Oliveira Bruno ABSTRACT The approach to archaeological collections, in this text, is guided by the problematization related to the longevity of the collections and their historical- cultural implications for the societies that are responsible for their constitutions, maintenance, and preservations, with special projection on some Brazilian issues in the contemporary and still that corresponds to the curatorial responsibilities. Keywords: collection; longevity, curatorship. A R T Í C U L O RESUMEN En este trabajo el abordaje de las colecciones arqueológicas está guiado por la problematización relacionada con la longevidad de las colecciones y sus implicaciones histórico-culturales para las sociedades que son responsables de su constitución, mantenimiento y preservación, con especial proyección sobre algunas cuestiones brasileñas en la actualidad, así como en lo que concerniente a las responsabilidades curatoriales. Palabras clave: colección; longevidad; curaduría. 9 ARCHAEOLOGICAL COLLECTIONS:RELEVANCES, PROBLEMS AND CHALLENGES FROM EVER AND FOREVER 9 ARCHAEOLOGICAL COLLECTIONS:RELEVANCES, PROBLEMS AND CHALLENGES FROM EVER AND FOREVER APRESENTAÇÃO Trata-se de referendar que são questões desde sempre e, mais ainda, para sempre e, portanto, precisamos identificá-las e propor estratégias para o 10 enfrentamento dos dilemas, pois ainda teremos um indefinido tempo pela frente para a construção de argumentos que reafirmem a importância dos acervos e para a necessária experimentação de estratégias para a ressignificação, salvaguarda e divulgação deles. enfrentamento dos dilemas, pois ainda teremos um indefinido tempo pela frente para a construção de argumentos que reafirmem a importância dos acervos e para a necessária experimentação de estratégias para a ressignificação, salvaguarda e divulgação deles. Entre muitas possibilidades para discutir a problemática que envolve os acervos5 nos dias de hoje e de uma forma geral, especificamente os acervos arqueológicos no Brasil, foram selecionados alguns dilemas para serem abordados neste texto, aqui organizados em sete segmentos de análises, a saber: 1. Acervos refletem atitudes individuais e coletivas que as sociedades têm implementado em nome do colecionismo desde os mais remotos tempos e que, paulatinamente, têm acompanhado processos sociais, refletindo distintas dimensões de poder; as mudanças dos tempos históricos; a produção do conhecimento que se renova sistematicamente; as diferenças territoriais; e as diversas características identitárias: o colecionismo como matriz simbólica e operacional. 2. Acervos têm servido para a compreensão de identidades culturais, temporalidades sócio-históricas e determinações territoriais, mas também operam como forças para as negociações culturais, para a administração dos sistemas de memória e para a opressão social e econômica entre distintos indivíduos e grupos sociais: a longevidade dos acervos. g 3. Acervos são apropriados por instituições formatadas com a finalidade de pesquisa, preservação e musealização, podendo ser sistematicamente ressignificados e, ainda, abandonados, roubados, destruídos e menosprezados: o uso dos acervos. 4. Acervos têm sido a força alavancadora para a constituição e fortalecimento de muitos ramos do conhecimento, ensejando a identificação de distinções tipológicas e sinergias de aproximações que explicam os ambientes e as sociedades ao longo tempo e do espaço geográfico; caracterizando perfis profissionais e contribuindo para experimentações interdisciplinares e multiprofissionais a sua volta: a pesquisa e a geração de conhecimento. 5. Acervos evidenciam indicadores de memórias e referências culturais e têm a vocação para servir à educação para o patrimônio, para os direitos humanos e para a identidade em todas as suas dimensões: a educação e o exercício da cidadania. 6. 5 Cabe sublinhar que, em outros países, o que entendemos por “acervo” é tratado por “coleção”, de acordo com as publicações acadêmicas e documentos normativos das mais diversas origens. Para a compreensão deste texto o termo acervo será utilizado como um conjunto de coleções. 6 A expressão “enquadramento brasileiro” não deixa de levar em consideração as expressivas diferenças regionais. Acervos arqueológicos… \| Maria Cristina Oliveira Bruno em nossa trajetória de território colonizado; em uma nação federativa e de dimensões continentais, com longos anos de governos autoritários e alguns períodos de democracia; e em um país que tem construído os seus caminhos sócio patrimoniais entre tradições e rupturas preservacionistas. Mas, de uma forma geral, também podemos aplicar entre nós a ideia de que acervos arqueológicos (e mesmo os de outras tipologias) desvelam problemas desde sempre e representam problemas para sempre, pois de alguma forma são responsáveis pelo desvelar do que há de muito significativo em nossas sociedades ao longo do tempo, como também não nos deixam esquecer de tudo que há de mais perverso na trajetória da humanidade. São expressões e indicadores de memórias. em nossa trajetória de território colonizado; em uma nação federativa e de dimensões continentais, com longos anos de governos autoritários e alguns períodos de democracia; e em um país que tem construído os seus caminhos sócio patrimoniais entre tradições e rupturas preservacionistas. Mas, de uma forma geral, também podemos aplicar entre nós a ideia de que acervos arqueológicos (e mesmo os de outras tipologias) desvelam problemas desde sempre e representam problemas para sempre, pois de alguma forma são responsáveis pelo desvelar do que há de muito significativo em nossas sociedades ao longo do tempo, como também não nos deixam esquecer de tudo que há de mais perverso na trajetória da humanidade. São expressões e indicadores de memórias. Essa reconhecida vida dupla dos acervos, atrelada aos meandros técnico-científicos curatoriais, dependentes da superação de muitos paradigmas científicos e, ao mesmo tempo, do convívio sócio comunitário, nos leva a considerar a importância das políticas públicas concebidas, realizadas e avaliadas sistematicamente no que se refere às projeções que podem ser consideradas a partir dos usos dos acervos. Projeções essas responsáveis pela abertura de horizontes para que a educação que conta com indicadores de memórias em sua pedagogia impulsione uma visão crítica para o exercício da cidadania. Nesse sentido, não se enquadra uma visão ingênua relativa ao desvelar e ao cuidar de acervos enquanto conjunto de indicadores de memórias. Ao contrário, o tratamento e a preservação de acervos estão subordinados às perspectivas políticas em suas distintas conjunções e abordagens: tanto o estudo, o tratamento e a preservação quanto a negligência e o abandono. Acervos arqueológicos… \| Maria Cristina Oliveira Bruno APRESENTAÇÃO Acervos e as suas necessidades correspondentes ao estudo e à preservação exigem procedimentos que identificamos como curatoriais e as suas perspectivas de extroversão impõem que esses procedimentos sejam qualificados como museológico-curatoriais e colaborativos: as ações interdisciplinares, multiprofissionais e processuais. 7. Acervos, quando reconhecidos como conjuntos de referências patrimoniais materiais e imateriais, servem à valorização das grandes e multifacetadas expressões de humanidade, mas, ao mesmo tempo, documentam importantes tragédias e opressões sócio-históricas: a vida dupla dos acervos. Os acervos arqueológicos, evidenciados e preservados ao longo dos séculos, permitem análises, entre outras, referentes aos sete segmentos acima elencados. No enquadramento brasileiro6, tais acervos exigem que essas análises sejam contextualizadas Acervos arqueológicos… \| Maria Cristina Oliveira Bruno 11 11 Donde o aparecimento de uma procura, que atribui um valor aos objetos que virtualmente são peças de colecções, e cria um mercado. Donde também a pressão exercida sobre o Estado para que torne possível o acesso a estes bens àqueles que não podem comprá-los nem o prazer estético, nem os conhecimentos históricos e científicos, nem o prestígio (POMIAN, 1984, p. 54)7. Com essas palavras o autor articula diversas questões inerentes ao universo dos acervos. Por um lado, sinaliza para os processos de seleção e de novos enquadramentos individuais e coletivos e, por outro, aponta para o que circunda essas questões, como as variáveis do mercado, as implicações em políticas públicas de Estado, as expectativas sociais em relação à apreciação estética e à obtenção de conhecimento. Cabe sublinhar que os vestígios, fragmentos, artefatos, resíduos, ruínas, monumentos, entre outros segmentos reconhecidos, estudados e valorizados pela Arqueologia estão imersos desde sempre nessas questões. p q A atitude humana de extrair da realidade8 elementos, fragmentos, coisas materiais, para protegê-los e com isso formar coleções e acervos, foi incorporada pelas sociedades ao longo do tempo. Isso contribuiu para a formatação de uma lógica de procedimentos inerentes ao estudo, à preservação e à extroversão dessas coisas valorizadas, lógica essa que está na base da organização de instituições especializadas. Tal lógica tem se sofisticado ao longo dos tempos, gerando um caleidoscópio de ramos de pesquisa e de produção de conhecimento, de profissões e de ações técnicas. Ela é responsável também pela elaboração de normas, legislações, manuais, entre outras conquistas que o trabalho sistemático sobre acervos tem gerado. A maturidade técnica e científica aqui indicada, originada a partir do reconhecimento de que acervos constituídos exigem tratamentos específicos, tem levado à construção de convicções de que a necessidade de proceder à cura desses conjuntos transformados em acervos exige a ação curatorial 9 ou, mais precisamente, está subordinada aos processos curatoriais. Trata-se, entretanto, de construções de convicções que trilharam e ainda trilham distintos percursos, permeadas por olhares provenientes de diversos campos de conhecimento, inseridas em contextos socioeconômicos com perfis muito distintos e com disponibilidades incomparáveis de recursos (financeiros, técnicos e científicos) para a realização das ações necessárias. Refletir e agir em relação a processos curatoriais exige a compreensão de que abordamos, sempre, cenários institucionais – públicos e privados – com enormes diferenças. Neste contexto de longevidade, cabe ênfase para o momento em que essas convicções passaram a identificar novas necessidades para as ações curatoriais em relação aos acervos. Refiro-me, em especial, à passagem entre os séculos XIX e XX, quando a projeção das exposições e ações educacionais passaram a ser consideradas e valorizadas de uma forma mais ampla no que se refere ao tratamento dos acervos. Assim, a extroversão trouxe novos paradigmas e desafios inéditos, considerando que a ação de comunicação passou a fazer sentido e impôs outros problemas àqueles que têm sido responsáveis por acervos. 7 Nesta citação foi respeitado o texto original em português de Portugal. q q q p p p 9 Embora nos últimos anos as expressões curadoria e processo curatorial tenham sido aplicadas em contextos muito alargados, aqui indicam o conjunto de ações voltadas aos processos de pesquisa, de tratamento e extroversão dos acervos. Acervos arqueológicos… \| Maria Cristina Oliveira Bruno A LONGEVIDADE QUE ENVOLVE AS QUESTÕES SOBRE ACERVOS Diversos autores(as) se debruçam na construção histórica sobre os acervos, especialmente, abordando a historicidade das instituições de guarda, notadamente, os museus. Da mesma forma, essas análises explicitam movimentos transnacionais; reiteram o papel de destaque dos processos de colonização que entrelaçam os diversos continentes em conjunturas culturais comuns e expressivos dramas humanitários; mobilizam fontes documentais em diferentes suportes, tanto de caráter privado quanto público, entre muitas outras facetas dessa edificação. Esses estudos apontam que o colecionismo, entendido como base geradora dos acervos e de muitas instituições museológicas, tem atravessado os séculos, atingindo o seu auge entre os séculos XIX e XX, já com reflexos de diásporas que implementaram estas tradições em diversos continentes, especialmente, a tradição de constituir instituições em torno de acervos. É possível reconhecer que as coleções e acervos, ao longo do tempo, evidenciam muitas reciprocidades e muitos antagonismos entre os universos privados e públicos das sociedades. Conforme aponta Krzysztof Pomian, ...uma colecção, isto é, qualquer conjunto de objetos naturais ou artificiais, mantidos temporária ou definitivamente fora do circuito das actividades económicas, sujeitos a uma proteção especial num local fechado preparado para este fim, e expostos ao olhar público (POMIAN, 1984, p. 53). Diz também que certas peças de colecção são fonte de prazer estético; que outras – e por vezes são as mesmas – permitem adquirir conhecimentos históricos e científicos. Enfim, observa-se que o facto de as possuir confere prestígio, enquanto testemunham o gosto de quem as adquiriu, ou as suas profundas curiosidades, ou ainda a sua riqueza ou generosidade, ou todas as qualidades conjuntamente. Não é o caso para espanto, então, que se encontrem pessoas que queiram apropriar-se de tais objetos e que para atingirem este fim sacrifiquem uma parte de suas fortunas; ou outras que, não podendo apropriar-se de tais objetos, queiram ter pelo menos o direito de olhá-los. Acervos arqueológicos… \| Maria Cristina Oliveira Bruno 12 cumprirem com essas finalidades e estão na base da construção dessas convicções. Refiro-me, sobretudo, aos museus10, considerando sua longa trajetória comprometida com heranças dos gabinetes de curiosidades, antiquários, estúdios, fóruns de debates, laboratórios de pesquisa, espaços educacionais, entre outras características. Heranças essas ainda presentes com sintomas muito nítidos na contemporaneidade, mas que têm se articulado com os novos olhares e novos argumentos subordinados à dinâmica dos processos museológicos e às perspectivas dos processos de musealização. A partir de um olhar retrospectivo é possível identificar que sob a identidade de Museu de Arqueologia acumula-se, nos dias de hoje uma multiplicidade de características museológicas que indica um passado comprometido com aspectos relevantes da história dos museus, no que diz respeito à importância do colecionismo para estas instituições, como também, a evidente parceria com diferentes fases das descobertas e da pesquisa arqueológica nos últimos séculos. Outro aspecto marcante dessas instituições está vinculado ao caráter universalista de seu conteúdo, constituído de coleções difusas no tempo e no espaço. Da mesma forma, pode-se afirmar que, nas últimas décadas, esses museus têm servido também para o tratamento patrimonial da memória local, elevando-a, algumas vezes, ao patamar das referências nacionais e universais. Os museus de Arqueologia existem em todos os continentes, ainda reconhecidos como centros de pesquisa, vinculados em sua maioria às universidades e responsáveis por parcelas significativas do patrimônio que tem sido preservado ao longo dos séculos (...). As coleções arqueológicas estão na gênese da história dos museus. Amparados em alguns séculos de investigação e interesse pelo passado, pelo exótico e pelo diferente, esses acervos foram constituídos, de uma certa forma, para diminuir a distância entre as sociedades que vivem em tempos distintos. Espelham, também, a colonização, o saque e a destruição de alguns povos por outros. Sobretudo, esses acervos, espalhados em museus de portes diferentes, podem sinalizar aspectos inerentes à longevidade e diversidade da herança patrimonial dos seres humanos (BRUNO, 1999, p. 35-36). A partir do século XX, é possível considerar que, para os acervos constituídos e organizados de acordo com a lógica dos museus, têm sido impostas novas rotas, percepções inéditas e responsabilidades que se renovam a cada momento. 10 A referência a museu não deixa de prestar atenção também a outros modelos de instituição responsáveis por acervos. Acervos arqueológicos… \| Maria Cristina Oliveira Bruno A MUSEALIZAÇÃO QUE ACOLHE A LONGEVIDADE DA TRAJETÓRIA DOS ACERVOS Os pontos, aqui realçados em função das ações curatoriais inerentes aos acervos, levam em consideração as instituições que ao longo dos séculos foram formatadas para 13 responsáveis; e, sobretudo, (iv) de encontro de uma função social mais nítida para os processos curatoriais. O tempo, em um certo sentido, tem legado possibilidades de mudanças expressivas nesse contexto e, de alguma forma, tem desvelado a imperiosa necessidade de transformações e rupturas. Esse tempo percorrido, no caso das constituições e tratamentos dos acervos arqueológicos no Brasil, tem recebido muita atenção – às vezes de forma progressiva e em outras com grandes hiatos – no que se refere à compreensão sobre as reciprocidades entre construção de conhecimento e produção de acervos; no que diz respeito à identificação de vulnerabilidades entre legislação preservacionista e ações curatoriais; como também no que tange à cumplicidade entre os modelos de desenvolvimento socioeconômico e as estratégias de uso qualificado dos bens patrimoniais. 11 É importante ressaltar que essa trajetória tem recebido atenção de inúmeros profissionais, nas diferentes regiões do país, resultando em muitas publicações que desvelam novos processos, identificam momentos de rupturas e analisam a “questão arqueológica” brasileira sob diferentes ângulos, como já referido na nota de rodapé 5 deste texto. Acervos arqueológicos… \| Maria Cristina Oliveira Bruno Há uma nítida convicção de que (i) os museus precisam não só proceder ao tratamento dos acervos a partir de ações de estudo, salvaguarda e comunicação, mas que essas ações precisam interferir na realidade, permitindo novos olhares e a elaboração de novos argumentos; (ii) as atividades museológicas devem ressignificar os acervos; (iii) a educação a partir dos indicadores de memórias deve ser um princípio ético dessas instituições; e (iv) todas as suas ações têm uma razão e um propósito políticos. Essa convicção ainda tem sido permeada pela constatação de perspectivas mais inovadoras, como por exemplo: a compreensão da relevância da reciprocidade entre acervos materiais e imateriais; o entendimento sobre a importância do ambiente digital e das múltiplas tecnologias nesse contexto de inovações; e a constatação de que as ações curatoriais são dependentes de atividades coletivas quer sejam interdisciplinares no âmbito de uma instituição, quer sejam comunitárias em contextos compartilhados. A longevidade em relação à convivência das sociedades com os acervos, aqui apenas pontuada com vistas à projeção de alguns aspectos, tem dado a oportunidade (i) de um repensar sobre questões éticas; (ii) de um reposicionamento técnico e científico para a projeção mais nítida da necessidade do emprego de verbas públicas em ações curatoriais; (iii) de buscas incessantes sobre novas metodologias de trabalho mais inclusivas e 14 A LONGEVIDADE, O FUTURO DOS ACERVOS E O PAPEL DA MUSEALIZAÇÃO Ao reconhecermos que os acervos aportam e potencializam características de longa duração e se espraiam por distintos territórios geográficos, as perspectivas aqui enunciadas nos levam a considerar que é fundamental que a sua perenidade seja garantida de forma a produzir ressignificações, conhecimentos novos, tratamentos adequados de salvaguarda e projeções múltiplas de comunicação. Nesse sentido, é fundamental valorizar a importância das abordagens museológico- curatoriais (conservação, documentação, exposição e ação educativa), planejadas e executadas a partir de estratégias que envolvam e consolidem cadeias operatórias de ações técnicas e científicas, em que vários profissionais precisam atuar de forma organizada e em sinergia. Essa cadeia operatória, por sua vez, implica em cumplicidades, reciprocidades e produções coletivas que, normalmente, nos acostumamos a chamar de processos interdisciplinares e multiprofissionais. Da mesma forma, é possível reconhecer que esses procedimentos podem ser compartilhados com grupos comunitários e diferentes segmentos da sociedade. Hoje, compreendemos que é possível submeter os acervos arqueológicos a novas questões abrindo perspectivas de ressignificação; sabemos que existem as potencialidades de abordagens de inéditos e importantes problemas para a contemporaneidade, como a discussão sobre os processos de decolonização, a projeção de argumentos de gênero, a popularização de nuances identitárias, o entendimento sobre as hierarquias sociais, entre muitas outras possibilidades. Cada vez mais verificamos a relevância de inserir os acervos em cenários interpretativos no âmbito de paisagens culturais e contextualizá-los nas dimensões das memórias individuais, coletivas e históricas. Os processos de musealização recebem as heranças historicamente constituídas em função da longevidade dos problemas dos acervos, buscando a sua projeção futura para o enfrentamento dos problemas contemporâneos. Nesse sentido, para a longevidade desses processos, é fundamental uma inflexão, um mergulho, um aprofundamento nos paradigmas que envolvem os conceitos e as práticas referentes à musealidade e que se refletem nas estratégias substanciais para a implementação de ações museológicas com vistas à constituição de processos de musealização, mais especificamente processos de Musealização da Arqueologia. q g Entre muitas ideias que giram em torno dos conceitos acima referidos, é possível abrir um caminho de reflexão que nos conduz à compreensão sobre musealidade como a potencialidade inerente às coisas, aos vestígios, às expressões, sujeita aos olhares, a percepções e às seleções, ou seja: o fruto de olhares que identificam e valorizam. No caso da musealidade aplicada à Arqueologia, é possível projetar a ideia de que esse olhar seletivo é derivado de premissas de pesquisa e contextualizado com preocupações interdisciplinares. g q p Nesta trajetória11, cujo início pode ser identificado no final do século XIX, que entrelaça pesquisa, ensino, tratamento preservacionista, processos de musealização, educação patrimonial e outras esferas de ações públicas e privadas, é possível verificar onze movimentos ao longo dos anos: (I) o tempo do pioneirismo e dos amadores que, atuando em outras frentes, se interessaram pelo passado e empreenderam buscas de seus vestígios; (II) os olhares estrangeiros voltados ao conhecimento do passado do território brasileiro que focaram diferentes regiões, capacitaram equipes e dialogaram com instituições; (III) as trincheiras que foram necessárias para que a legislação preservacionista alcançasse os vestígios arqueológicos, protegendo-os enquanto bens patrimoniais; (IV) a formação de equipes de estudantes e pesquisadores, que iniciaram os estudos sistemáticos em grandes áreas do território nacional, produzindo explicações sobre as sociedades que precederam os períodos da colonização e mesmo avançando para outros momentos da trajetória do país; (V) a realização de estudos acadêmicos em larga escala, confrontando teorias, linhas metodológicas, estratégias interdisciplinares e refletindo as diversas frentes de formações profissionais e de estudos pós-graduados; (VI) o despertar para a importância das conexões entre estudos, acervos arqueológicos e processos museológicos, educacionais e de difusão científica; (VII) as reações aos impactos ambientais e à legislação preservacionista, permitindo a explosão de atividades de pesquisa, a expansão das opções profissionais para além das instituições públicas, alcançando a valorização do mercado e a convivência e cumplicidade com os grandes projetos de desenvolvimento do país; (VIII) a projeção das atividades museológicas e de educação patrimonial em diferentes contextos comunitários e socioeconômicos, com ações internas e externas às instituições de pesquisa; (IX) a mobilização de estudantes e profissionais em torno de causas comuns a partir da participação em associações, redes e grupos de pesquisa; (X) a conscientização em relação à necessidade de atenção aos acervos e à reavaliação sobre as necessárias conexões entre pesquisa, salvaguarda e ensino, referentes aos procedimentos arqueológicos e suas cumplicidades com as teorias, metodologias e funções das ações museológicas; (XI) o olhar de investigação orientado para temas marginalizados pelas pesquisas, o acolhimento a novos acervos desprezados pelos estudos normativos e a atuação comunitária e colaborativa como base para ações que geram acervos. Esses movimentos podem ser compreendidos em uma escala cronológica. Devem ser percebidos em suas tradições e rupturas e em ações de aproximações e estranhamentos, mas, em especial, precisam ser reconhecidos e interpretados para que 15 seja possível ampliar a importância da Arqueologia como campo de ensino e pesquisa e os acervos arqueológicos como itens estruturantes de nossa memória identitária. São esses os desafios para que a longevidade da trajetória dos acervos possa se ealmente acolhida pelos processos de musealização. Acervos arqueológicos… \| Maria Cristina Oliveira Bruno preservados, podem alçar o patamar de herança cultural. Da mesma forma, esses indicadores podem ser sistematicamente ressignificados, mas também podem trilhar o destino dos abandonos patrimoniais. preservados, podem alçar o patamar de herança cultural. Da mesma forma, esses indicadores podem ser sistematicamente ressignificados, mas também podem trilhar o destino dos abandonos patrimoniais. p Reiteramos abordagem precedente quando colocamos, O interesse está centrado na decodificação dos elementos que constituem os processos de Musealização da Arqueologia em seus domínios teóricos e estratégias metodológicas. Não se trata da apresentação de mais uma tendência e sim da indicação de um equilíbrio mais adequado entre os campos da Arqueologia e Museologia no que tange às ações de preservação, valorização, extroversão e educação dos contextos culturais que têm nos bens arqueológicos o eixo gerador de ações patrimoniais. De acordo com Shanks e Tylley (1992), pode-se compreender por musealização a elaboração de um sistema estético para criar significados. Já Waldisa Rússio Guarnieri (1990) aponta que, ao projetarmos as intenções de documentalidade e fidelidade quando musealizamos os objetos e artefatos, estamos procurando construir informações, o que pressupõe a transmissão de conhecimento afetivo / cognitivo), a realização do registro (daquilo que foi identificado) e o acesso aos mecanismos da memória (sistematização de ideias). Entre as colocações desses autores, pode-se inferir que os processos de musealização estão vinculados à valorização e à sistematização dos sentidos e significados extraídos das referências culturais que são alvo da atenção museológica (BRUNO, 2013/2014, p. 7). Com isso, queremos sublinhar que tanto a musealidade quanto a musealização são dependentes de ações que, por sua vez, são subordinadas a princípios éticos, conjunturas socioculturais e domínios de técnicas e tecnologias. Enquanto a musealidade está na gênese do processo, a musealização pode ser compreendida como a metodologia que consubstancia o processo, com a potencialidade de gerar conhecimento, valorizar indicadores de memórias e contribuir para a compreensão sobre o nosso contexto histórico e, sobretudo, impulsionar novas ações e reações no presente. Os acervos, em especial os acervos arqueológicos no Brasil, nos trazem problemas e preocupações desde sempre e, com certeza, nos conduzem a enfrentamentos e tentativas de superação de problemas para sempre. Acervos arqueológicos… \| Maria Cristina Oliveira Bruno A LONGEVIDADE, O FUTURO DOS ACERVOS E O PAPEL DA MUSEALIZAÇÃO Os resultados dessas percepções e seleções podem ou não ser interpretados a partir de contextos históricos, circunstâncias geográficas, especificidades inerentes aos problemas científicos projetados, entre outros fatores, que devem contribuir para as explicitações das camadas estratigráficas que constituem temporalidades socioculturais. Esses indicadores de musealidades permitem o enquadramento no universo dos vestígios, coleções e acervos, e incorporados como bens patrimoniais e uma vez Acervos arqueológicos… \| Maria Cristina Oliveira Bruno 16 Acervos arqueológicos… \| Maria Cristina Oliveira Bruno Nesse cenário delineado pelo passado, desenhado no presente e projetado para o futuro, é possível vislumbrar que os processos de Musealização da Arqueologia reúnem fortes potencialidades para o estabelecimento de necessárias sinergias interpessoais, muito necessárias para o desvelar das estratigrafias do abandono, ainda muito nítidas nos contextos da Arqueologia no Brasil. q g Ao terminar este texto, quando tive a especial possibilidade de compartilhar algumas reflexões, voltei à leitura do primeiro artigo que escrevi em coautoria com a arqueóloga e grande parceira Solange Bezerra Caldarelli, publicado em 1982 – “Arqueologia e Museologia: Experiências de um trabalho integrado. Pesquisas e Exposições do Instituto de Pré-História” – na Revista de Pré-História. Nesse artigo, relatamos as nossas experiências entrelaçadas entre a Arqueologia e Museologia, com diferentes comunidades, e sobretudo, depositamos o início da construção de nossas convicções e dos nossos sonhos profissionais. Por isso, agradeço muito à Solange por ter respeitado e encorajado os caminhos da Musealização da Arqueologia. Dedico este texto à memória de Paulo Duarte, por tudo que ele representa ainda hoje no que se refere às políticas públicas direcionadas à preservação patrimonial; e às novas gerações de estudantes e profissionais, que têm demonstrado que é possível conjugar o verbo esperançar! Acervos arqueológicos… \| Maria Cristina Oliveira Bruno 17 17 REFERÊNCIAS BIBLIOGRÁFICAS BRUNO, Maria Cristina Oliveira. O Museu do Instituto de Pré-História: um museu a serviço da pesquisa científica. 1984. Dissertação (Mestre em História Social). Faculdade de Filosofia, Letras e Ciências Humanas, Universidade de São Paulo, São Paulo. 1984. BRUNO, Maria Cristina Oliveira. Musealização da Arqueologia: um estudo de modelos para o Projeto Paranapanema. 1995. Tese (Doutorado em Arqueologia). Museu de Arqueologia e Etnologia, Universidade de São Paulo, São Paulo. 1995. BRUNO, Maria Cristina Oliveira. Museologia: A luta pela perseguição ao abandono. 2000. Tese (Livre-Docência). Museu de Arqueologia e Etnologia. São Paulo. 2000. BRUNO, Maria Cristina Oliveira. Arqueologia e antropofagia: a musealização de sítios arqueológicos. Revista do Patrimônio Histórico e Artístico Nacional. Museus: antropofagia da memória e do patrimônio. Iphan: Rio de janeiro, n° 31, p. 234-247, 2005. BRUNO, Maria Cristina Oliveira Bruno. Musealização da Arqueologia: caminhos percorridos. Revista de Arqueologia. Sociedade de Arqueologia Brasileira. São Paulo, vol. 26, n° 2, vol. 27, n° 1, p. 4-15, 2013/2014. CALDARELI, Solange Bezerra; BRUNO, Maria Cristina Oliveira. Arqueologia e Museologia: Experiências de um trabalho integrado. Pesquisas e Exposições do Instituto de Pré-História. Revista do Instituto de Pré-História, Universidade de São Paulo, São Paulo, vol. 111, n° 4, p. 143-191, 1982. DUARTE, Paulo. Fontes de Pesquisa Pré-Histórica. Separata de Estudos de Pré-História Geral e Brasileira p. 347-442. Instituto de Pré-História da Universidade de São Paulo, São Paulo, 1970. MORAES WICHERS, Camila de A. Museus e Antropofagia do Patrimônio Arqueológico: (des) caminhos da prática brasileira. 2010.Tese (Doutoramento em Museologia). Universidade Lusófona de Humanidades e Tecnologias, Portugal. Lisboa, 2010. MORAES WICHERS. Camila de. Patrimônio Arqueológico Paulista: proposições e provocações arqueológicas. Tese (Doutorado em Arqueologia). Museu de Arqueologia e Etnologia da Universidade de São Paulo. São Paulo, 2011. MORAES WICHERS, Camila de A. Dois enquadramentos, um mesmo problema: os desafios da relação entre museus, sociedade e patrimônio arqueológico. Revista de Arqueologia. Sociedade de Arqueologia Brasileira. São Paulo, vol.26, n° 2. vol. 27, n° 1, p.16-39, 2013/2014. POMIAN, Krzysztof. Coleção. Enciclopédia Einaudi Memória – História. Lisboa, Imprensa Nacional – Casa da Moeda vol.1, p.51-86, 1984. SALADINO, Alejandra. IPHAN, Arqueólogos e Patrimônio Arqueológico Brasileiro: um breve panorama. Revista de Arqueologia. Sociedade de Arqueologia Brasileira. São Paulo, vol.26, n° 2, vol.27, p. 40-57, 2013/2014. 18 18 Acervos arqueológicos… \| Maria Cristina Oliveira Bruno
W4376610672.txt	https://journals.univie.ac.at/index.php/voebm/article/download/7895/8057	de		Digitale Barrierefreiheit von Repositorien	Mitteilungen der Vereinigung Österreichischer Bibliothekarinnen & Bibliothekare	2,023	cc-by	4,841	Digitale Barrierefreiheit von Repositorien Christine Bazalka, Karin Kostrhon, Angelika Mucha, Leo Urlesberger Zusammenfassung: Thema dieses Beitrags ist die Barrierefreiheit von Repositorien, mit besonderem Fokus auf Nutzer:innen mit motorischen Beeinträchtigungen. Zur Erhebung des Status Quo und möglicher Verbesserungen wurden Expert:innen- und Nutzer:inneninterviews sowie automatisierte und manuelle Tests von 25 Bibliotheksrepositorien durchgeführt. Die Einschätzungen der Interviewpartner:innen und die Testergebnisse bieten nicht nur einen Einblick in die aktuelle Situation, sondern auch Anregungen hinsichtlich Sensibilisierung, Schulungsunterlagen für Nutzer:innen und Bibliothekar:innen sowie Integration von Barrierefreiheits-Checks in den Arbeitsalltag. Der Beitrag fasst die Ergebnisse eines im Rahmen des Universitätslehrgangs Library and Information Studies (Grundlehrgang) umgesetzten Projekts zusammen. Schlagworte: digitale Barrierefreiheit, Repositorien, motorische Beeinträchtigung, Leitfadeninterviews, Accessibility-Tests, Sensibilisierung, Universitätsbibliotheken Digital Accessibility of Repositories Abstract: This article focuses on the accessibility of repositories, with an emphasis on users with physical disabilities. Expert and user interviews as well as automated and manual tests of 25 library repositories were conducted to learn about the status quo and to suggest possible improvements. The assessments of the interviewees and the test results present an insight into the current situation and offer suggestions regarding raising awareness, providing instruction material for users and librarians and integrating accessibility checks into the workflow. The article summarizes the results of a project for the study programme Library and Information Studies. Keywords: digital accessibility, repositories, physical disability, guided interview, accessibility check, raising awareness, university libraries DOI: https://doi.org/10.31263/voebm.v76i1.7895 Dieses Werk ist – exkl. einzelner Logos und Abbildungen – lizenziert unter einer Creative-Commons-Lizenz Namensnennung 4.0 International-Lizenz Dieser Beitrag entstand infolge der Projektarbeit „Digitale Barrierefreiheit von Repositorien. Anregungen zur Gestaltung barrierefreier Repositorien mit Schwerpunkt auf motorischen Beeinträchtigungen“, die wir während des Universitätslehrgangs Library and Information Studies 2021/2022 an der ÖNB verfassten. Auftraggeberin war Dr.in Susanne Blumesberger, Leiterin der VÖB-AG Barrierefreiheit in Bibliotheken 1. Fragestellungen Mithilfe von Expert:innen- und Nutzer:inneninterviews sowie manueller und automatisierter Barrierefreiheitstests von Repositorien wollten wir insbesondere herausfinden, – was die wichtigsten Aspekte von digitaler Barrierefreiheit für Menschen mit motorischen Beeinträchtigungen sind – welche Barrieren bei bibliothekarischen Repositorien existieren – inwieweit manuelle und/oder automatisierte Tests von Webseiten dazu beitragen können, Barrierefreiheit von Repositorien festzustellen, und ob diese auch im bibliothekarischen Alltag und ohne IT-Fachwissen durchführbar sind 2. Studieren mit motorischer Beeinträchtigung Laut Studierenden-Sozialerhebung1 waren in Österreich im Jahr 2019 12,2 % bzw. 39.100 Studierende von einer studienerschwerenden Beeinträchtigung betroffen. Bei 2,6 % davon wirkte sich eine Mobilitäts- oder motorische Beeinträchtigung einschränkend auf das Studium aus. Motorische Einschränkungen können die Fein- und/oder die Grobmotorik betreffen; die möglichen Ursachen sind vielfältig, informiert die Universität Wien auf ihrer Website für barrierefreie Lehre.2 Als motorische Einschränkungen, die sich im Studium auswirken können, werden genannt: – akute Einschränkungen der Schreibhand (z.B. durch Unfall, Sehnenscheidenentzündung oder Bruch) – permanente Einschränkungen (z.B. Spasmen, Lähmungen, Fehlen von Gliedmaßen, Zittern, Muskelschwäche etc.) – schubhaft verlaufende Einschränkungen (z.B. bei Rheuma oder Gicht) oder – eingeschränktes Sprechvermögen (z.B. durch Multiple Sklerose oder Schlaganfall) Im Studienalltag kann das mit Einschränkungen beim Schreiben, Sprechen, Arbeiten unter Zeitdruck oder bei der Benutzung von technischen Geräten verbunden sein. 3. Gesetzliche Rahmenbedingungen und Standards für digitale Barrierefreiheit Auf gesetzlicher Ebene wird die Umsetzung digitaler Barrierefreiheit mit der Web-Zugänglichkeits-Richtlinie der Europäischen Union konkretisiert, die Vorschriften für Websites und mobile Anwendungen öffentlicher Stellen beinhaltet.3 Das österreichische Web-Zugänglichkeits-Gesetz (WZG) ist seit 2019 in Kraft und ist die Umsetzung der EU-Web-Zugänglichkeits-Richtlinie in österreichisches Recht.4 Die zugrundeliegende EU-Norm orientiert sich an den Standards der Web Content Accessibility Guidelines (WCAG) und legt einen „harmonisierten europäischen Standard“ fest.5 Barrierefreie Webinhalte müssen laut WCAG Kriterien in vier grundlegenden Kategorien erfüllen6: Wahrnehmbarkeit – – – – Bilder und andere Nicht-Text-Inhalte sollen mit Text beschrieben werden Multimediale Inhalte sollen mit Untertiteln und Alternativen wie Bildbeschreibungen versehen sein Inhalte sollen auch mit assistierenden Technologien wie Screenreadern wahrnehmbar sein Inhalte sollen – etwa durch ausreichenden farblichen Kontrast – für möglichst viele Menschen leicht wahrnehmbar sein Bedienbarkeit – – – Webinhalte sollen grundsätzlich auch nur mit der Tastatur steuerbar sein Nutzer:innen sollen genügend Zeit zum Lesen und Nutzen der Inhalte haben Nutzer:innen sollen genügend Unterstützung beim Lesen und Navigieren erhalten – – Webinhalte sollen keine Anfälle oder physischen Reaktionen hervorrufen Alternative Eingabegeräte sollen gut nutzbar sein Verständlichkeit – – – Text soll lesbar und verständlich sein Inhalte sollen auf vorhersagbare Art präsentiert werden Nutzer:innen sollen dabei unterstützt werden, Fehler zu vermeiden oder zu korrigieren Robustheit – Inhalte sollen mit derzeit genutzten und künftig verfügbaren assistierenden Werkzeugen oder Technologien kompatibel sein 4. Computer- und Internetnutzung mit motorischen Beeinträchtigungen Die möglichen Ursachen und Manifestationen von motorischen Beeinträchtigungen sind vielfältig. Dadurch ergeben sich auch unterschiedliche Arten der Computer- und Internetnutzung, wie etwa Hellbusch7 im Detail beschreibt. Im Folgenden stellen wir eine Auswahl an Geräten und assistierenden Technologien vor, die je nach individuellem Bedarf genutzt werden können: Ist die Bewegungsfähigkeit der Arme, Hände und/oder Finger reduziert, können alternative Eingabegeräte zum Einsatz kommen. Neben Kompakt- oder Einhandtastaturen können etwa Großfeldtastaturen oder Bildschirmtastaturen genutzt werden. Trackball, Joystick- oder Kopfmaus können für Menschen mit motorischen Einschränkungen besser nutzbare Alternativen zur herkömmlichen „Computermaus“ darstellen. Auch Augen- oder Mund-Steuerungssysteme sind je nach Art der motorischen Einschränkung mögliche Optionen. Bei Spracheingabesystemen wird gesprochener Text der Nutzer:innen in Befehle für den Computer umgewandelt. Auch Betriebssysteme selbst verfügen über eine Reihe von Bedienungshilfen, die je nach individuellem Bedarf aktiviert werden können, zum Beispiel die Möglichkeit der Einfingerbedienung, Bildschirmlupen, Optionen zur alternativen Kontrastund Farbdarstellung oder Sprachausgaben.8 Bei der Nutzung von Internetangeboten durch Menschen mit motorischen Beeinträchtigungen kommt einigen Richtlinien der WCAG ganz besondere Bedeutung zu, auf die wir zum Teil auch in unseren Repositorientests Bezug nehmen. Bei den deutschen Übersetzungen und Erläuterungen orientieren wir uns an der deutschen BIK-Initiative9, deren BIK BITV-Test seit 2005 das etablierte Prüfverfahren in Deutschland ist: – Guideline 2.1 Keyboard Accessible: Make all functionality available from a keyboard. Success criterion: 2.1.1 Keyboard (Level A) Die Bedienung soll geräteunabhängig möglich sein. Das bedeutet: Sie muss sowohl mit der Maus als auch mit der Tastatur möglich sein. Auf die Tastaturbedienbarkeit sind zum Beispiel viele motorisch eingeschränkte oder blinde Menschen angewiesen. – Guideline 2.4 Navigable: Provide ways to help users navigate, find content, and determine where they are. Success criteria: 2.4.1 Bypass Blocks (Level A); 2.4.3 Focus Order (Level A); 2.4.7 Focus Visible (Level AA) Verschiedene Inhaltsbereiche wie Navigation, Suche oder Seiteninhalt sollen von Nutzer:innen assistiver Technologien übersprungen werden können. Der Seitenaufbau soll unabhängig von der Darstellung deutlich werden. Wenn die Webseite mit der Tastatur bedient wird, soll die Reihenfolge, in der Links, Formularelemente und Objekte angesteuert werden, schlüssig und nachvollziehbar sein. Auch, wenn eine Website via Tastatur, beispielsweise über die Tab-Taste, navigiert wird, muss jederzeit klar sein, wo sich der Cursor gerade befindet. Das Element, das gerade angesteuert wird, hat dabei Fokus. Der Tastaturfokus soll mindestens genau so deutlich hervorgehoben werden wie der Mausfokus. Versteckte Sprunglinks10 sollen bei Fokuserhalt eingeblendet werden. Zusammenfassend lässt sich jedenfalls sagen, dass digitale Barrierefreiheit das gesamte World Wide Web und auf Nutzer:innenseite verschiedenste Arten der Beeinträchtigung bzw. Nutzungsgewohnheiten betrifft. Damit wollen wir explizit auf einen der Leitsätze des WAI Bezug nehmen: „Web accessibility is essential for people with disabilities and useful for all.“ 5. Erhebung, Teil 1: Tests ausgewählter Repositorien Im Rahmen der Projektarbeit haben wir insgesamt 25 Repositorien getestet und uns dabei auf die – gemessen an ihrem Bestand im Jahr 201911 – größten Universitätsbibliotheken im deutschsprachigen Raum, mit Schwerpunkt auf Österreich, beschränkt. Damit soll zumindest eine gewisse Vergleichbarkeit der Ergebnisse gewährleistet sein, da hier ähnliche gesetzliche Rahmenbedingungen bestehen. Die Liste umfasst folgende Repositorien: BOK:ePub (UB BOKU Wien) • BORIS (UB Bern) • edoc (UB Berlin) • edoc (UB Basel) • ePLUS (UB Salzburg) • GRO.publications (SUB Göttingen) • JKU ePUB (UB Linz) • KUGscholar (UB KU Graz) • LARA (ZHB Luzern) • MedUni Wien ePub (UB MedUni Wien) • miami (ULB Münster) • Netlibrary (UB Klagenfurt) • Phaidra (UB Wien) • Phaidra (UB Vetmeduni Wien) • pub.mdw (ub.mdw) • Publikationsserver (ULB Tirol) • Publikationsserver (UB Frankfurt a. M.) • Refubium (UB FU Berlin) • Repository (UB Mozarteum) • reposiTUm (UB TU Wien) • Research Collection (ETH-Bibliothek) • TU Graz Forschungsportal (UB TU Graz) • unipub (UB Graz) • WU Research (UB WU Wien) • ZORA (ZB/UB Zürich). Im Rahmen der Projektarbeit haben wir automatisierte Tests und manuelle Tests durchgeführt – diese können aufgrund der Komplexität der geltenden Vorgaben und der großen Zahl an Testoptionen Tests durch Expert:innen oder Nutzer:innen nicht ersetzen, aber – so unser Denkansatz – zumindest eine Vorstellung davon vermitteln, welche Aspekte hinsichtlich digitaler Barrierefreiheit relevant sein können und wie man sie auch im bibliothekarischen Alltag erkennen kann. 5.1 Automatisierte Tests Wir haben mit WAVE (Web Accessibility Evaluation Tool) gearbeitet, um einige grundlegende Aussagen über die digitale Barrierefreiheit der Startseiten der Repositorien machen zu können. WAVE testet viele Aspekte, die für digitale Barrierefreiheit von grundlegender Bedeutung sind, darunter Bilder und Alternativtexte, Strukturen von Websites, Formulare, Tabellen oder Kontraste. Die Testergebnisse werden in WAVE in sechs Kategorien dargestellt, die dann wiederum weiter spezifiziert werden: Errors, Contrast Errors, Alerts, Features, Structural Elements und ARIA (Accessible Rich Internet Applications). Näher untersucht haben wir die Kategorie „Errors“, da damit der drängendste Handlungsbedarf aufgezeigt wird. Darunter sind in WAVE Zugänglichkeitsbarrieren zu verstehen, die sich unmittelbar für die Nutzer:innen bemerkbar machen und mit großer Wahrscheinlichkeit gegen die WCAG 2 Level A/AA verstoßen. Von insgesamt 100 Errors, die auf den Startseiten von WAVE gefunden wurden, entfallen allein 23 auf die Kategorie „Missing alternative text“, gefolgt von „Empty link“ mit 20 und „Linked image missing alternative text“ mit 18. Nicht vorhandene Alternativtexte, die insbesondere – aber nicht nur – für sehbeeinträchtigte Menschen wichtig wären, sind demnach gemeinsam mit leeren Links die häufigste Fehlerart. In den Browsern Chrome und Firefox werden im Durchschnitt pro getesteter Seite 4,0 Errors angezeigt, wobei der Minimalwert 0, der Maximalwert 11 beträgt. Die Zahl der durchschnittlichen Contrast Errors pro Seite schwankt je nach Browser geringfügig zwischen 5,9 (Chrome) und 6,2 (Firefox). Der Minimalwert liegt bei 0, der Maximalwert bei 39. WAVE ist nicht zuletzt deshalb niederschwellig, weil es zu den Einzelergebnissen auch für technische Lai:innen relativ gut verständliche Hintergrundinformationen und Lösungsoptionen bereithält und auf die korrelierenden WCAG-Paragrafen verweist. Man kann dadurch nicht jedes der aufgezeigten Probleme selbst lösen, aber man kann sicher ein besseres Verständnis dafür entwickeln. 5.2 Manuelle Tests Eingabemöglichkeiten lassen sich im Wesentlichen in zwei Kategorien einteilen: Eingabe mittels Zeigegerät (z. B. Computermaus) oder Eingabe über eine Tastaturschnittstelle.12 Wenn die Verwendung einer herkömmlichen Computermaus wegen starker motorischer Einschränkungen nicht möglich ist, und eine Steuerung des Mauszeigers auch mit alternativen Geräten nicht funktioniert, rücken Tastaturschnittstellen in den Fokus: Diese werden nicht nur mit Tastaturen im engeren Sinn bedient, sondern interagieren z. B. mit Spracheingaben oder Spezialtastaturen. Für die seiteninterne Navigation stehen dabei nur einige Tasten zur Verfügung – die Tabulatortaste (Tab) etwa wird für die Navigation zwischen Links und Steuerelementen verwendet und ist damit für Tastaturnutzer:innen, die keine Maus verwenden können, zentral. Sie können z. B. mittels Tab-Taste durch eine Webseite navigieren („tabben”), indem sie von Element zu Element springen. Die Bedienbarkeit der Repositorien mittels Tastatur haben wir so getestet: – einen Suchprozess in sechs Einzelschritte zerlegt und diese bewertet – erfasst, wie häufig die Tab-Taste gedrückt werden muss, um erstmals einen Fokus zu sehen – die visuelle Darstellung des Fokus bewertet – Uploadformulare, soweit frei verfügbar, auf ihre Tastaturbedienbarkeit geprüft und bewertet Der Suchprozess konnte in allen Fällen zu einem Abschluss gebracht werden, es gab aber bei Einzelschritten häufig Unwägbarkeiten oder auch schwerer wiegende Probleme. Fast überall fehlte etwa ein beim Tabben sichtbarer Skip-Link, der es ermöglichen würde, direkt vom Einstiegspunkt zur Suche zu springen – stattdessen muss z.B. durch die Navigationsleisten in Kopf- und Fußzeile getabbt werden, was langwierig ist, zumal sich dieser Schritt während eines Suchprozesses oft mehrmals wiederholt. In rund 50 % der Fälle war der Fokus nicht uneingeschränkt über den gesamten Prozess sichtbar. Fünf der acht getesteten Upload-Formulare mussten negativ bewertet werden, da die Aktionsfelder „PDF hochladen“ sowie “PDF, ZIP hochladen” mit der Tastatur nicht ansteuerbar waren.13 5.3 Fazit der Tests All unsere Tests ergaben Mängel bei der Barrierefreiheit: Nur zwei der 25 getesteten Repositorien (8 %) wurde durch den WAVE-Test kein Fehler ausgewiesen.14 Beim Test der Tastaturbedienbarkeit konnten alle Suchvorgänge positiv bewertet werden, die Spannweite bei der Sichtbarkeit des Fokus und der Nutzer:innenfreundlichkeit war allerdings erheblich. Da wir uns in den einzelnen Testschritten auf so unterschiedliche Aspekte konzentriert haben, erscheint uns eine Synthese der Ergebnisse nicht sinnvoll. Im Rahmen des Testprozesses sind uns aber einige, unserer Meinung nach erwähnenswerte Punkte aufgefallen: – Gerade bei technisch relativ einfach umzusetzenden Verbesserungen, konkret z.B. Alternativtexte oder durchgehende Fokussichtbarkeit, wäre eine baldige Beseitigung der Hürden wünschenswert. – Auch das Bereitstellen von Informationen zur digitalen Barriere- – – freiheit an möglichst prominenter Stelle auf der Website erscheint uns eine wichtige und relativ einfach umzusetzende Maßnahme, die nicht nur einen konkreten Informationsbedarf abdecken, sondern auch zur Bewusstseinsbildung beitragen kann. Für Repositorien stellt sich die Frage nach der Prüfung der von Studierenden oder Forschenden hochgeladenen Ressourcen. Hier gäbe es unserer Ansicht nach zwei mögliche Ansätze: 1. Kostenlose Schnelltests wie den PDF Accessibility Checker15, auf die deutlich sichtbar verwiesen wird. 2. Eine automatisierte Prüfung der hochgeladenen Dateien hinsichtlich Barrierefreiheit. Bestimmte Tests könnten auch im bibliothekarischen Arbeitsalltag eingesetzt werden, etwa zur Prüfung von Kontrasten. Eine Übersicht über einfache Evaluationsmöglichkeiten bietet die WAI, zudem eine Liste spezifischer Anwendungen. Auch die Forschungsförderungsgesellschaft hat eine Liste mit hilfreichen Tools erstellt. 6. Erhebung, Teil 2: Interviews In diesem Teil soll auf die Interviews eingegangen werden, die im Rahmen der Projektarbeit durchgeführt wurden. Insgesamt wurden neun leitfadengestützte Interviews mit zwei Personengruppen durchgeführt. – Expert:innen: 8 Personen, die beruflich mit digitaler Barrierefreiheit und/oder Repositorien zu tun haben (Bibliotheken, Verbände, Organisationen) – Nutzer:innen: 3 Personen mit motorischen Einschränkungen Die erste Personengruppe (Expert:innen) umfasst acht Personen, die auf unterschiedliche Weise beruflich mit dem Thema digitale Barrierefreiheit zu tun haben. Darunter fallen z.B. Personen aus dem Bibliotheksbereich, die für den Betrieb und die Wartung von Repositorien zuständig sind, aber auch Menschen, die in Verbänden, Organisationen oder Agenturen mit digitaler Barrierefreiheit befasst sind. Die Interviews mit diesen Personen dienten insbesondere dazu, den technischen Blickwinkel auf das Themenfeld Repositorien sowie ganz allgemein Computerbedienung für Menschen mit motorischen Beeinträchtigungen einzufangen und abzubilden. Die zweite Personengruppe (Nutzer:innen) umfasst drei Personen mit motorischen Einschränkungen. Das Ziel ihrer Befragung bestand darin, die Sichtweise von motorisch eingeschränkten Personen und den daraus resultierenden Barrieren bei der Nutzung von Repositorien oder anderen Bibliotheksservices nachzuzeichnen. Die Interviews wurden, in Abstimmung mit den befragten Personen, wahlweise physisch oder online durchgeführt, aufgezeichnet und anschließend transkribiert. Alle Personen wurden ersucht, eine Einverständniserklärung zur Weiterverarbeitung der erhobenen Daten auszufüllen und erhielten im Vorfeld der Interviews die Leitfäden zur Vorbereitung. Sofern Personen eine namentliche Nennung im Projektbericht wünschten, wurde dies berücksichtigt, andernfalls erfolgte eine Anonymisierung. Die Leitfäden der jeweiligen Interviews orientieren sich inhaltlich an unseren zuvor gestellten Forschungsfragen, wurden aber an jedes Interview angepasst und frei geführt. Im nächsten Teil soll nun kurz ein Überblick über die Themen in den Interviews gegeben werden. 6.1 Themen in den Interviews Folgende Fragen und Themengebiete versuchten wir im Zuge der Interviews zu erörtern: 1. Wie sieht die Situation von Menschen mit motorischen Beeinträchtigungen aus? Welche Barrieren tun sich in der Handhabung von PCs/Webseiten/allgemeiner Texteingabe auf? Welche Erfahrungen werden seitens der Nutzer:innen eingebracht? 2. Was sind wichtige Aspekte von digitaler Barrierefreiheit, sowohl aus Sicht von Nutzer:innen als auch Expert:innen? Wie beschäftigen sich die Expert:innen mit dem Thema im beruflichen Kontext und wo sind Verbesserungsvorschläge erwünscht? 3. Welche technischen Hintergründe gilt es bei der Umsetzung von digitaler Barrierefreiheit auf Webseiten von (Universitäts-)Bibliotheken zu beachten? 4. Wie ist es um die Barrierefreiheit des Repositoriums PHAIDRA bestellt? Welche Probleme tun sich dabei für Nutzer:innen auf, welche Schritte müssen noch gemacht werden und wie werden sie von Expert:innen eingeschätzt? 5. Was sind aus Expert:innensicht Anforderungen an Schulungsunterlagen? 6. Was ist der politische bzw. gesellschaftliche Rahmen von digitaler Barrierefreiheit? 6.2 Zusammenfassung der Interviewaussagen aus der Nutzer:innengruppe Zunächst ist zu sagen, dass sich Verallgemeinerungen zu Nutzer:innen mit motorischen Einschränkungen nur schwer treffen lassen. Begründet liegt dies nicht zuletzt darin, dass Personen mit motorischen Einschränkungen keineswegs eine homogene Gruppe darstellen und teilweise eine Vielzahl an Einschränkungen bei der Computernutzung auftreten kann. Darüber hinaus sind die von uns befragten Nutzer:innen nur eingeschränkt mit Problemen bei der Nutzung von Webseiten konfrontiert, die nicht auch von anderen behinderten oder nichtbehinderten Personen erfahren werden können. Insbesondere wurde in den Interviews thematisiert, dass ein schlechter Aufbau und mangelhafte Strukturierung sowie fehlende Möglichkeiten zur Tastaturbedienung Barrieren für Nutzer:innen mit motorischen Einschränkungen darstellen. Diese Punkte sind besonders schwerwiegend bei der Verwendung von Sprachsteuerung, aber auch von Screenreadern:16 – Zeitdruck auf Webseiten oder in Apps durch Zeitbeschränkungen – Einsatz von Sprachsteuerung bei nicht dafür optimierten Webformularen – Exkludierende Hardware – Eine nicht ausreichende Größe der Bedienungselemente – fehlende Alternativen für Multipoint- oder pfadbasierte Gesten Zusammenfassend lässt sich sagen, dass aus den Interviews deutlich wurde, dass eine gesamtheitliche Betrachtung des Themas digitale Barrierefreiheit notwendig ist und von uns sowie unseren Interviewpartner:innen als sinnvoll erachtet wird.17 6.3 Zusammenfassung der Interviewaussagen aus der Expert:innengruppe In diesem Abschnitt sollen die wesentlichen Interviewaussagen aus der Personengruppe der Expert:innen zusammengefasst werden. Insgesamt sind hier vier Punkte zentral: Als wichtigster Punkt in allen Expert:inneninterviews wird die Sensibilisierung genannt. Eine persönliche Auseinandersetzung mit dem Thema digitale Barrierefreiheit kann allen empfohlen werden (z. B. durch Nut- zung von Screenreadern oder mittels Tastaturbedienung) und kann als erster gelungener Schritt in Richtung Sensibilisierung gesehen werden. Auf einer breiteren Ebene könnte dies auch durch Schulungen, Informationsmaterialien und klare Handlungsrichtlinien geschehen. Die Sensibilisierung sollte sich nicht nur auf Bibliothekar:innen beschränken, sondern auch für unterschiedliche Nutzer:innen-Rollen zugeschnitten werden (wie beispielsweise Schulungsunterlagen für Nutzer:innen zur Contentgestaltung, in Bereichen der IT-Entwicklung, etc.), um ein grundlegendes Verständnis für die Kriterien von digitaler Barrierefreiheit zu schaffen. Die befragten Expert:innen, die an Hochschulen tätig sind und sich mit Metadaten beschäftigen, nutzen in ihrer jeweiligen Institution unterschiedliche Metadatenstandards (z.B. Dublin Core bei einer/einem Expert:in und UWMETADATA bei zwei anderen). Die Erarbeitung klarer und einheitlicher Metadatenstandards auf Repositorien stellt einen wichtigen Punkt dar. Metadaten sind besonders bei der Verwendung von assistiven Technologien wie Screenreadern sehr wichtig, um eine Grundlage für eine erste barrierefreie Navigation und Nutzung zu schaffen. Einen besonders wichtigen Punkt hinsichtlich der Barrierefreiheit von Repositorien stellen die darin hochgeladenen Inhalte dar, da diese nicht auf ihre Barrierefreiheit geprüft werden können. Hier ist man auf die Sensibilisierung der Nutzer:innen hinsichtlich der Erstellung von barrierefreien Dokumenten angewiesen. Um diese Bewusstseinsbildung bei Nutzer:innen zu ermöglichen, sollten möglichst einfache und klare Guidelines oder Handlungsempfehlungen für die Erstellung barrierefreier Inhalte zur Verfügung gestellt werden. Das Repositorium PHAIDRA verweist z.B. an prominenter Stelle auf Guidelines zur Erstellung barrierefreier Dokumente. Idealerweise sollten solche Guidelines nicht erst vor dem Formular zum Upload von (Forschungs-) Daten an die Nutzer:innen herangetragen werden, sondern es sollte bereits in den frühen Phasen von Contenterstellung auf barrierefreie Gestaltung von Dokumenten verwiesen werden. Wir haben uns bereits vor der Durchführung der Interviews die Frage gestellt, wie mögliche Schulungsunterlagen zur Umsetzung von digitaler Barrierefreiheit in Repositorien aufgebaut sein könnten. Wir haben zu dieser Frage von unseren Interviewpartner:innen eine Vielzahl an Antworten erhalten. Punkte, die unseren Gesprächspartner:innen wichtig waren, sind Stepby-Step-Anleitungen, die Auflistung von eindeutigen Beispielen und Kontakt zu qualifizierten Ansprechpersonen. Ein Wiki-Format würde sich hier nach Aussage der Expert:innen besonders empfehlen, müsste aber regelmäßig aktualisiert werden. Der Austausch, der etwa in Form eines Forums passieren könnte, bildete ebenfalls einen wichtigen Punkt in den Interviews. 6.4 Fazit der Interviews Als Schlussfolgerung aus unseren Interviews schlagen wir folgende Empfehlungen vor, die uns sinnvoll erscheinen, um digitale Barrierefreiheit im bibliothekarischen Kontext ganzheitlich betrachten, Probleme der eigenen Institution erkennen und sich in dem Bereich weiterbilden zu können: 1. Maßnahmen zur Sensibilisierung sind die wesentliche Voraussetzung für die Umsetzung von digitaler Barrierefreiheit. Sensibilisierung kann sowohl durch Gespräche mit Nutzer:innen als auch durch das eigenständige Austesten von assistiven Technologien, im Idealfall unter Einbeziehung von Interessenverbänden und Beratungseinrichtungen, erfolgen. Andererseits bieten sich Schulungen sowie die Entwicklung und Bereitstellung von Informationsmaterialien an, wobei klar sein sollte, dass eine kontinuierliche Weiterentwicklung notwendig ist. Der Zugang zu Informationen ist, weiterführend gedacht, eine der wichtigsten Maßnahmen zur Sensibilisierung. Hier bietet es sich an, ein institutionsübergreifendes Wiki zu schaffen, in dem alle gesetzlichen Hintergründe leicht zu finden sind, bereits vorhandene Richtlinien und Guidelines (wie WCAG) vorhanden und ausreichend kommentiert sind und es eine ausführliche Beispielsammlung gibt, anhand derer man Dokumente barrierefrei gestalten oder selbst Testungen der eigenen Webseite durchführen kann. Es sollte auch ein Workflow enthalten sein, mit dessen Hilfe Bibliothekar:innen möglichst einfach Testungen des eigenen Repositoriums durchführen können (inklusive Verweis auf aktuelle Testungssoftware, ausführlicher Anleitung, etc.). Um Hilfestellung im Arbeitsalltag zu bieten, müssen die im Wiki enthaltenen Informationen stets aktuell und auf bibliothekarische Bedürfnisse zugeschnitten sein, was natürlich einen gewissen Betreuungsaufwand bedeutet. Wir würden vorschlagen, dass jene Bibliothekar:innen, die das Wiki betreuen, von ihren jeweiligen Institutionen dafür genügend Arbeitszeit zur Verfügung gestellt bekommen. Wir möchten auch das Abhalten von regelmäßigen Schulungen vorschlagen, die losgelöst von der jeweiligen Institutionsebene für alle Bibliothekar:innen österreichweit getragen werden. Als Orientierung könnte dabei die Kommission Kundenorientierter und inklusiver Services des Deutschen Bibliotheksverband dienen, die in Zusammenarbeit mit der AG Barrierefreiheit versucht, diese Thematik mit Seminaren und Schulungsangeboten zu vermitteln. 2. Eine standardmäßige Einbindung von Menschen mit Beeinträchtigungen ist aus unserer Sicht besonders wichtig, um Anforderungen ermitteln zu können, Barrieren aufzuzeigen und eine inklusive Umsetzung zu ermöglichen. 3. In der bibliothekarischen Welt in Österreich wurde mit der Gründung der VÖB-AG Barrierefreiheit in Bibliotheken im Jahr 2020 ein wichtiger Grundstein gelegt, um sich institutionsübergreifend mit dem Thema zu beschäftigen. Wir schlagen eine Stärkung und längerfristige Weiterführung dieser vorerst auf zwei Jahre angelegten AG vor, um eine zielführende Auseinandersetzung mit dem komplexen und sich stetig entwickelnden Thema der digitalen Barrierefreiheit zu ermöglichen. Idealerweise sollte jede größere Bibliothek in der AG selbst vertreten sein, um eine breite und sinnvolle Befassung mit Barrierefreiheit zu ermöglichen. 4. Entwicklung neuer Strategien beim Erwerb von Repositoriumslizenzen: Viele österreichische Hochschulen kaufen die Software für ihre Repositorien zu. Barrierefreiheit sollte hier schon im Erwerbungsprozess mitgedacht werden.18 Um die Verhandlungsposition in Bezug auf digitale Barrierefreiheit zu stärken und gemeinschaftlich gegenüber großen Softwareanbietern aufzutreten, könnte die Vermittlerrolle des OBVSG, der ja für seine Mitglieder bereits ein Repositoriumssystem anbietet, genutzt werden. 7. Abschließende Überlegungen und Ausblick Eine wesentliche Erkenntnis dieses Projekts ist für uns, dass digitale Barrierefreiheit möglichst gesamtheitlich gedacht werden sollte. Eine separate Betrachtung von Nutzer:innen mit bzw. ohne Beeinträchtigungen erscheint ebenso wie eine Trennung der Nutzer:innen- Gruppe nach Art der Beeinträchtigung nur bedingt zielführend – vielmehr sollte Barrierefreiheit, analog und digital, möglichst umfassend interpretiert, berücksichtigt und umgesetzt werden, damit die Rahmenbedingungen möglichst vielen Menschen gerecht werden. Unter dem Stichwort „Universal Design“ wird diese Überlegung bereits umgesetzt. Unsere Interviews lieferten wichtige und interessante Einblicke, auch wenn sich daraus keine allgemein gültigen Aussagen ableiten lassen. Weiterführend wäre nach unserer Ansicht eine umfangreiche Datenerhebung an den österreichischen Bibliotheken sinnvoll, um die Nutzer:innenerfahrungen hinsichtlich digitaler Barrierefreiheit breiter abbilden und als Grundlage für künftige IT-Projekte heranziehen zu können. Abschließend wollen wir auf die jährliche Konferenz des Österreichischen Behindertenrates hinweisen, in deren Zentrum im September 2021 „Inklusive Digitalisierung“ stand. Ein Thema war dabei die partizipative Gestaltung von technischen Innovationen. Dazu wurde nicht zuletzt festgehalten, dass Barrierefreiheit bereits im Planungsprozess ein Thema sein müsste; dazu wäre es auch notwendig, Menschen mit Behinderungen selbstverständlich in die Entwicklung von Produkten und Dienstleistungen einzubinden. Es gelte, sich auf Benutzer:innen zu konzentrieren und diese zu involvieren. Christine Bazalka MA Universität Wien, Bibliotheks- und Archivwesen christine.bazalka@univie.ac.at Karin Kostrhon karin.kostrhon@posteo.at Angelika Mucha BA TU Wien, Bibliothek angelika.mucha@tuwien.ac.at Leo Urlesberger MA leo@urlesberger.at Literatur Magdalena Andrae, Susanne Blumesberger, Sonja Edler, Julia Ernst, Sarah Fiedler, Doris Haslinger, Gerhard Neustätter, Denise Trieb, Barrierefreiheit für Repositorien. Ein Überblick über technische und rechtliche Voraussetzungen. Mitteilungen der Vereinigung Österreichischer Bibliothekarinnen und Bibliothekare 73 (2020), Nr. 2, 259–277, https://doi.org.10.31263/ voebm.v73i2.3640. Silvia Antoneanu, Mihaela Hubert, Digitale Datenarchive/Repositorien und Barrierefreiheit. Erstellung von Schulungsmaterialien und Schulungskonzepten für barrierefreie Inhalte. Projektbericht im Rahmen des Grundlehrgangs “Library and Information Studies” Jahrgang 2019/20/21 an der Universität Wien (Wien 2019), https://phaidra.univie. ac.at/open/o:1165547. Christine Bazalka, Karin Kostrhon, Angelika Mucha, Leo Urlesberger, Digitale Barrierefreiheit von Repositorien. Anregungen zur Gestaltung barrierefreier Repositorien mit Schwerpunkt auf motorischen Beeinträchtigungen. Projektbericht im Rahmen des Grundlehrgangs “Library and Information Studies” Jahrgang 2021/22 an der Universität Wien (Wien 2022), https://phaidra.univie.ac.at/o:1602693. B. I. K. BITV-Test, BIK BITV-Test \| Verzeichnis der Prüfschritte, https://www.bitvtest.de/bitv_test/das_testverfahren_im_detail/pruefschritte.html (29.08.2022). Susanne Blumesberger, Sonja Edler, Eva Gergely, Doris Haslinger, Denise Trieb, Guidelines zur Erstellung barrierearmer Inhalte für Repositorien (Wien 2022), https://phaidra.univie.ac.at/open/o:1430148. Bundesamt für Statistik, Die zehn grössten Bibliotheken der Schweiz gemäss Angebot – 2013–2019 \| Tabelle. Bundesamt für Statistik (2020), https://www.bfs.admin.ch/asset/de/13547580. Deutsche Bibliotheksstatistik – Variable Auswertung, https://www.bibliotheksstatistik.de/ (11.08.2022). Sonja Fiala, Kennzeichnung barrierefreier Dateien – eine Zusammenstellung am Beispiel MARC21 und MODS (Innsbruck 2019), https://doi.org/10.25651/1.2019.0014. Sonja Fiala, Christina Huggle, Metadatenmapping: Die Gegenüberstellung verschiedener Metadatenschemata am Beispiel UWMETADATA>MODS 3.6 (Innsbruck 2019), https://doi.org/10.25651/1.2019.0013. Jan Eric Hellbusch, Kerstin Probiesch, Barrierefreiheit verstehen und umsetzen: Webstandards für ein zugängliches und nutzbares Internet. 1. Aufl. (Heidelberg 2011). Andreas Jeitler, Mark Wassermann, Empfehlungen für barrierefreie Repositorien e-Infrastructures Austria, Cluster L: Projektübergreifende Fragestellungen (Klagenfurt 2016), https://phaidra.univie. ac.at/o:459805. Julia Jungwirth, Iris Mayrhofer, Sylvia Nechvatal, Julia Zimmermann, Repositorien und Barrierefreiheit. Projektbericht im Rahmen des Grundlehrgangs Library and Information StudiesJahrgang 2018/19 an der Universität Wien (Wien 2019), https://phaidra. univie.ac.at/o:1030333. Alexandra Marcaccio, Sara Clarke, Alison Wetheral, Learning About Real Experiences From Real Users: A Blueprint for Participatory Accessibility Testing. Partnership: The Canadian Journal of Library and Information Practice and Research 17 (2022), Nr. 1, 1–21, https://doi.org/10.21083/partnership.v17i1.6658. Sabina Misoch, Qualitative Interviews. 2. Aufl. (Berlin/München/Boston 2014), https://doi.org/10.1515/9783110354614. Nationaler Aktionsplan Behinderung 2022–2030 – Österreichischer Behindertenrat, https:// www.behindertenrat.at/2022/05/stellungnahme-zum-entwurf-des-nap-2022-2030/ (28.08.2022). Österreichweite Demonstration für die Menschenrechte von Menschen mit Behinderungen – Österreichischer Behindertenrat, https://www.behindertenrat.at/2022/07/ oesterreichweite-demonstration/ (26.08.2022). Jonas Poskowsky, Sonja Heißenberg, Sarah Zaussinger, Julia Brenner, beeinträchtigt studieren – best2 Datenerhebung zur Situation Studierender mit Behinderung und chronischer Krankheit (Berlin 2018), https://www.fachportal-paedagogik.de/literatur/vollanzeige.html?FId=1149041 (26.08.2022). Richtlinie (EU) 2016/2102 des Europäischen Parlaments und des Rates vom 26. Oktober 2016 über den barrierefreien Zugang zu den Websites und mobilen Anwendungen öffentlicher Stellen (Text von Bedeutung für den EWR ), OJ L 327 (2016), https://eur-lex.europa.eu/eli/dir/2016/2102/ oj/deu. Web Accessibility Initiative (WAI), Evaluating Web Accessibility Overview, https:// www.w3.org/WAI/test-evaluate/ (09.08.2022). Web Accessibility Initiative (WAI), WCAG 2.1 at a Glance, https://www.w3.org/WAI/standards-guidelines/wcag/glance/ (29.08.2022). W3C, Web Content Accessibility Guidelines (WCAG) 2.1, https://www.w3.org/TR/WCAG21/ (29.08.2022). W3C, Web Accessibility Initiative (WAI), Accessibility Principles. Web Accessibility Initiative (WAI), https://www.w3.org/WAI/fundamentals/accessibility-principles/ (11.08.2022). Sarah Zaussinger, Andrea Kulhanek, Berta Terzieva, Martin Unger, Zur Situation behinderter, chronisch kranker und gesundheitlich beeinträchtigter Studierender. Quantitativer Teil der Zusatzstudie zur Studierenden-Sozialerhebung 2019 (Wien 2019), https:// irihs.ihs.ac.at/id/eprint/5603. Interviewtranskripte Expertin 1, 220710_Interview 3. Interviewt von Karin Kostrhon, Angelika Mucha. Transkription, Wien am 10.07.2022. Expertin 2, 220718_Interview 5. Interviewt von Karin Kostrhon, Angelika Mucha. Transkription, Wien am 11.07.2022. Experte 3, Nutzer 3, 220726_Interview 7. Interviewt von Angelika Mucha, Leo Urlesberger. Transkription, Wien am 26.07.2022. Experte 4, 220729_Interview 9. Interviewt von Angelika Mucha. Transkription, Wien am 29.07.2022. Sonja Fiala, 220701_Interview 2. Interviewt von Angelika Mucha, Leo Urlesberger. Transkription, Wien am 01.07.2022. Raman Ganguly, 220711_Interview 4. Interviewt von Angelika Mucha, Leo Urlesberger. Transkription, Wien am 11.07.2022. Sandra Hermann, 220630_Interview 1. Interviewt von Angelika Mucha, Leo Urlesberger. Transkription, Wien am 30.06.2022. Christian Kienesberger, 220728_Interview 8. Interviewt von Christine Bazalka, Karin Kostrhon, Angelika Mucha, Leo Urlesberger. Transkription, Wien am 28.07.2022. Steffi Proßegger, 220721_Interview 6. Interviewt von Karin Kostrhon, Angelika Mucha. Transkription, Wien am 21.07.2022. Diese Transkripte sind unter https://phaidra.univie.ac.at/o:1601870 zu finden. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Vgl. Zur Situation behinderter, chronisch kranker und gesundheitlich beeinträchtigter Studierender. Quantitativer Teil der Zusatzstudie zur Studierenden-Sozialerhebung 2019 (2019). Siehe https://barrierefreielehre.univie.ac.at/ (aufgerufen: 02.09.2022). Richtlinie (EU) 2016/2102 des Europäischen Parlaments und des Rates vom 26. Oktober 2016 über den barrierefreien Zugang zu den Websites und mobilen Anwendungen öffentlicher Stellen (Text von Bedeutung für den EWR ), 327 OJ L § (2016), online unter http://data.europa.eu/eli/dir/2016/2102/oj/deu. BGBl. I Nr. 59/2019. Vgl. European Telecommunications Standards Institute, EN 301 549 V3.2.1 Accessibility requirements for ICT products and services (Brüssel 2018), online unter https://www.etsi.org/deliver/etsi_en/301500_301599/301549/03.02.01_60/ en_301549v030201p.pdf, (29.08.2022). Vgl. W3C Web Accessibility Initiative (WAI), WCAG 2.1 at a Glance, Web Accessibility Initiative (WAI), online unter https://www.w3.org/WAI/standards-guidelines/wcag/ glance/, (29.08.2022). Vgl. Jan Eric Hellbusch, Kerstin Probiesch, Barrierefreiheit verstehen und umsetzen: Webstandards für ein zugängliches und nutzbares Internet (Heidelberg 2011). Weitere Informationen und anschauliche Beispiele finden sich z.B. hier: https:// www.barrierefrei-kommunizieren.de/datenbank/ und https://www.lifetool.at/beraten/was-bieten-wir-an/arbeitsplatzanpassung/. Umfassende Einblicke in unterschiedliche Nutzungsvarianten ermöglicht auch die Website der Web Accessibility Initiative (WAI), https://www.w3.org/WAI/people-use-web/. B. I. K. BITV-Test, BIK BITV-Test \| Verzeichnis der Prüfschritte, online unter https:// www.bitvtest.de/bitv_test/das_testverfahren_im_detail/pruefschritte.html, (29.08.2022). Mit Sprunglinks (Skip Links) können Nutzer:innen assistierender Technologien zum Hauptinhalt einer Seite springen. Deutsche Bibliotheksstatistik – Variable Auswertung, online unter https://www. bibliotheksstatistik.de/, (11.08.2022); Bundesamt für Statistik, Die zehn grössten Bibliotheken der Schweiz gemäss Angebot – 2013–2019 \| Tabelle, Bundesamt für Statistik, 21.07.2020, online unter https://www.bfs.admin.ch/asset/de/13547580. Jan Eric Hellbusch, Kerstin Probiesch, Barrierefreiheit verstehen und umsetzen. Webstandards für ein zugängliches und nutzbares Internet (Heidelberg 2011). ULB Tirol Publikationsserver, UB Salzburg ePLUS, UB Linz JKU ePUB, UB Klagenfurt Netlibrary, UB Mozarteum Repository. Die Repositorien und damit auch die Formulare wurden alle vom gleichen Anbieter bereitgestellt. BOK:ePub, MedUni Wien ePub Kann aber nicht uneingeschränkt empfohlen werden, da er nur mit Windows kompatibel ist. Vgl. Christine Bazalka, Karin Kostrhon, Angelika Mucha, Leo Urlesberger, Digitale Barrierefreiheit von Repositorien. Anregungen zur Gestaltung barrierefreier Repositorien mit Schwerpunkt auf motorischen Beeinträchtigungen. Projektbericht im Rahmen des Grundlehrgangs “Library and Information Studies” Jahrgang 2021/22 an der Universität Wien (Wien 2022), online unter https://phaidra.univie.ac.at/o:1602693, S. 22. Ebda., S. 22. Siehe Jeitler, Wassermann, Empfehlungen für barrierefreie Repositorien, 10. Hier wird angeregt, bei Ausschreibung und Auftragsvergabe die existierenden Richtlinien zur Barrierefreiheit einzufordern.
https://openalex.org/W2743062246	https://europepmc.org/articles/pmc5580596?pdf=render	English	null	Spatio-Temporal Distribution of Vector-Host Contact (VHC) Ratios and Ecological Niche Modeling of the West Nile Virus Mosquito Vector, Culex quinquefasciatus, in the City of New Orleans, LA, USA	International journal of environmental research and public health/International journal of environmental research and public health	2,017	cc-by	11,331	Spatio-Temporal Distribution of Vector-Host Contact (VHC) Ratios and Ecological Niche Modeling of the West Nile Virus Mosquito Vector, Culex quinquefasciatus, in the City of New Orleans, LA, USA Mohamed F. Sallam 1,2,* ID , Sarah R. Michaels 3, Claudia Riegel 3, Roberto M. Pereira 1, Wayne Zipperer 4, B. Graeme Lockaby 5 and Philip G. Koehler 1 1 Department/School Entomology and Nematology Department, University of Florida, Gainesville, FL 32611, USA; rpereira@uﬂ.edu (R.M.P.); pgk@uﬂ.edu (P.G.K.) 1 Department/School Entomology and Nematology Department, University of Florida, Gainesville, FL 32611, USA; rpereira@uﬂ.edu (R.M.P.); pgk@uﬂ.edu (P.G.K.) p pg 2 College of Science, Ain Shams University, Cairo 11566, Egypt g y gyp 3 City of New Orleans Mosquito & Termite Control Board, 2100 Leon C. Simon, New Orleans, LA 70122, U srmichaels@nola.gov (S.R.M.); criegel@nola.gov (C.R.) g g g 4 Research Forester, USDA Forest Service, P.O. Box 110806, Gainesville, FL 32611-0806, USA; wzipperer@fs.fed.us pp 5 Center for Environmental Studies at the Urban-Rural Interface (CESURI), School of Forestry & Wildlife Sciences Auburn University Auburn AL 36849 USA; lockabg@auburn edu 5 Center for Environmental Studies at the Urban-Rural Interface (CESURI), School of Forestry & Wildlife Sciences, Auburn University, Auburn, AL 36849, USA; lockabg@auburn.edu , y, , , ; g Correspondence: mwsallam2009@aol.com or sallam.mohamed@epa.gov * Correspondence: mwsallam2009@aol.com or sallam.mohamed@epa.gov Received: 22 June 2017; Accepted: 5 August 2017; Published: 8 August 2017 Abstract: The consistent sporadic transmission of West Nile Virus (WNV) in the city of New Orleans justiﬁes the need for distribution risk maps highlighting human risk of mosquito bites. We modeled the inﬂuence of biophysical and socioeconomic metrics on the spatio-temporal distributions of presence/vector-host contact (VHC) ratios of WNV vector, Culex quinquefasciatus, within their ﬂight range. Biophysical and socioeconomic data were extracted within 5-km buffer radii around sampling localities of gravid female Culex quinquefasciatus. The spatio-temporal correlations between VHC data and 33 variables, including climate, land use-land cover (LULC), socioeconomic, and land surface terrain were analyzed using stepwise linear regression models (RM). Using MaxEnt, we developed a distribution model using the correlated predicting variables. Only 12 factors showed signiﬁcant correlations with spatial distribution of VHC ratios (R2 = 81.62, p < 0.01). Non-forested wetland (NFWL), tree density (TD) and residential-urban (RU) settings demonstrated the strongest relationship. The VHC ratios showed monthly environmental resilience in terms of number and type of inﬂuential factors. The highest prediction power of RU and other urban and built up land (OUBL), was demonstrated during May–August. This association was positively correlated with the onset of the mosquito WNV infection rate during June. Spatio-Temporal Distribution of Vector-Host Contact (VHC) Ratios and Ecological Niche Modeling of the West Nile Virus Mosquito Vector, Culex quinquefasciatus, in the City of New Orleans, LA, USA These ﬁndings were conﬁrmed by the Jackknife analysis in MaxEnt and independently collected ﬁeld validation points. The spatial and temporal correlations of VHC ratios and their response to the predicting variables are discussed. Keywords: West Nile virus; Culex quinquefasciatus; habitat suitability; New Orleans; distribution risk International Journal of Environmental Research and Public Health 1. Introduction West Nile virus (WNV) was ﬁrst reported in 1999 in New York City, NY, USA. By 2000 the disease has spread throughout the northeastern USA [1–3]. The virus reached Louisiana in the fall of 2001, when a dead crow in Jefferson Parish was identiﬁed as being infected with WNV [4]. By 2003, WNV www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2017, 14, 892; doi:10.3390/ijerph14080892 www.mdpi.com/journal/ijerph 2 of 20 Int. J. Environ. Res. Public Health 2017, 14, 892 infections occurred in 60 of the 64 Louisiana’s parishes. In the New Orleans metropolitan areas (Orleans and Jefferson Parishes) focal transmission activity occurs principally during mid-July [4]. Culex quinquefasciatus Say, as the main vector, with Cx. salinarius Coquillett possibly acting as a secondary vector, were incriminated in the WNV outbreak in southern Louisiana during 2002 [5–7]. The former mosquito species, with a feeding preference for mammals, was responsible for enzootic/epidemic transmission, especially in urban and sub-urban settings [7–14]. The primary mosquito vector showed biological and ecological resilience in space and time based on the available environmental resources. This resilience may inﬂuence the spatio-temporal distribution of the WNV vector, which may or may not bring them to the vicinity of both reservoir host(s) and human populations. Eventually this will affect the ampliﬁcation and transmission cycles of WNV in areas under risk. In New Orleans, the conﬂuence of availability of competent mosquito vector(s), susceptible reservoir host(s), suitable natural systems and climate for both mosquitoes and host(s) enabled the autochthonous transmission of WNV with hundreds of human cases and major mortality of wild native and exotic birds [2,4]. Nonetheless, the transmission dynamics of WNV in terms of space and time in relationship to the biology, ecology of mosquito vector(s), and their biophysical systems remains unclear. In fact, the distribution, blood-feeding preference, ﬂight range and vectorial capacity of mosquito vectors are very critical inputs for predicting the transmission cycle of this disease. Furthermore, mosquito vectors often shift their feeding preference seasonally or spatially, depending on the availability of the blood meal source. For example, Cx. quinquefasciatus showed an opportunistic preference for blood meal. In peninsular Florida, it is responsible for an epizootic cycle and sustaining the virus circulation within reservoir host bird(s) [15,16]. However, it has been incriminated with the enzootic/epidemic transmission cycle of WNV in urban and sub-urban areas in Louisiana due to feeding preference to humans and other mammals [4,6,7,17–19]. 1. Introduction Currently, most species distribution models for mosquitoes are based on hydrological and meteorological data [15,16,20–22]. Some models include socio-environmental predictors in terms of vegetation or urban and sub-urban areas [23]. With respect to WNV, models have used either used data points of WNV cases and mosquito vectors instead of the ﬂight range of the mosquito vectors around their hosts or predicted the distribution risk of WNV on regional scale. Prediction models for WNV and Zika virus (ZIKV) transmission potential were generated for their mosquito vectors in regard to their ﬂight range around recorded positive cases highlighting their response to surrounding biophysical systems such as climate and non-climate factors [24,25]. Although previous models are useful, their ﬁndings did not adequately account for the comprehensive response of vector-host contact (VHC) ratios to climate and non-climate variables such as land use-land cover (LULC) and Digital Elevation Models (DEM) and the overall inﬂuence on arbovirus transmission potential [26,27]. Mosquito density reﬂect neither the likelihood of biting risk, which is caused by mosquito vector, nor the transmission potential as a function of biting rate. The VHC explains the ratio between collected mosquito density and human population census, which reﬂects areas under risk of increased biting rate by mosquito vector. The lack of available vaccines for WNV and consistent development of insecticide resistance for mosquito vector populations jeopardize public health in affected areas. Additionally, the focal and sporadic locally-transmitted cases justify the necessity to generate prediction models that identifies areas under risk of infective biting rates in order to target during surveillance and control activities. In our model, we evaluated the spatio-temporal distribution of VHC ratios in response to: (i) future climate scenarios during 2011–2030, (ii) LULC, (iii) socioeconomic, and (iv) DEM systems. Our correlative models were generated within the flight range of WNV vector in the city of New Orleans, LA (NOLA). The spatio-temporal VHC ratios were estimated utilizing data records on female gravid mosquito and human population census per block during 2015. The spatio-temporal resilience of VHC ratios to their predicted biophysical systems was characterized. This allowed developing prediction risk maps for the WNV vector presence using the Maximum Entropy (MaxEnt) tool, emphasizing the human population 3 of 20 Int. J. Environ. Res. Public Health 2017, 14, 892 under risk of infective mosquito bites. Since the local economy in NOLA is primarily driven by tourism, management of arbovirus diseases has a significant economic implications. 1. Introduction Arbovirus transmission has the potential to jeopardize the tourism industry, making NOLA surveillance and control programs very important to the economic and ecological health of the city. 2.2.1. Mosquito Sampling and Socioeconomic Data Density of wild-collected female gravid Cx. quinquefasciatus was estimated using Center for Disease Control (CDC) gravid traps (John W. Hock Company, Gainesville, FL, USA) at 37 permanent locations in the City of New Orleans. Traps were placed outdoors at the beginning of April through the last week of December during 2015. Traps were setup on the ground and operated for 18–20 h using a 12-v battery. Mosquito collections were transported to the laboratory facility at The NOLA Mosquito Control Board for further identiﬁcation to the species level using the taxonomic keys of Darsie and Ward [28]. Because the density of collected mosquitoes reﬂects neither the risk of vector contact with host nor disease transmission rates, the vector-host contact (VHC) ratios were calculated within 5-km buffer radii around the sampling localities [25,27,29]. These buffer radii reﬂect the foraging activity of both ovipositing and newly emerged host seeking mosquitoes from their breeding sites. Moreover, these buffer radii demonstrate human populations under risk of bites of host seeking mosquito and disease transmission. Although the sampled mosquitoes in the current study were gravid and not host seeking, their density gives insight regarding the wild mosquito populations within 5-km radii around sampling sites. The VHC ratios were estimated utilizing spatial and temporal density of WNV vector and human population census within ﬂight buffer radii (~5-km) of this mosquito vector. For the spatial analysis, density of the mosquito vector (total number of mosquito vector/season/5-km) was estimated for each sampling site, whereas in the temporal analysis, density was estimated on a monthly basis. Data on human population census/housing block was imported from the NOLA census records of 2015 (data.nola.gov), and clipped within the buffer radii around vector sampling sites. Extraction of both mosquito density and human population census were conducted using the Arc toolbox in ArcGIS ver. 10.1 (Esri, Redlands, CA, USA). Accordingly, the spatio-temporal ﬂuctuation of VHC ratios were estimated in response to their predicted biophysical systems within the ﬂight buffer radii. 2.1. Study Area New Orleans, Louisiana (NOLA) lies on the Mississippi River, near the Gulf of Mexico with a total area of 1084 km2 inhabited by almost 1,262,888 people, with an average density of 1165/km2. NOLA is sub-tropical with an annual high temperature of 25 ◦C, an annual low of 16.8 ◦C, and average annual precipitation of 162.3 cm. Average highest precipitation occurs in July (17.9 cm). 2.2. Data Layers 2.2. Data Layers 2.2.1. Mosquito Sampling and Socioeconomic Data 2.2.2. Bioclimatic Data In order to demonstrate the spatio-temporal ﬂuctuation of VHC ratios in response to future climate scenarios in NOLA, we utilized climate data from 2011–2030 (Table 1). Utilization of future climate scenarios not only highlights the sampling time frame of WNV vector during 2015, but also projects the future distribution of VHC ratios. Bioclimatic data layers were obtained from the General Circulation Models (GCMs) for the optimistic IPCC Special Range of Emission Scenarios (SRES A1B). The A1B represents a medium and balanced scenario for the emission rate produced by Green House Gases (GHG). This scenario was imported from the data base at Centro International de Agricultura Tropical (CIAT) (http://www.ccafs-climate.org/data/) [30]. We also used the data based on the global 4 of 20 Int. J. Environ. Res. Public Health 2017, 14, 892 circulation model CSIRO-Mk3.5.0. For projections, a spatial resolution of 30 arc sec (~1 km) was applied. The layers were clipped to match dimensions of NOLA and saved as ASCII grids using Model Builder in ArcGIS ver. 10.1 (Esri, Redlands, CA, USA). Table 1. Proposed thirty-three variables in prediction model of WNV mosquito vectors in the city of New Orleans, LA. Table 1. Proposed thirty-three variables in prediction model of WNV mosquito vectors in the city of New Orleans, LA. 2.2.2. Bioclimatic Data Variable Variable Name Data Source Units Alt Elevation in meters WorldClim 1 Meter Aspect Aspect ratio Generated 2 Degrees Bio01 Annual Mean Temperature WorldClim 1 Degree Celsius Bio02 Mean Diurnal Range (Mean of monthly (max temp −min temp)) WorldClim 1 Degree Celsius Bio03 Isothermality (BIO2/BIO7) (* 100) WorldClim 1 Dimensionless Bio04 Temperature Seasonality (standard deviation * 100) WorldClim 1 Degree Celsius Bio05 Max Temperature of Warmest Month WorldClim 1 Degree Celsius Bio06 Min Temperature of Coldest Month WorldClim 1 Degree Celsius Bio07 Temperature Annual Range (BIO5-BIO6) WorldClim 1 Degree Celsius Bio08 Mean Temperature of Wettest Quarter WorldClim 1 Degree Celsius Bio09 Mean Temperature of Driest Quarter WorldClim 1 Degree Celsius Bio10 Mean Temperature of Warmest Quarter WorldClim 1 Millimeter Bio11 Mean Temperature of Coldest Quarter WorldClim 1 Millimeter Bio12 Annual Precipitation WorldClim 1 Millimeter Bio13 Precipitation of Wettest Month WorldClim 1 Millimeter Bio14 Precipitation of Driest Month WorldClim 1 Millimeter Bio15 Precipitation Seasonality (Coefﬁcient of Variation) WorldClim 1 Fraction Bio16 Precipitation of Wettest Quarter WorldClim 1 Millimeter Bio17 Precipitation of Driest Quarter WorldClim 1 Millimeter Bio18 Precipitation of Warmest Quarter WorldClim 1 Millimeter Bio19 Precipitation of Coldest Quarter WorldClim 1 Millimeter Curvature Curvature Generated 2 Degrees DFL Deciduous forest land USGS 3 Integer values FW Forested wetland USGS 3 Integer values Hill shade Hill shade Generated 2 Degrees ICS Industrial and commercial services USGS 3 Integer values NFWL Non-forested wetland USGS 3 Integer values OUBL Other urban and build-up land USGS 3 Integer values Population census Population census per block NOLA 5 No. household/block RU Residential and urban settings USGS 3 Integer values SCLRE Streams, canals, lakes, reservoirs and estuaries USGS 3 Integer values Slope Slope Generated 2 Degrees TD Tree density Lewis et al. 4 No. trees/area 1 WorldClim Global Climate database v1.4, available at: http://www.ccafs-climate.org/data/ (accessed on 7 March 2016); 2 Digital elevation model using the surface spatial analyst tool in Arc tool box of ArcGIS ver. 10.1; 3 USGS available at: http://water.usgs.gov/GIS/dsdl/ds240/ (accessed on 3 March 2016); 4 Lewis et al. (In Review) [31]; 5 data.nola.gov (accessed on 7 March 2016) All layers of variables data used in producing species distribution model gridded to ~1 km spatial resolution and projected into World Geodetic System (WGS) 1984. The DEMs representing slope, aspect ratio, curvature, and hill shade were highlighted in previous studies to predict land geomorphology, temporary water accumulation and probable breeding sites for mosquitoes [25,32–35]. 2.2.3. Land Use-Land Cover Data (LULC) 2 2 3 Land Use-Land Cover Data (LULC) Since mosquitoes depend on the human population as a source of blood meal [36], an urban areas layer was included as a predictor (Table 1). Urban areas were categorized into three classes to represent degree of urbanization: (1) residential and urban, in which housing predominates in two different forms; (2) industrial and commercial services, which represents industrial settings and fewer housing structures; and (3) other urban and built-up land with the least housing and human populations. ( ) Since mosquitoes depend on the human population as a source of blood meal [36], an urban areas layer was included as a predictor (Table 1). Urban areas were categorized into three classes to represent degree of urbanization: (1) residential and urban, in which housing predominates in two different forms; (2) industrial and commercial services, which represents industrial settings and fewer housing structures; and (3) other urban and built up land with the least housing and human populations Vegetation at the site level (data from USDA Forest Service and Tulane University), representing resting places and sugar meal sources for adult mosquitoes, was included in our model [37–39]. Additionally, vegetation reﬂected habitat quality for nesting birds [40,41], which are reservoir hosts of WNV. Therefore, four classes of vegetation types were extracted from the USGS and literature [31] to build up our model: (1) non-forested wetland, (2) forest wetland, (3) deciduous forestland, and (4) tree density. and (3) other urban and built-up land with the least housing and human populations. Vegetation at the site level (data from USDA Forest Service and Tulane University), representing resting places and sugar meal sources for adult mosquitoes, was included in our model [37–39]. Additionally, vegetation reflected habitat quality for nesting birds [40,41], which are reservoir hosts of WNV. Therefore, four classes of vegetation types were extracted from the USGS and literature [31] to build up our model: (1) non-forested wetland, (2) forest wetland, (3) deciduous forestland, and (4) tree density. Streams canals lakes reservoirs and estuaries of different sizes (> and ≤1 km) were also included Streams, canals, lakes, reservoirs and estuaries of different sizes (> and ≤1 km) were also included to represent permanent water bodies, as possible breeding sites for Cx. quinquefasciatus. All LULC data layers were imported from a US Geological Survey (USGS) data set. These data were built during the 1970s and 1980s and updated and released during 2007 [42]. 2.2.2. Bioclimatic Data Accordingly, these land surface indicators were generated from a 30 arc-seconds DEM to be included in our investigation. Land geomorphology and temporary water accumulations within 1 km were not highlighted in our study, because of two reasons: (i) resolution of all data layers were resampled to match worldclim data (~1 km), (ii) Cx quinquefasciatus has been reported to ﬂy for longer distances within ~5 km seeking suitable host(s) and breeding sites. Int. J. Environ. Res. Public Health 2017, 14, 892 5 of 20 2.3. Variables Selection A total of 33 bioclimatic, LULC, socioeconomic and DEM data layers (Table 1) were clipped to NOLA and extracted within each 5-km buffer radii around mosquito sampling sites in preparation for collinearity analysis to: (i) reduce redundancy between inﬂuential factors, and (ii) select the signiﬁcant explanatory variables to be included in MaxEnt (Figure 2) [25,27]. In this regard, a stepwise linear regression model (RM) was carried out for three purposes: (i) to test the spatial dependency of vector-host contact ratios on their predicting variables within the ﬂight range radii of this mosquito vector around their sampling sites, (ii) to characterize monthly resilience of Cx. quinquefasciatus to their predicting variables, (iii) to overcome redundancy and exclude the linearly correlated variables. This analysis was carried out using JMP pro statistical package ver. 10.0.0 [43]. The minimum corrected Akaik Information Criterion (AICc) and R2 values were used to select the signiﬁcant predicting variables (p < 0.05) [25,44–47]. 2.2.3. Land Use-Land Cover Data (LULC) 2 2 3 Land Use-Land Cover Data (LULC) For comparison and conﬁrmation of the USGS data, areas of LULC from zoning district data layers were imported from the City of New Orleans Enterprise GIS Database during 2016. Streams, canals, lakes, reservoirs and estuaries of different sizes (> and ≤1 km) were also included to represent permanent water bodies, as possible breeding sites for Cx. quinquefasciatus. All LULC data layers were imported from a US Geological Survey (USGS) data set. These data were built during the 1970s and 1980s and updated and released during 2007 [42]. For comparison and confirmation of the USGS data, areas of LULC from zoning district data layers were imported from the City of New Orleans Enterprise GIS Database during 2016. Although sampling sites were randomly selected to represent all LULC classes within the buffer Although sampling sites were randomly selected to represent all LULC classes within the buffer radii, urban areas were extensively highlighted in our study to predict human population under risk of increased biting rate (Figure 1, Table 2). Additionally, the area percentages of each LULC class to the total sampled areas within 5-km buffer radii were estimated (Table 2). Although sampling sites were randomly selected to represent all LULC classes within the buffer radii, urban areas were extensively highlighted in our study to predict human population under risk of increased biting rate (Figure 1, Table 2). Additionally, the area percentages of each LULC class to the total sampled areas within 5-km buffer radii were estimated (Table 2). Figure 1. The seven LULC classes and sampling sites in NOLA. Figure 1. The seven LULC classes and sampling sites in NOLA. Figure 1. The seven LULC classes and sampling sites in NOLA. Figure 1. The seven LULC classes and sampling sites in NOLA. 6 of 20 Int. J. Environ. Res. Public Health 2017, 14, 892 Table 2. Number of traps and area percentage of LULC classes within 5-km buffer radii in NOLA. LULC Class Area % LULC Class No. Traps/Class Deciduous forest 7.18 2 Forested wetland 8.01 2 Industrial and commercial services 9.56 4 Non forested wetland 7.72 1 Other urban and built-up land 4.61 3 Residential-Urban 28.29 25 Streams, canals, lakes, reservoirs and estuaries 24.93 0 Table 2. Number of traps and area percentage of LULC classes within 5-km buffer radii in NOLA. 2.4. Habitat Suitability Modeling of WNV Vector The maximum likelihood of habitat suitability for Cx. quinquefasciatus was modeled using MaxEnt software v. 3.3 (http://www.cs.princeton.edu/~schapire/maxent/, New York, NY, USA), [48–50]. This analysis uses the occurrence records of mosquito vectors in association with the selected predicting variables from RM to generate suitability risk maps. Accordingly, the Jackknife test was used to evaluate the permutation importance of independent variables in our model. The generated risk probability was categorized into ﬁve classes using the natural area breaks in ArcGIS utilizing WNV minimum infection rate (0.8/1000) recorded by Godsey et al. (2005) [5]: very low (0–0.2), low (>0.2–0.4), medium (>0.4–0.6), high (>0.6–0.8), and very high (>0.8). The 37 collection sites were used as spatio-temporal replicates for the mosquito vector distribution. These records were randomly partitioned for model evaluation into two subsamples: 75% of the records were used for training and building the model, and 25% of the records were used for testing the model’s accuracy. The duplicate records of WNV mosquito vectors within ~1-km of the same cell size were excluded [51]. During data training, a matrix of spatial correlations between sampling points and their associated predicting variables were created. Accordingly, the habitat suitability maps were created for sampled and unsampled areas based on the habitat similarity between sampled and unsampled regions. Five replicate runs were assigned in running the model to generate the average, maximum, minimum and median of the distribution range of mosquito vectors. Prediction models were evaluated using the cross-validation method, by systematically removing each data point from our training data set and predicting the removed point based on the remaining data points. Two thresholds have been used to examine the performance accuracy of our model [49]: (i) the extrinsic omission was evaluated at a ﬁxed threshold (10 percentile training presence), and (ii) the area under the curve (AUC) of the receiver operating characteristics (ROC). 7 of 20 of 19 Int. J. Environ. Res. Public Health 2017, 14, 892 Int J Environ Res Public Health 2017 14 89 Int. J. Environ. Res. Public Health 2017, 14, 892 7 of 19 Figure 2. West Nile Virus transmission model, and expected outcomes in response to proposed predicting variables. Figure 2. West Nile Virus transmission model, and expected outcomes in response to proposed predicting variables. Figure 2. West Nile Virus transmission model, and expected outcomes in response to proposed predicting variables. Figure 2. 3. Results 3.1. Variables Selection 3.1. Variables Selection 2.4. Habitat Suitability Modeling of WNV Vector West Nile Virus transmission model, and expected outcomes in response to proposed predicting variables. 8 of 20 Int. J. Environ. Res. Public Health 2017, 14, 892 To increase the potential of the habitat suitability model and maximize the sampling effort of extracted mosquito vectors, a separate ASCII ﬁle generated from the 5-km buffer zones was included and weighted to the corresponding VHC ratios [25,52]. Accordingly, the habitat suitability of the gravid mosquito vector will be predicted in regard to their ﬂight range around human hosts, breeding habitats and in response to their predicting variables. Therefore, the generated risk map reﬂects the association between Cx. quinquefasciatus, available avian or human blood meal, and water habitats. The MaxEnt evaluates different correlations between the presence records of extracted mosquito vector data and their predicting variables within the sampled areas utilizing logistic regression analysis. In our study, we weighted the presence records to VHC ratios 5-km around sampling localities and attached these as ASCII bias ﬁles. Since the habitat suitability for WNV transmission generated by MaxEnt was produced at the threshold of WNV minimum infection rate, our risk maps highlighted human populations under risk of infective mosquito bites. 2.5. Model Validation To evaluate the generated risk probability maps, a total of 18 independent ﬁeld validation points were sampled biweekly. The validation points were randomly selected to represent areas with high mosquito and human population density. Female mosquitoes (~4–100) sampled from each locality were pooled according to their date of collection in order to be used for WNV testing. The sampling sites were identiﬁed as WNV positive as long as one pool was reported positive from the same locality during the season 3.1.1. Spatial Analysis The vector-host contact ratios showed variation in their response to the 32 variables used in RM. The increase in VHC ratios showed signiﬁcant correlations with 12 predicting variables (AICc = 505.01, R2 = 81.62, p < 0.01) (Table 3). The LULC related variables were found to be the key predictors, especially non-forested wetland (NFWL) (r(13) = 1.91, AICc = 505.01, R2 = 81.62, p < 0.01). This land cover type alongside with tree density (TD) positively correlated with the vector-host contact ratios. However, the increase in residential and urban settings shared a reduced negative inﬂuence on the spatial distribution of WNV mosquito vector (Table 4). Table 3. Predicting variables used in building up the spatial and temporal models. Model Variables Test AUC R2 AICc Spatial Model Bio 1, 2, 4, 6, 7, 11, 13, 17, 18, NFWL, RU, TD 0.71 0.82 505.01 April Bio 11, 12, 14, 15, 8 0.77 0.75 79.05 May Alt, Bio 8, 11, 12, 14, 15, NFWL 0.74 0.85 71.53 June Alt, Bio 11, 14, 2, 8, OUBL 0.73 0.81 80.57 July bio14, 15, 8, 9, OUBL 0.77 0.83 73.28 August bio11, 12, ,15, 8, 9, OUBL, RU 0.8 0.93 60.67 September Bio 8, 11, 12 0.79 0.58 88.26 October Bio 1, 8, 12 0.75 0.55 81.30 November Bio 2, 8 0.78 0.52 84.34 December Bio 8, 12 0.71 0.42 80.95 Table 3. Predicting variables used in building up the spatial and temporal models. Three of the predictors were related to seasonal precipitation and temperature variables namely: precipitation of wettest month (Bio13), and precipitation of driest (Bio17) and warmest quarters (Bio18). The coefﬁcient estimates in RM demonstrated a negative association between WNV mosquito vector and Bio17 and Bio18. Additionally, six temperature related variables correlated with VHC 9 of 20 Int. J. Environ. Res. Public Health 2017, 14, 892 ratios namely: mean annual temperature (Bio1), mean diurnal range (Bio2), temperature seasonality (Bio4), minimum temperature of coldest month (Bio6), temperature annual range (Bio7), and mean temperature of coldest quarter (Bio11). Temperature related variables had varied correlations with VHC ratios. Although the increased contact ratios showed a positive association with warm sampling localities (Bio1, Bio4, and Bio11), Bio2, Bio6 and Bio7 showed negative correlations (Table 4). Table 4. Percent contribution of predicting variables on spatial distribution of WNV mosquito vector during 2015 in City of New Orleans, LA. 3.1.1. Spatial Analysis I J E i R P bli H l h 14 892 1 f 19 Table 4. Percent contribution of predicting variables on spatial distribution of WNV mosquito vector during 2015 in City of New Orleans, LA. Variable Linear Regression Analysis % Contribution (Jackknife’s Test) Coefﬁcient R2 p AICc Bio 1 3.08 0.65 8 635.73 1.1 Bio 11 4.74 0.46 4 722.22 8.8 Bio 13 0.77 0.79 10 532.48 ** 0.1 Bio 17 -0.24 0.08 2 834.61 * 0.1 Bio 18 -0.25 0.73 9 579.02 ** 37.2 Bio 2 -0.83 0.81 12 514.95 * 0.2 Bio 4 0.07 0.11 3 828.82 ** 1.6 Bio 6 -2.99 0.49 5 711.32 * 0.1 Bio 7 -1.74 0.57 6 676.71 * 0.3 NFWL 1.91 0.82 13 505.01 28.9 RU -1.24 0.60 7 662.57 20.9 TD 0.60 0.80 11 523.11 0.7 * Signiﬁcant at p < 0.05; Signiﬁcant at p < 0.01; Best predictor, signiﬁcant (p < 0.01). Int. J. Environ. Res. Public Health 2017, 14, 892 15 of 19 Table 4. Percent contribution of predicting variables on spatial distribution of WNV mosquito vector during 2015 in City of New Orleans, LA. Variable Linear Regression Analysis % Contribution (Jackknife’s Test) Coefficient R2 p AICc Bio 1 3.08 0.65 8 635.73 1.1 Bio 11 4.74 0.46 4 722.22 8.8 Bio 13 0.77 0.79 10 532.48 0.1 Bio 17 -0.24 0.08 2 834.61 * 0.1 Bio 18 -0.25 0.73 9 579.02 ** 37.2 Bio 2 -0.83 0.81 12 514.95 * 0.2 Bio 4 0.07 0.11 3 828.82 ** 1.6 Bio 6 -2.99 0.49 5 711.32 * 0.1 Bio 7 -1.74 0.57 6 676.71 * 0.3 NFWL 1.91 0.82 13 505.01 28.9 RU -1.24 0.60 7 662.57 20.9 TD 0 60 0 80 11 523 11 0 7 3.1.2. Temporal Analysis * Signifi Mean temperature of wettest quarter (Bio8) shared reduced prediction power with Bio11 through the whole season and reached its maximum in December. However, a negative correlation was recorded with the mean temperature of the driest (Bio9) and wettest (Bio8) quarters during July–August and September, respectively. Table 5. Percent contribution of predicting variables on temporal distribution of vector-host contact ratios of WNV in the City of New Orleans, LA. 3.1.2. Temporal Analysis * Signifi The temporal distribution of WNV mosquito vector showed ecological resilience in terms of month-to-month response to their predicting variables. This resilience was demonstrated as the temporal changes of predicting variables and did not affect monthly vector-host contact ratios (20.16 ± 0.02) (Figure 3). 3.1.2. Temporal Analysis The temporal distribution of WNV mosquito vector showed ecological resilience in terms of month- to-month response to their predicting variables. This resilience was demonstrated as the temporal changes of predicting variables and did not affect monthly vector-host contact ratios (20.16 ± 0.02) (Figure 3). Figure 3. Monthly mosquito WNV infection rate in correlation with vector-host contact ratios in NOLA. 0 1 2 3 4 5 6 7 8 9 10 11 12 19.80 19.90 20.00 20.10 20.20 20.30 20.40 20.50 20.60 20.70 Mosquito WNV infection rate Vector-Host Contact ratio Month VHC ratio Mosquito WNVinfection rates Figure 3. Monthly mosquito WNV infection rate in correlation with vector-host contact ratios in NOLA. Figure 3. Monthly mosquito WNV infection rate in correlation with vector-host contact ratios in NOLA. Figure 3. Monthly mosquito WNV infection rate in correlation with vector-host contact ratios in NOLA. Generally, temperature related variables were the key factors in predicting monthly distribution of WNV mosquito vector (Table 5). These variables were manifested as: mean temperature of coldest (Bio11), wettest (Bio8) and driest (Bio9) quarters, mean annual temperature (Bio1), and mean diurnal range (Bio2). The distribution of WNV mosquito density was shown to be positively associated with the increase of all temperature parameters during sampling time. The increase in density of WNV Generally, temperature related variables were the key factors in predicting monthly distribution of WNV mosquito vector (Table 5). These variables were manifested as: mean temperature of coldest (Bio11), wettest (Bio8) and driest (Bio9) quarters, mean annual temperature (Bio1), and mean diurnal Int. J. Environ. Res. Public Health 2017, 14, 892 10 of 20 range (Bio2). The distribution of WNV mosquito density was shown to be positively associated with the increase of all temperature parameters during sampling time. The increase in density of WNV mosquito vector during April–September was found to be positively associated with Bio11. This correlation was demonstrated as maximum prediction probability of vector-host contact ratios during September (r(4) = 0.96, AICc = 88.26, R2 = 58.49, p < 0.01). This ﬁnding was conﬁrmed by the percent contribution of (Bio11) generated by the Jackknife’s test for this month (88.5%) (Table 5). 3.1.2. Temporal Analysis * Signifi Month Linear Regression Model % Contribution (Jackknife’s Test) Variable Coefﬁcient R2 p AICc April Bio 11 1.5671996 0.7468 6 79.0541 73.6 Bio 12 −0.1904792 0.6252 5 83.8586 ** 1.7 Bio 14 0.580292 0.4581 3 85.311 * 0.8 Bio 15 0.9505776 0.5683 4 83.3859 * 1.9 Bio 8 1.549815 0.292 2 88.5009 ** 21.9 May Alt 0.2054192 0.6929 5 74.2539 * 2.2 Bio 11 1.2170901 0.8465 8 71.5334 51.1 Bio 12 −0.1428952 0.7531 7 77.6814 ** 1.7 Bio 14 −0.0756836 0.5338 3 77.4397 * 0.9 Bio 15 0.8079636 0.6232 4 75.555 1.2 Bio 8 1.9098991 0.3773 2 81.4792 15.4 NFWL 1.6682811 0.726 6 75.5791 27.6 June Alt 0.2648213 0.8139 7 80.5656 1.6 Bio11 1.0789617 0.7693 6 81.1173 57.9 Bio14 −1.0029246 0.5241 3 87.6001 1.1 Bio2 1.4828419 0.5889 4 87.3124 0.5 Bio8 2.3433611 0.2854 2 94.4485 38.8 OUBL 1.5141391 0.6708 5 85.5914 0.1 July Bio14 −0.6971647 0.5415 3 86.6717 2.1 Bio15 1.5743201 0.7435 5 79.5723 1.5 Bio 8 2.4390573 0.3209 2 93.1952 31 Bio 9 −0.6412268 0.8334 6 73.2771 65.3 OUBL 1.9151789 0.6321 4 84.6186 0.1 August Bio 11 1.0996856 0.8687 6 65.7127 35.1 Bio12 −0.0854343 0.6302 4 82.893 1.2 Bio15 1.5403677 0.8289 5 68.006 0.3 Bio8 1.8883324 0.1999 2 95.2837 9.7 Bio9 −0.7676054 0.9091 7 61.4863 * 16.1 OUBL 2.9633858 0.4018 3 91.2107 1.3 RU 0.2968421 0.9294 8 60.6681 36.3 September Bio 8 −0.1073224 0.2915 2 95.6155 10.1 Bio 11 0.9622917 0.5849 4 88.2612 88.5 Bio 12 −0.1073224 0.4927 3 90.1222 1.4 October Bio 1 1.2929084 0.5465 4 81.2964 56.6 Bio 8 1.431466 0.1947 2 89.6387 37.7 Bio 12 −0.0983597 0.4436 3 83.2546 5.7 November Bio 2 0.9214511 0.5155 3 84.3401 0.1 Bio 8 1.6743075 0.2768 2 91.4944 99.9 December Bio 8 1.591142 0.1993 2 85.9884 ** 84.9 Bio 12 −0.0493324 0.4161 3 80.9488 15.1 * Signiﬁcant at p < 0.05; ** Signiﬁcant at p < 0.01; Best predictor, signiﬁcant (p < 0.01). Table 5. Percent contribution of predicting variables on temporal distribution of vector-host contact ratios of WNV in the City of New Orleans, LA. 11 of 20 Int. J. Environ. Res. Public Health 2017, 14, 892 Precipitation related variables, LULC and elevation showed less inﬂuence in predicting Cx. quinquefasciatus distributions and VHC ratios. 3.2. Habitat Suitability Modeling of West Nile Virus Vector 3.2. Habitat Suitability Modeling of West Nile Virus Vector 3.1.2. Temporal Analysis * Signifi Precipitation variables showed negative inﬂuence on the distribution of WNV mosquito vector during April–September, especially annual precipitation (Bio12) and precipitation of the driest month (Bio14). However, the seasonal precipitation (Bio15), during April–August, demonstrated a positive correlation with the distribution of Cx. quinquefasciatus. The prediction power of LULC was demonstrated during May–August. Unlike the spatial model, both “Other Urban and Built-up Land” (OUBL) and “Residential-Urban” (RU) settings were found to be positively associated with vector-host contact ratios and the onset of mosquito WNV infection rates during June (Table 5, Figure 3). Similarly, elevation shared a subtle positive prediction power during May and June. Spatial and Temporal Analysis A total of 37 sampling points were included in both spatial and temporal models. Nine and 28 points were used for testing and training the habitat suitability models, respectively. For the spatial model, the average predictive performance was found to be high with an AUC value of 0.85 and 0.71 for training and testing occurrence records respectively, with a standard deviation of 0.07. The speciﬁcity of the model was demonstrated as the fractional predicted area. This area at a 10-percentile training presence was found to be 0.36, which were classiﬁed as signiﬁcantly no better than random (p < 0.05). The average likelihood of predicting very high risk areas was ~107 km2, which is ~9.87% of the total area of the city of New Orleans (Figure 4). The Jackknife test conﬁrmed the ﬁndings we retrieved from RM analysis. The LULC related variables (NFWL, RU, and TD) signiﬁcantly maximized the predictability of vector-host contact (50.5%). The highest training gain was shared with precipitation related variables (37.4%). Temperature related variables shared a reduced training gain (12.1%) in our model (Table 4). Although RM showed a negative correlation between Cx. quinquefasciatus and residential-urban settings, the maximum likelihood of the vector-host contact ratio was predicted at urban areas with less housing structures with an AUC training gain of 0.68 (Figure 5). For the monthly habitat suitability model, the average predictive performance for the nine months was found to be high with an AUC value of 0.82 and 0.76 for training and testing occurrence records, respectively (Table 3). The fractional predicted area at a 10-percentile training presence was found to be no better than random (p < 0.05). The average likelihood of predicting very high suitable habitat was almost ~10.87% of the total area of NOLA. This habitat suitability ranged from ~13.8 and 8.13% during the early and late seasons. The percentage contribution of predicting variables generated by the Jackknife test for each month is summarized in (Table 5). Int. J. Environ. Res. Public Health 2017, 14, 892 Int J Environ Res Public Health 2017 14 892 12 of 20 12 f 19 Figure 4. Distribution risk maps for WNV in NOLA representing average, median, maximum, and minimum habitat suitability and sampling localities. Average Risk Probability Median Risk Probability Max. Risk Probability Min. Risk Probability Figure 4. Distribution risk maps for WNV in NOLA representing average, median, maximum, and minimum habitat suitability and sampling localities. Spatial and Temporal Analysis Average Risk Probability Median Risk Probability Average Risk Probability Min. Risk Probability Max. Risk Probability senting average, median, maximum, and minimum habitat suitability and sampling localities. LA representing average, median, maximum, and minimum habitat suitability and sampling localities. 4. Distribution risk maps for WNV in NOLA representing average, median, maximum, and minimum habita Figure 4. Distribution risk maps for WNV in NOLA representing average, median, maximum, and minimu WNV in NOLA representing average, median, maximum, and minimum habitat suitability and sampling lo maps for WNV in NOLA representing average, median, maximum, and minimum habitat suitability and sa 13 of 20 Int. J. Environ. Res. Public Health 2017, 14, 892 Figure 5. Response curve of spatial distribution of VHC ratio to predicting variables in the Jackknife test. . Model Validation Figure 5. Response curve of spatial distribution of VHC ratio to predicting variables in the Jackknife test. J. Environ. Res. Public Health 2017, 14, 892 13 o Figure 5. Response curve of spatial distribution of VHC ratio to predicting variables in the Jackknife test. Figure 5. Response curve of spatial distribution of VHC ratio to predicting variables in the Jackknife test. Model Validation Figure 5. Response curve of spatial distribution of VHC ratio to predicting variables in the Jackknife test. Figure 5. Response curve of spatial distribution of VHC ratio to predicting variables in the Jackknife test. 4. Discussion In our model, we generated habitat suitability estimates for the spatio-temporal likelihood of VHC ratios in NOLA. Since the relationship between mosquito population density and human hosts is important in determining the infective biting rate and transmission risk of arboviruses, density of Cx. quinquefasciatus was linked to the human population census in order to generate risk maps for areas under risk of increased VHC. Additionally, we predicted the habitat suitability for the likelihood of VHC within ﬂight ranges of Cx. quinquefasciatus (~5-km) around their sampling sites [29,53]. The signiﬁcant explanatory variables were evaluated and selected using minimum AICc values using stepwise RM. The potentiality of the current prediction model was proven by the high AUC and R2 values produced by MaxEnt and RM. These thresholds indicate that occurrence records were likely assigned a higher probability of presence than background sites. Additionally, the generated risk map was validated using 18 independent ﬁeld collected sampling points and tested for mosquito WNV infection rates during the season. g The human population in NOLA is centralized in the western areas of the city. However, the likelihood of VHC ratios demonstrated heterogeneous distributions in this side (Figure 4). Although the monthly predicting variables showed some variations, especially the climate, in terms of their percent contribution, the number of these variables declined gradually toward the end of the season. However, the changes in these predicting variables had a consistent inﬂuence on the distribution of VHC likelihood of the same high risk areas in the west side of the city. This may reﬂect the temporal resilience of this mosquito vector to their predicting climate variables in these habitats. This resilience gives the WNV vector the ability to develop and survive in close vicinity to WNV reservoir bird host(s), which was conﬁrmed by the positive WNV mosquito pools during our study [29,53]. However, multi-year mosquito data are recommended to be included in further investigations. During June and early August 2002, WNV was identiﬁed in pools of Cx. quinquefasciatus mosquitoes in southeastern Louisiana with the possibility of Cx. salinarius acting as a secondary vector [6]. Although other mosquito vectors were incriminated in ampliﬁcation and transmission potentials, the selective feeding preference on both human and avian blood and vectorial capacity experiments emphasized that Cx. quinquefasciatus is the competent vector in transmitting WNV in LA. The Cx. A total of 715 m 3.3. Model Validation 3.3. Model Validation mosquito pools were WNV positive during June–December, confirming our results in the temporal analysis. Thirteen and five collection sites were recorded as WNV positive and negative, respectively, representing different habitat suitability classes (Figure 6). A total of 715 mosquito pools were tested for WNV representing 18 collection sites. Only 30 mosquito pools were WNV positive during June–December, conﬁrming our results in the temporal analysis. Thirteen and ﬁve collection sites were recorded as WNV positive and negative, respectively, representing different habitat suitability classes (Figure 6). A total of 715 mosquito pools were tested for WNV representing 18 collection sites. Only 30 mosquito pools were WNV positive during June–December, confirming our results in the temporal analysis. Thirteen and five collection sites were recorded as WNV positive and negative, respectively, representing different habitat suitability classes (Figure 6). Figure 6. Average habitat suitability of infective mosquito showing field validation points. Discussion Figure 6. Average habitat suitability of infective mosquito showing field validation points. Figure 6. Average habitat suitability of infective mosquito showing ﬁeld validation points. ssion Figure 6. Average habitat suitability of infective mosquito showing field validation points. Figure 6. Average habitat suitability of infective mosquito showing ﬁeld validation points. Int. J. Environ. Res. Public Health 2017, 14, 892 14 of 20 14 of 20 4. Discussion The varied correlations between temperature-related variables and VHC explain the association of WNV mosquito distribution with warmer areas during the coldest months. This was conﬁrmed by the negative correlation between VHC and Bio6, which represents the minimum temp of the coldest months. Similarly, Bio6 predicts timing and distribution of the nesting/roosting bird hosts during early spring. In a ﬁeld study, severe winter caused delay in birds nesting, which explains the variation in timing of host feeding shifts. The VHC showed a negative correlation with precipitation during the driest (Bio17) and warmest (Bio18) quarters [25,55,56]. This negative correlation may be attributed to the ﬂush of limited numbers of mosquitoes from breeding habitats due to rainfall during dry and wet seasons. Subsequently, this reduced the abundance/distribution of WNV mosquito vector during these periods. q g p In the temporal analysis, we attempted to characterize the ecological resilience of WNV mosquito vector in response to seasonal temperature and precipitation related variables, in association with other LULC variables. This resilience was demonstrated by a decrease in the number of variables that predicted VHC ratios during the late season. The lagged inﬂuence of mean temperature of the coldest quarter (Bio11) had a positive association with the increased development/distribution of WNV vector during April–September [25]. This was manifested as maximum prediction probability of VHC ratios during September (R2 = 58.49, p < 0.01). This lagged inﬂuence may cause the increase in the development rate of mosquito vectors in affected areas during the ampliﬁcation phase of the WNV pathogen during April–May. Meanwhile, neotropical bird migrants, mainly passerines, tend to nest during April–May or roost through July, which is crucial for the ampliﬁcation phase [55,59]. Accordingly, this may accelerate the disease transmission during June–September. Moreover, blood meal preference may shift from birds to mammals including humans that are temporally associated with distribution of nesting/roosting birds, thereby enhancing human risk of arboviruses [55,56,58]. This shift in mosquito feeding preference may be inﬂuenced by the temperature-related variables during dry and wet seasons, i.e., negative correlations between VHC and temperature-related variables during dry and wet seasons. The increase in these temperatures reﬂects the reduction in water bodies that may allow breeding and nesting habitats for mosquito vectors and birds, respectively. Similarly, the negative inﬂuence of precipitation variables during April–September, especially annual precipitation (Bio12) and precipitation of the driest month (Bio14), shared a reduced inﬂuence toward the prediction of distributions of Cx. 4. Discussion quinquefasciatus mosquito is well known as exophilic and exophagic and the breeding habitats range from ditches, woodland pools, and freshwater marshes of a semi-permanent or permanent nature [25]. As much as Cx. quinquefasciatus maintains and ampliﬁes WNV within reservoir host bird(s) [15,16], it is responsible for the urban transmission cycle of WNV in southern and southeastern parts of the USA [17–19]. In the spatial analysis, the RM demonstrated a signiﬁcant association between NFWL and VHC ratios (R2 = 82, p < 0.01). The NFWL habitats were dominant in the eastern side of the city with signiﬁcantly low human population census. Although this side has not been extensively sampled (No. traps = 1), it is worthy to be highlighted in further investigations to understand the temporal association between WNV vector and reservoir host(s). Other LULC related variables such as TD, OUBL and RU showed a reduced contribution in predicting the likelihood of VHC. These LULC habitats provide both sugar and blood meals, and are favorable to WNV maintenance by enhancing maintenance and ampliﬁcation phases between mosquito vector and their nesting/roosting reservoir bird hosts, especially the passerines [17]. This ﬁnding was conﬁrmed by the selective feeding preference of this mosquito vector in NOLA, their contribution in both enzootic and epidemic transmission cycle of WNV [5,17,54–57], and the extended transmission season due to the milder climatic conditions of the Gulf Coast as manifested in mosquito WNV infection rates (Figure 3). Similar reports of increases in mosquito WNV infection rates have been made in 2005 and 2006 in Chicago [58]. Moreover, both the Jackknife test in MaxEnt (28.9%) and temporal analysis emphasized the association between LULC types during ampliﬁcation and early transmission phases (May–August) (Tables 3 and 5). Although no WNV human cases were included in our model, the transmission potential still exists due to the high VHC ratios during September and high suitable habitats. In addition, the lack Int. J. Environ. Res. Public Health 2017, 14, 892 15 of 20 of data on vector competence, survival rate, and the gonotrophic cycle period for NOLA prevented inclusion of this information in our model. However, the high risk probability was generated utilizing WNV minimum infection rates (0.8/1000) [5]. The climate variables shared reduced contributions in predicting likelihood of VHC in the spatial analysis [55,56]. However, climate was demonstrated as the key predicting factor in determining temporal distribution of WNV vectors. 5. Conclusions In the current study we modeled the spatio-temporal distribution of VHC ratios in response to future climate scenario, LULC, human population census, and DEM. Vector-host contact (VHC) ratios were estimated as a potential entomological indicator for the likelihood of biting rate and transmission potential of WNV. The VHC ratios were estimated within 5-km buffer zones around mosquito sampling sites representing their average ﬂight range utilizing mosquito density and population census/house block. The likelihood of VHC ratio was ﬁrst predicted in response to the biophysical systems using stepwise multiple regression model (RM). Accordingly, we used the signiﬁcant predicting variables from RM to highlight the spatio-temporal distribution of areas under risk of increased VHC emphasizing the likelihood of infective mosquito bites. p g q The interaction between these different biophysical systems showed heterogeneous inﬂuences on the spatio-temporal distribution of VHC ratios. In the spatial analysis, 12 variables were associated with the distribution of VHC ratios, and NFWL showed the highest prediction gain (R2 = 81.62). Although NFWL has not been sampled extensively during our study (1), to complete our objective, this variable needs to be highlighted rigorously in a separate investigation. Seasonal precipitation- and annual temperature-related variables shared reduced signiﬁcant associations with VHC ratios. The average likelihood of predicting very high risk areas was ~107 km2, which is ~9.87% of the total area of NOLA. Although neither the temporal distribution of VHC ratios nor their estimates were signiﬁcantly changed from month-to-month (except during September), their monthly response showed resilience to the number and type of the inﬂuential factors. The highest VHC ratio was reported during September, which was associated with the peak positive WNV mosquito pools. Seasonal temperature-related variables showed the highest inﬂuence on monthly likelihood of VHC in comparison with seasonal precipitation, LULC and DEM variables. This ﬁnding was conﬁrmed by both RM (R2 = 58.49) and Jackknife’s test (88.5%). The inﬂuence of LULC on likelihood of VHC was demonstrated during May–August. The NFWL showed a positive association with the increased VHC ratio during May, with no positive WNV mosquito pools. Meanwhile, both OUBL and RU settings were associated with the onset of mosquito WNV infection rates during June. This may reﬂect the distribution of WNV vector in close vicinity to reservoir host(s) during May, during the virus ampliﬁcation phase, and the virus beginning to build up inside mosquito bodies during June–August. 4. Discussion quinquefasciatus. This may help stimulate blood feeding during these times. The seasonal precipitation (Bio15) during April–August increased both available water habitats and distribution of WNV vector. Our ﬁndings showed heterogeneity in the temporal distribution of Cx. quinquefasciatus in response to LULC during May–August. This reﬂects the inﬂuence of some LULC classes on monthly ﬂux and distribution patterns of mosquito populations. The heterogeneity in monthly distributions of mosquito vectors can have a large impact on virus ampliﬁcation during the early season, especially when it is in close vicinity to reservoir bird hosts. Although no WNV positive mosquito pools were reported during April and May, NFWL was positively correlated with VHC during May (R2 = 72.6). This may reﬂect the association between nesting/roosting reservoir bird hosts and VHC ratios in May. During June–August, WNV seemed to build inside mosquito bodies to the detectable level that can cause potential transmission. Both OUBL and RU settings were associated with the onset of mosquito WNV infection rates during June. Other models reported that WNV transmission was either associated with forested and urban land [18,60] or socioeconomic status [61]. However, these correlations may vary spatially or temporally [62]. 16 of 20 16 of 20 Int. J. Environ. Res. Public Health 2017, 14, 892 Some previous studies improved our understanding about biology and ecology of Cx. quinquefasciatus and epidemiology [16,21,63]. Nevertheless, their ﬁndings did not highlight the interaction between different systems and their overall inﬂuence on mosquito vector distribution at a local scale. Moreover, these models predicted the geographic distribution of WNV vectors utilizing mosquito density as sampling points rather than the ﬂight range area and vector-host contact ratios. Land geomorphology and topography were used in predicting suitable habitats for mosquito breeding [25,34,35,62]. Four potential indicators were rigorously investigated: aspect ratio, slope, land surface curvature and hill shade [25,34,35,62]. The sampled gravid mosquito vectors reﬂect the proximity of water habitats to collected samples. Although these indicators showed potentiality in predicting WNV mosquito vectors in other areas [25,35], only relative high altitude demonstrated temporal inﬂuence on increased VHC ratios during May and June. References 1. Centers for Disease Control and Prevention. Guidelines for surveillance, prevention and control of West Nile virus infection—United States. MMWR Morb. Mortal. Wkly. Rep. 2000, 49, 25–28. 1. Centers for Disease Control and Prevention. Guidelines for surveillance, prevention and control of West Nile virus infection—United States. MMWR Morb. Mortal. Wkly. Rep. 2000, 49, 25–28. 2. Steele, K.E.; Linn, M.J.; Schoepp, R.J.; Komar, N.; Geisbert, T.W.; Manduca, R.M.; Calle, P.P.; Raphael, B.L.; Clippinger, T.L.; Larsen, T.; et al. Pathology of fatal west Nile virus infections in native and exotic birds during the 1999 outbreak in New York City, New York. Vet. Pathol. 2000, 37, 208–224. [CrossRef] [PubMed] 2. 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Conclusions During September–December, reduced numbers of positive WNV mosquito pools were recorded, which may explain the reduced viremia in wild Cx. quinquefasciatus. The independent ﬁeld collected sampling points were consistent with both likelihood of VHC ratios and spatio-temporal distribution of increased VHC ratios. However, multi-year mosquito, spatial projections of LULC and human population census data are recommended to be included in further investigations. Moreover, due to data limitation of reservoir host(s), human cases, mosquito vector survival rates, vector capacity parameters, and gonotrophic periods, we did not have the chance to include these variables in our study. 17 of 20 17 of 20 Int. J. Environ. Res. Public Health 2017, 14, 892 Acknowledgments: Authors would like to express their deep gratitude to City of New Orleans for the logistic support, especially mosquito surveillance and data entry team. Author Contributions: Sarah R. 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https://openalex.org/W1507384449	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0127561&type=printable	English	null	Bone Marrow Mesenchymal Cells Improve Muscle Function in a Skeletal Muscle Re-Injury Model	PloS one	2,015	cc-by	8,056	RESEARCH ARTICLE Bone Marrow Mesenchymal Cells Improve Muscle Function in a Skeletal Muscle Re- Injury Model Bruno M. Andrade1,2☯, Marcelo R. Baldanza1☯, Karla C. Ribeiro2, Anderson Porto1, Ramon Peçanha1, Fabio S. A. Fortes2, Gisele Zapata-Sudo3, Antonio C. Campos-de- Carvalho2, Regina C. S. Goldenberg2, João Pedro Werneck-de-Castro1* 1 Laboratório de Biologia do Exercício, Instituto de Biofísica Carlos Chagas Filho e Escola de Educação Física e Desportos, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil, 2 Instituto de Biofísica Carlos Chagas Filho, Centro de Ciências e Saúde, Universidade Federal do Rio de Janeiro, Bloco G, Ilha do Fundão, Rio de Janeiro, RJ, Brasil, 3 Departamento de Farmacologia Básica e Clínica, Universidade Federal do Rio de Janeiro, Centro de Ciências e Saúde, Bloco J, Ilha do Fundão, Rio de Janeiro, Brasil ☯These authors contributed equally to this work. * joaopedrowerneck@yahoo.com.br ☯These authors contributed equally to this work. * joaopedrowerneck@yahoo.com.br Data Availability Statement: All relevant data are within the paper. Data Availability Statement: All relevant data are within the paper. Funding: The study was supported by research grants from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), FAPERJ (Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. OPEN ACCESS Citation: Andrade BM, Baldanza MR, Ribeiro KC, Porto A, Peçanha R, Fortes FSA, et al. (2015) Bone Marrow Mesenchymal Cells Improve Muscle Function in a Skeletal Muscle Re-Injury Model. PLoS ONE 10 (6): e0127561. doi:10.1371/journal.pone.0127561 Skeletal muscle injury is the most common problem in orthopedic and sports medicine, and severe injury leads to fibrosis and muscle dysfunction. Conventional treatment for succes- sive muscle injury is currently controversial, although new therapies, like cell therapy, seem to be promise. We developed a model of successive injuries in rat to evaluate the therapeu- tic potential of bone marrow mesenchymal cells (BMMC) injected directly into the injured muscle. Functional and histological assays were performed 14 and 28 days after the injury protocol by isometric tension recording and picrosirius/Hematoxilin & Eosin staining, re- spectively. We also evaluated the presence and the fate of BMMC on treated muscles; and muscle fiber regeneration. BMMC treatment increased maximal skeletal muscle contraction 14 and 28 days after muscle injury compared to non-treated group (4.5 ± 1.7 vs 2.5 ± 0.98 N/cm2, p<0.05 and 8.4 ± 2.3 vs. 5.7 ± 1.3 N/cm2, p<0.05 respectively). Furthermore, BMMC treatment increased muscle fiber cross-sectional area and the presence of mature muscle fiber 28 days after muscle injury. However, there was no difference in collagen deposition between groups. Immunoassays for cytoskeleton markers of skeletal and smooth muscle cells revealed an apparent integration of the BMMC within the muscle. These data suggest that BMMC transplantation accelerates and improves muscle function recovery in our ex- tensive muscle re-injury model. Academic Editor: Eva Mezey, National Institutes of Health, UNITED STATES Academic Editor: Eva Mezey, National Institutes of Health, UNITED STATES Received: November 10, 2014 Accepted: April 16, 2015 Published: June 3, 2015 Copyright: © 2015 Andrade 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: © 2015 Andrade 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. ☯These authors contributed equally to this work. * joaopedrowerneck@yahoo.com.br Bone Marrow Cells Improve Muscle Function associated with the extent of residual scar tissue in the muscle [2] induced by a previous dam- age. The SKM healing process comprises degeneration and inflammation, regeneration, and fi- brosis in a complex and well-orchestrated series of events [3,4]. SKM regenerative capacity is due to the presence of a tissue-specific population of myogenic stem cells termed satellite cells, so-called due to its peripheral location on the skeletal muscle myofibre where it lies between the sarcolemma of the myofibre cell and its surrounding basal lamina [5]. Competing Interests: The authors have declared that no competing interests exist. However, depending on the severity and frequency of the injury, the regeneration process of skeletal muscle is very slow and incomplete, resulting in strength loss, fibrosis and a high rate of re-injury at the site of the prior injury [3]. Fibrosis is triggered mainly by the expression of transforming growth β (TGF- β) which stimulates fibroblast proliferation and collagen synthe- sis; and, concomitantly, inhibits both satellite cell proliferation and differentiation into myo- tubes [6,7]. Therefore, complete recovery of injured skeletal muscle appears to be hindered by fibrosis and usually leads to incomplete muscle healing [3]. The resultant disorganized scar tis- sue that often replaces damaged myofibers may contribute to muscle injuries recurrence [8]. Therapeutic intervention to treat skeletal muscle injury is controvertial. Common treat- ments for SKM injuries are RICE (rest, ice, compression, and elevation), nonsteroidal anti-in- flammatory drugs, pulsed ultrasound and immobilization. However, none of them treats the main problem, e.g. SKM cell loss and scar tissue formation. Also, routine treatments do not im- prove satellite cells number, proliferation and differentiation. Therefore, efforts to develop treatments to promote faster and more complete recovery after muscle injury should be fo- cused on the enhancement of muscle regeneration and/or the prevention of fibrosis. Because fast and complete repair of the injured muscle is the obvious target, especially in athletes, attempts should be made to find clinically feasible modalities to enhance the prolifera- tion phase of muscle regeneration. Cell transplantation has been used to overcome the loss of cells induced by lesions in many organs such as liver [9],, heart [10,11] and skeletal muscle [12,13]. Introduction Skeletal muscle (SKM) injuries are very commonly diagnosed in sports medicine and, more im- portant, the incidence of re-injury remains very high [1]. The risk of recurrent injury is 1 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0127561 June 3, 2015 Furthermore, in SKM injuries, a variety of cell types have been tested: myoblasts, which is the most used cell type [14], muscle-derived stem cells [15], hematopoietic stem cells [16,17], bone marrow-derived human mesenchymal stem cells [18], adipose-derived stem cells [13] and BMMC [12,19]. BMMC have been reported to differentiate in vitro into contractile myotubes or convert into myogenic linage in vivo in response to physiological stimuli [17,20– 22]. The search for new therapeutic alternatives requires the development of adequate animal models of muscle lesion. In this perspective, the availability of an animal model that mimics the characteristics of human muscle injury is crucial to study the pathophysiological mecha- nisms of regeneration and possible therapies. Many animal models of muscle injury have been proposed such as injection of cardiotoxin [23,24], mdx transgenic mice, crioinjury, manual clamping, laceration, strain and induced by exercise but none reproduces re-injuries which are the most common type of damage in the orthopedic field. Thus, the main purpose of this study was to investigate whether BMMC injected directly into damaged muscles could improve muscle function in a rat model with repetitive muscle injuries. Cell transplantation and experimental groups Three days after the injury protocol the rats were randomized for treatment with BMMC or ve- hicle (non-treated group). Under anesthesia, the soleus muscle was exposed and 3 X 106 labeled BMMC were injected with a tuberculin syringe along the border and the center of the scar tis- sue. To improve retention, cells were suspended in 40 μl of a three-dimensional gel of reconsti- tuted basement membrane (Matrigel Matrix BD Biosciences, USA) and BSS in a proportion of 1:3 [10,13]. Non-treated rats received only Matrigel in BSS (vehicle). After treatment, the leg was stitched closed, and the animals were monitored for 48hs. Cell isolation and culture procedures The isolation and primary culture of BMMC from femoral and tibial bones of donor rats were performed as described by Fidelis-de-Oliveira et al. [25]. BMMC were cultured in high glucose DMEM supplemented with 10% fetal bovine serum (GIBCO-BRL), 2 mM L-glutamine (Sigma), and antibiotics (100 U/ml penicillin G and 100 μg/ml streptomycin; GIBCO-BRL). BMMC were grown in 75 cm2 flasks and maintained at 37°C in a 5% CO2 incubator for 1 week during which medium was changed, at least twice, washing away all floating hematopoietic cells. At ~ 80–90% confluence, the cells were detached from the culture flasks with 0.25% tryp- sin-EDTA (Sigma) and replated. After the third replating, BMMC were resuspended in DMEM without serum, and labeled with 5μg/ml Hoescht 33342 (Sigma- Aldrich) for 20 min- utes. The cells were rinsed and centrifuged six times in balanced salt solution (BSS) to remove unbound Hoescht 33342 and kept in warm DMEM for a few minutes before transplantation. This labeling procedure was very efficient, ensuring ~90–95% labeling of cell nuclei. Cell viabil- ity was tested after labeling and was never lower than 95%. These cells are capable of differenti- ation in osteoblasts and adypocites. Muscle re-injured model Animals were anesthetized with ketamine (50 mg/kg) and xylazine (5 mg/kg ip). An incision was made in the skin overlying the lateral portion of the gastrocnemius muscle. The soleus muscle of the right limb was exposed along its entire length and lacerated (crunched 40 times) through 50% of its width and 100% of its thickness with a surgical blade type Basket (0.6mm). Twenty crunches were repeated 7 and 14 days after the first surgical procedure. The contralat- eral soleus was used as control muscle. Methods Animals Male inbred Wistar rats (200–250g) were obtained from the Centro de Pesquisa Gonçalo Muniz (Fiocruz-Bahia/Brasil). Animals were housed at controlled temperature (23°C) with daily exposure to a 12:12-h light-dark cycle and free access to water and standard rat chow. Iso- genic rats were used as donors and recipients of bone marrow-derived stromal cells. The Rio de 2 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0127561 June 3, 2015 Bone Marrow Cells Improve Muscle Function Janeiro Federal University Institutional Committee for Animal Use in Research (CEUA—CCS —EEFD 07) approved this study, which was in accordance with the International Guiding Principles for Biomedical Research Involving Animals (Geneva, Switzerland). The surgery was done under anesthesia with ketamine (50 mg/kg, i. p.) and xylazine (5mg/kg, i. p.) and the ani- mals were killed by cervical dislocation after being anesthetized. All efforts were made to minimize suffering. Tissue architecture and collagen deposition Soleus muscles were fixed in 10% buffered formalin and embedded in paraffin. Longitudinal and cross sections of 5μm thickness were obtained and mounted on glass slides. Slides were dewaxed and rehydrated with distilled water. Thereafter the samples were stained with heama- toxilin and eosin to provide an overall view of the tissue and with picrosirius stain for collagen deposition evaluation. For the picrosirius staining procedure the slides were incubated in 0.2% Phosphomolybdic acid (Sigma-Aldrich Ltd) for 1 minute following incubation at room tem- perature for 90 minutes in sirius red solution (0.6g in 600 mL of saturated picric Acid), (Sigma- Aldrich Ltd). The slides were then incubated in 0.01N Hydrochloric Acid (Sigma-Aldrich Ltd) for 2 minutes, dehydrated, cleared in xylene, and mounted with Entellan. Slides were examined in a light Axiovert 100 microscope (Zeiss). For fibrosis quantification (BMMC, 14d n = 3 / 28d n = 4 and vehicle, 14d n = 6 / 28d n = 5), 20 random fields (20X original magnification) were taken. The acquisition was standardized using ImagePro 5.0 and no modifications in the im- ages were done after acquisition. Images were analyzed using ImagePro 5.0 to quantify the red area and the total area. The area of fibrosis was considered the ratio between the red area / total area. Centronucleated fibers and fiber cross sectional area quantifications In order to quantify skeletal muscle hypertrophy, we measured muscle fiber cross-sectional area (BMMC, n = 4 and non-treated, n = 3) 28 days after muscle injury as previously described [26]. Hematoxylin and eosin stained images taken on the microscope were analyzed on Image J program and the area of 500 fibers per soleus was measured. We counted the number of cen- tronucleated regenerating myofibers at 28 days after muscle injury (BMMC, n = 4 and non- treated, n = 3) in 103 fibers as previously described [13]. At least ten fields were analyzed for each muscle, and the average number of regenerating myofibers per 103 total fibers was com- pared between groups. Nuclei with no discernible surrounding cytoplasm were discarded. Maximal tension developed in isolated soleus muscle Fourteen and twenty-eight days after the last injury, BMMC (14d n = 6 / 28d n = 9) and non- treated rats (vehicle- 14d n = 6/ 28d n = 7) rats were anesthetized with isoflurane and soleus muscles were dissected for isometric tension recording. Muscles were mounted in vertical chambers filled with Ringer solution (NaCl 135 mM, KCl 5.0 mM, MgCl2 1.0 mM, Na2HPO4 1.0 mM, NaHCO3 25 mM, CaCl2 1.25 mM, glucose 5 mM) and continuously oxygenated with carbogen gas (95% O2 /5% CO2) at 37°C. One muscle end was attached to a force transducer 3 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0127561 June 3, 2015 Bone Marrow Cells Improve Muscle Function (Grass, model FT-03) and the other end to a hook fixed at the bottom of the experimental chamber. Soleus muscles were field stimulated (Grass S88) at a rate of 0.2 Hz with pulses of 2 ms duration. The transducer’s electrical signal was amplified (Cyberamp, Axon Instruments), digitized (Digidata 1200, Axon Instruments), displayed and stored on a computer using Axo- scope software (Axon Instruments). After stabilization (30 min), maximal isometric tension was obtained by stimulation at 50 Hz. The amplitude of maximal contractile response was nor- malized to the cross-sectional area (CSA) of the muscle using the equation: Tension (N/cm2) = Force/CSA. CSA (cm2) was obtained by the formula: weight (g)/length (cm) 1.06 g/cm3 (muscle density) (54). PLOS ONE \| DOI:10.1371/journal.pone.0127561 June 3, 2015 Muscle function Our repetitive injury model resulted in great muscle function impairment. Soleus isometric de- veloped force decreased ~70% at day 14 (2.5 ± 0.98 N/cm2, p<0.001; Fig 1A) and remained ~33% below non-injured muscles at day 28 (5.7 ± 1.3 N/cm2, p<0.001; Fig 1B) days after inju- ry, when compared with the contralateral uninjured muscle (8.4 ± 0.95 N/cm2). BMMC treat- ment increased muscle force at both 14 (4.5 ± 1.7 vs. 2.5 ± 0.98 N/cm2, p<0.01) and 28 (8.4 ± 2.3 vs. 5.7 ± 1.3 N/cm2, p<0.001) days after injury compared to the vehicle group (non- treated). Moreover, no statistical difference was found between the BMMC-treated and normal muscles at day 28 (8.4 ± 2.3 vs. 8.7 ± 0.68 N/cm2, respectively; Fig 1A). These results demon- strate that BMMC can improve muscle function even after severe damage produced by the re- petitive injury protocol employed. Statistical analyses Values are presented as mean ± SD. Differences between the BMMC-treated, vehicle-treated and the contralateral normal muscles in all time points were evaluated by one-way ANOVA followed by Tukey´s post hoc test. Values were considered different when P<0.05. Bone Marrow Cells Improve Muscle Function Fig 1. Maximal isometric tension of soleus muscle after BMMC treatment. (A) Maximal isometric force in normal (non-injured), BMMC and vehicle- treated groups 14 days after injury. (B) Same as in (A) except that animals were analyzed 28 days after injury. Normal group values are from contralateral non-injured muscle. Values are means ± SD. *** p<0.001 * p<0.05. BMMC (14d n = 6 and 28d n = 9) and vehicle-treated rats (14d n = 6 and 28d n = 7) Fig 1. Maximal isometric tension of soleus muscle after BMMC treatment. (A) Maximal isometric force in normal (non-injured), BMMC and vehicle- treated groups 14 days after injury. (B) Same as in (A) except that animals were analyzed 28 days after injury. Normal group values are from contralateral non-injured muscle. Values are means ± SD. *** p<0.001 * p<0.05. BMMC (14d n = 6 and 28d n = 9) and vehicle-treated rats (14d n = 6 and 28d n = 7) doi:10.1371/journal.pone.0127561.g001 doi:10.1371/journal.pone.0127561.g001 Axiocam HR charge-coupled device videocamera (Zeiss) was used to obtain differential inter- ference contrast and fluorescence images from regions of the injured soleus muscle displaying nuclear and cytoskeletal markers. Images were overlaid and processed using Photoshop 7 soft- ware (Adobe). Immunohistochemistry/Identification of cell transplanted Soleus muscles were taken from BMMC (n = 5) and non-treated (n = 3) animals 28 after re- peated injuries. In order to perform immunohistochemistry, muscles were cryoprotected, cooled in liquid nitrogen, and 10μm consecutive cryostat sections were obtained. Tissue sec- tions were stained with antibodies against smooth muscle myosin heavy chain (MHC (G-4) 1:100—Santa Cruz Biotechnology—sc-6956) and troponin I (1:100—clone 5C5, Sigma). Goat anti-mouse secondary antibodies were used as follows: IgG FITC conjugate (1:50—catalog no. F-2012, Sigma) and IgM FITC conjugate (1:50—catalog no. F-9259, Sigma). Thereafter, sec- tions were washed with PBS and mounted with PPD solution (89% glycerol, 10% PBS and 1% p-phenylene diamine—PPD). Grafted BMMC were identified by Hoescht 33342-labeled nuclei excited at 351 nm by UV laser. An Axiovert 135 microscope coupled to a high-resolution 4 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0127561 June 3, 2015 Collagen content, number of centronucleated myofibers and soleos cross-sectional area Soleus muscle collagen content and the presence of transplanted BMMC were carefully exam- ined in postmortem histological evaluation. Normal muscles presented parallel fibers with multiple nuclei located in the periphery and a perfect arrangement of myofibrils giving rise to muscle striations (Fig 2A1 and 2A2). Twenty-eight days after injury, although the vast majority 5 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0127561 June 3, 2015 Bone Marrow Cells Improve Muscle Function Fig 2. Histological sections stained by heamatoxylin & eosin. (A1) H&E staining image of a rat non-injured soleus muscle 28 days after surgery; (A2) same as in A1 except that vehicle-treated muscle is shown; (A3) same as in A1 but BMMC-treated animal is presented; (A2, A3 and A4) Details of A1, A2 an A3 animals are shown in greater magnification, respectively; (A3) Picrossírius staining image of a rat non-injured soleus muscle 28 days after surgery;; (B3) same as in A3 except that vehicle-treated muscle is shown; (C3) same as in A3 but BMMC-treated animal is presented and (A4, B4 and C4) Details of A3, B3 and C3 animals are shown in greater magnification, respectively; Histological analysis reveals that both groups submitted to muscle injury present wide spread collagen deposition (A3-C4) independent of treatment. Note the central aligned disposition of the muscle fiber nuclei indicating recent muscle fiber regeneration (arrows in B2). Bars correspond to 300 μm in A1-C1 and A3-C3 and correspond to 50μm in A2-C2 and A4-C4. Fig 2. Histological sections stained by heamatoxylin & eosin. (A1) H&E staining image of a rat non-injured soleus muscle 28 days after surgery; (A2) same as in A1 except that vehicle-treated muscle is shown; (A3) same as in A1 but BMMC-treated animal is presented; (A2, A3 and A4) Details of A1, A2 and A3 animals are shown in greater magnification, respectively; (A3) Picrossírius staining image of a rat non-injured soleus muscle 28 days after surgery;; (B3) same as in A3 except that vehicle-treated muscle is shown; (C3) same as in A3 but BMMC-treated animal is presented and (A4, B4 and C4) Details of A3, B3 and C3 animals are shown in greater magnification, respectively; Histological analysis reveals that both groups submitted to muscle injury present wide spread collagen deposition (A3-C4) independent of treatment. Note the central aligned disposition of the muscle fiber nuclei indicating recent muscle fiber regeneration (arrows in B2). Bone Marrow Cells Improve Muscle Function Table 1. Quantification of collagen content. 14 days 28 days Normal (n=9) Non-treated (n=6) BMMC (n=3) Normal (n=9) Non-treated (n=5) BMMC (n=4) 0.14 ± 0.02 0.34 ± 0.04* 0.29 ± 0.03* 0.14 ± 0.02 0.30 ± 0.04* 0.26 ± 0.04* Quantiﬁcation of collagen content in longitudinal sections by picrosirius staining in BMMC-treated and non-treated groups soleus muscle 14 and 28 days after injury. Normal group values are contralateral non-injured muscle. Values are means ± SD of collagen area/total area ratio. * p<0,001 compared to normal. Table 1. Quantification of collagen content. 14 days 28 days Normal (n=9) Non-treated (n=6) BMMC (n=3) Normal (n=9) Non-treated (n=5) BMMC (n=4) 0.14 ± 0.02 0.34 ± 0.04* 0.29 ± 0.03* 0.14 ± 0.02 0.30 ± 0.04* 0.26 ± 0.04* Quantiﬁcation of collagen content in longitudinal sections by picrosirius staining in BMMC-treated and non-treated groups soleus muscle 14 and 28 days after injury. Normal group values are contralateral non-injured muscle. Values are means ± SD of collagen area/total area ratio. * p<0,001 compared to normal. Table 1. Quantification of collagen content. Table 1. Quantification of collagen content. Quantiﬁcation of collagen content in longitudinal sections by picrosirius staining in BMMC-treated and non-treated groups soleus muscle 14 and 28 days after injury. Normal group values are contralateral non-injured muscle. Values are means ± SD of collagen area/total area ratio. * p<0,001 compared to normal. Quantiﬁcation of collagen content in longitudinal sections by picrosirius staining in BMMC-treated and non-treated groups soleus muscle 14 and 28 days after injury. Normal group values are contralateral non-injured muscle. Values are means ± SD of collagen area/total area ratio. * p<0,001 compared to normal. doi:10.1371/journal.pone.0127561.t001 doi:10.1371/journal.pone.0127561.t001 2A3–2C4). There was no difference in the amount of collagen deposition after 28 days of treat- ment in vehicle- and cell-treated groups (Fig 2 and Table 1). We also assessed collagen deposi- tion on soleus cross sections and still found no difference between BMMC and non-treated groups (0.26 ± 0.06 vs 0.31 ± 0.08, respectively). At 28 days after injury, the BMMC-treated had fewer fibers with centrally located nuclei compared to non-treated rats (Fig 3A) suggesting a late stage of regeneration with more mature muscle fibers. Furthermore, BMMC increased muscle fiber cross-sectional area compared to non-treated muscles (580.1 ± 55.1 μm2 vs 390.0 ± 68.3 μm2, respectively; Fig 3B). Collagen content, number of centronucleated myofibers and soleos cross-sectional area Bars correspond to 300 μm in A1-C1 and A3-C3 and correspond to 50μm in A2-C2 and A4-C4. Fig 2. Histological sections stained by heamatoxylin & eosin. (A1) H&E staining image of a rat non-injured soleus muscle 28 days after surgery; (A2) same as in A1 except that vehicle-treated muscle is shown; (A3) same as in A1 but BMMC-treated animal is presented; (A2, A3 and A4) Details of A1, A2 and A3 animals are shown in greater magnification, respectively; (A3) Picrossírius staining image of a rat non-injured soleus muscle 28 days after surgery;; (B3) same as in A3 except that vehicle-treated muscle is shown; (C3) same as in A3 but BMMC-treated animal is presented and (A4, B4 and C4) Details of A3, B3 and C3 animals are shown in greater magnification, respectively; Histological analysis reveals that both groups submitted to muscle injury present wide spread collagen deposition (A3-C4) independent of treatment. Note the central aligned disposition of the muscle fiber nuclei indicating recent muscle fiber regeneration (arrows in B2). Bars correspond to 300 μm in A1-C1 and A3-C3 and correspond to 50μm in A2-C2 and A4-C4. doi:10.1371/journal.pone.0127561.g002 of the muscle presented as normal muscle tissue arrangement, few areas with disarranged tissue organization, sites of inflammation and fibers with centralized nuclei were still present in the vehicle group (Fig 2B1 and 2B2) and BMMC-treated groups (Fig 2C1 and 2C2). Quantitative analysis of picrosirius staining revealed a significant increase in the amount of fibrosis in both groups at both time points when compared to control muscle (Table 1). This fi- brosis was predominantly perivascular and interstitial in most of the tissue of both groups (Fig 6 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0127561 June 3, 2015 Muscle and vessel markers in transplanted BMMC One of the proposed mechanisms for cell therapy benefit is the differentiation of transplanted cells into host tissue cells, e.g. muscle fiber and/or vessel cells in skeletal muscle tissue. Thus, we stained skeletal muscle tissue with troponin I (Fig 4) or smooth muscle myosin (Fig 5) to deter- mine the fate of the transplanted cells previously stained with Hoestch 33342 and found labeled cells in all treated skeletal muscle although in few muscle fibers and vessels. Merged images of Fig 3. Centronucleated muscle fiber and cross-sectional area determination 28 days after muscle injury. (A) Number of centronucleated muscle fibers per 103 fibers in normal (non-injured), vehicle- and BMMC-treated groups; (B) muscle fiber cross-sectional area in same groups as A; (C) Cross-sectional image a normal soleus muscle stained with H&E; (D) Same as in C but vehicle muscle is shown and (E) same as in C but BMMC soleus muscle is presented. Normal group values are from contralateral non-injured muscle. Values are means ± SD. *** p<0.001 * p<0.05. BMMC (n = 4) and vehicle-treated rats (n = 3). Centronucleated muscle fibers are shown by arrow reads. doi:10.1371/journal.pone.0127561.g003 Fig 3. Centronucleated muscle fiber and cross-sectional area determination 28 days after muscle injury. (A) Number of centronucleated muscle fibers per 103 fibers in normal (non-injured), vehicle- and BMMC-treated groups; (B) muscle fiber cross-sectional area in same groups as A; (C) Cross-sectional image a normal soleus muscle stained with H&E; (D) Same as in C but vehicle muscle is shown and (E) same as in C but BMMC soleus muscle is presented. Normal group values are from contralateral non-injured muscle. Values are means ± SD. *** p<0.001 * p<0.05. BMMC (n = 4) and vehicle-treated rats (n = 3). Centronucleated muscle fibers are shown by arrow reads. doi:10.1371/journal.pone.0127561.g003 doi:10.1371/journal.pone.0127561.g003 7 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0127561 June 3, 2015 Bone Marrow Cells Improve Muscle Function Fig 4. Skeletal muscle troponin immunolocalization in soleus muscle treated with BMMC 28 days after repeated injuries. (A) and (D) Differential interference contrast images; (B) and (E) overlay images of Hoestch (in blue) and troponin I (green); (C) and (F) troponin I staining alone. Green fluorescence indicates typical striated skeletal muscle labelling pattern while nuclei are shown by blue Hoescht staining. Arrows denote nuclei position. Bars correspond to 20μm. Fig 4. Skeletal muscle troponin immunolocalization in soleus muscle treated with BMMC 28 days after repeated injuries. Muscle and vessel markers in transplanted BMMC (A) and (D) Differential interference contrast images; (B) and (E) overlay images of Hoestch (in blue) and troponin I (green); (C) and (F) troponin I staining alone. Green fluorescence indicates typical striated skeletal muscle labelling pattern while nuclei are shown by blue Hoescht staining. Arrows denote nuclei position. Bars correspond to 20μm. doi:10.1371/journal.pone.0127561.g004 Hoestch and troponin I staining indicate that donor cells engrafted skeletal muscle (Fig 4). Nu- clei of transplanted cells were located either in the peripheral (Fig 4A–4C) or central position (Fig 4D–4F) in skeletal muscle fibers. Regarding the distribution of Hoescht-labeled nuclei in blood vessel structures, Fig 5 shows sectioned vessels harboring typical heavy myosin smooth muscle labelling pattern of the tunica media with concomitant nuclei Hoescht staining. A dis- continuous labelling of the vessel smooth muscle marker can be readily noticed in the space oc- cupied by the nuclear compartment indicating that the labelled nuclei could belong to the smooth muscle cells (Fig 5). Hoestch and troponin I staining indicate that donor cells engrafted skeletal muscle (Fig 4). Nu- clei of transplanted cells were located either in the peripheral (Fig 4A–4C) or central position (Fig 4D–4F) in skeletal muscle fibers. Regarding the distribution of Hoescht-labeled nuclei in blood vessel structures, Fig 5 shows sectioned vessels harboring typical heavy myosin smooth muscle labelling pattern of the tunica media with concomitant nuclei Hoescht staining. A dis- continuous labelling of the vessel smooth muscle marker can be readily noticed in the space oc- cupied by the nuclear compartment indicating that the labelled nuclei could belong to the smooth muscle cells (Fig 5). Altogether, immunostaining data suggest that transplanted BMMC could engraft into host muscle and express both myofiber and vessel markers. However, not all Hoestch-stained nuclei were present in skeletal muscle or vessel cells. We carefully counted 267 labeled nuclei of three BMMC-treated soleus and determine their position in the host tissue. Among the 267 stained nuclei, the majority (73%) was found in myofibers or smooth muscle cells (195 out of 267). On the other hand, 27% of the labeled nuclei were found to be in the interstitial space. PLOS ONE \| DOI:10.1371/journal.pone.0127561 June 3, 2015 Bone Marrow Cells Improve Muscle Function and 30% of the normal value on day 14 and 28 after muscle injuries, respectively. In the cell treated group muscle tension development was significantly higher than in the vehicle group at both time points and recovery to control muscle tension values was observed 28 days after cell injection (Fig 1). This demonstrates that local injection of bone marrow mesenchymal cells (BMMC) significantly improves functional capacity of soleus muscle 14 and 28 days after re- injuries in comparison to vehicle-treated group. It is possible that longer observation times would result in complete recovery of tension development even in the vehicle group; this re- mains to be investigated and will allow distinction between an accelerated repair mechanism after cell therapy and a gain of function improvement induced by the cells. Muscle injury and, mostly, re-injuries are a challenge for sports medicine and orthopedics [1,2]. Despite the great regenerative capacity of skeletal muscle, completely lacerated muscle re- covers only approximately 50% of their strength and 80% of their ability to shorten [27]. Anti- inflammatory drugs, ultrasound therapy, ice and superficial heat do not target the main prob- lem of muscle damage: cell loss. Cell transplantation suits this requirement and has been useful in muscle damage treatment. In the current work we injected the adherent fraction of bone marrow mononuclear cells, a population of CD34-/CD45- cells [10,11], the BMMC, directly into damaged soleus muscles. Our group has shown that these adherent bone marrow cells are capable of differentiating in mesenchymal lineage such as adipogenic, osteogenic and chrondo- genic [28]. To optimize retention, BMMC were injected in matrigel, which is a reconstituted basement membrane, containing predominantly laminin-1, collagen IV, and perlecan, a hepa- rin sulfate proteoglican (Matrigel) that supports cells adhesion and can prevent cell loss due to diffusion [29]. BMMC have been reported to contribute to muscle regeneration [20] and improve func- tional capacity [12,19,30] after muscle lesion. More important, Palermo et al (2005) demon- strated that bone marrow cells migrate to skeletal muscle in response to physiologic stress [21]. The differentiation and fate of transplanted donor-derived cells in skeletal muscle repair is still a controversial issue. In contrast to Natsu et al (2004), we did find transplanted cells in host muscle tissue, as described by others [17,20,21,23,31–35]. PLOS ONE \| DOI:10.1371/journal.pone.0127561 June 3, 2015 Discussion Our model of repeated injuries in rat soleus muscle markedly impaired muscle function and caused wide spread scar tissue formation. Maximal developed tension was reduced to about 66 Fig 5. Immunolocalization of smooth muscle myosin as vessel marker in soleus muscle treated with BMMC 28 days after repeated injuries. A) and (D) Differential interference contrast images; (B) and (E) overlay images of Hoestch (in blue) and smooth muscle myosin (green) staining; (C) and (F) smooth muscle myosin alone. Green fluorescence indicates typical vessel shape labelling pattern while nuclei are shown by blue Hoescht staining. Arrows denote nuclei position. Bars correspond to 20μm. d i 10 1371/j l 0127561 005 Fig 5. Immunolocalization of smooth muscle myosin as vessel marker in soleus muscle treated with BMMC 28 days after repeated injuries. A) and (D) Differential interference contrast images; (B) and (E) overlay images of Hoestch (in blue) and smooth muscle myosin (green) staining; (C) and (F) smooth muscle myosin alone. Green fluorescence indicates typical vessel shape labelling pattern while nuclei are shown by blue Hoescht staining. Arrows denote nuclei position. Bars correspond to 20μm. doi:10.1371/journal.pone.0127561.g005 doi:10.1371/journal.pone.0127561.g005 8 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0127561 June 3, 2015 Blue nuclei indicate that BMMC, previously stained with the Hoestch dye, were present in the damage tissue and some of these cells showed immunoreactivity for smooth muscle myosin and skeletal muscle troponin (Figs 4 and 5). Matziolis et al (2006) also performed a crunch model, but lesion was induced by only seven manual clamping one week before cell transplant. Unfortunately, they did not show any imuno and/or histological analisys [12]. However, we cannot exclude a paracrine effect of the transplanted cells inducing functional improvement. Given that the frequency of donor cells in host tissue 28 days after muscle cell injections is low, we cannot rule out other possible benefi- cial effects of BMMC treatment such as a paracrine mechanism. Indeed, we recently demon- strate that soluble factors secreted by BMMC improve cardiac function in infarcted rat hearts [25]. Also, we reported that mesenchymal cells from adipose tissue improve muscle function even with no cell found in the host tissue 4 weeks after injetion [13]. The functional improvement did not correlate with scar tissue formation. We found that in- jection of BMMC in crunched muscles did not decrease the amount of fibrosis and scar forma- tion at the re-injured site (Table 1 and Fig 2). Despite its high regeneration capacity, muscle lesion always leads to extracellular matrix expansion, collagen deposition and formation of a fi- brotic scar. This process is strictly related to the level of muscle contusion. Moreover, successive injuries would exacerbate scar formation. In this context, both mononuclear and mesenchymal cells form bone marrow did not reverse liver fibrosis [28,36] and cells from adipose tissue were not capable of decreasing fibrosis in SKM laceration model [13]. Accordingly, antifibrotic ther- apy has been tested after muscle injury [35,37–44] and was able to improve cell-based therapies [45–47]. 9 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0127561 June 3, 2015 Bone Marrow Cells Improve Muscle Function During muscle regeneration, new skeletal muscle fiber exhibit their nuclei centrally located and then finally matures to periphery nucleated fibers [3]. Thus the number of fiber with cen- trally located nuclei imputes an immature muscle and/or in regeneration phase. The matura- tion constitutes the third and last phase of the degeneration/regeneration cycle and is characterized by fiber hypertrophy and gradual recovery functional properties of the muscle [3,48]. Treatment with BMMC increased soleus muscle cross-sectional area and decreased the number of fiber with centrally located nuclei suggesting a more mature phenotype of treated muscles. Therefore, increasing the pool of cells capable of differentiating into skeletal muscle cells is a promising tool to help in the treatment of serious and recidivous/recurrent injuries as in sports, aging and car accidents. Taken together, our results suggest that bone marrow in- jected cells might contribute to myotube formation process (repairing damaged muscle fibers) and/or form new muscle fibers regardless of fibrosis. Alternatively, as mentioned above, para- crine secretion of growth and angiogenic factors might result in improved function through hypertrophy and angiogenesis [49]. In conclusion, bone marrow stromal cells accelerate muscle regeneration and restore sol- eous muscle function despite scar tissue development in a severe re-injury protocol which is commonly found in recreational and elite sports due to insufficient regeneration. Author Contributions Conceived and designed the experiments: BMA MRB JPSWC RCSG. Performed the experi- ments: BMA MBR AP RP FSAF KCR. Analyzed the data: BMA JPSWC RCSG ACCC. Contrib- uted reagents/materials/analysis tools: GZS ACCC RCSG JPSWC. Wrote the paper: BMA MBR. PLOS ONE \| DOI:10.1371/journal.pone.0127561 June 3, 2015 References 1. Zemper ED. Track and field injuries. Medicine and sport science. 2005; 48:138–51. doi: 10.1159/ 000084287 PMID: 16247256. 2. Croisier JL, Forthomme B, Namurois MH, Vanderthommen M, Crielaard JM. Hamstring muscle strain recurrence and strength performance disorders. Am J Sports Med. 2002; 30(2):199–203. Epub 2002/ 03/26. PMID: 11912088. 3. Charge SB, Rudnicki MA. 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https://openalex.org/W2584627048	https://repository.ubn.ru.nl//bitstream/handle/2066/190014/190014.pdf	English	null	Identification of TP53BP2 as a Novel Candidate Gene for Primary Open Angle Glaucoma by Whole Exome Sequencing in a Large Multiplex Family	Molecular neurobiology	2,017	cc-by	5,723	Identification of TP53BP2 as a Novel Candidate Gene for Primary Open Angle Glaucoma by Whole Exome Sequencing in a Large Multiplex Family Micheal, S.; Saksens, N.T.M.; Hogewind, B.F.T.; Khan, M.I.; Hoyng, C.B.; Hollander, A.I. den 2018, Article / Letter to editor (Molecular Neurobiology, 55, 2, (2018), pp. 1387-1395) Doi link to publisher: https://doi.org/10.1007/s12035-017-0403-z Version of the following full text: Publisher’s version Downloaded from: http://hdl.handle.net/2066/190014 Download date: 2024-10-24 Note: To cite this publication please use the final published version (if applicable). Mol Neurobiol (2018) 55:1387–1395 DOI 10.1007/s12035-017-0403-z Identification of TP53BP2 as a Novel Candidate Gene for Primary Open Angle Glaucoma by Whole Exome Sequencing in a Large Multiplex Family Shazia Micheal1,2 & Nicole T.M. Saksens1 & Barend F. Hogewind1 & Muhammad Imran Khan3 & Carel B. Hoyng1 & Anneke I. den Hollander1,3 Shazia Micheal1,2 & Nicole T.M. Saksens1 & Barend F. Hogewind1 & Muhammad Imran Khan3 & Carel B. Hoyng1 & Anneke I. den Hollander1,3 Received: 11 May 2016 /Accepted: 12 January 2017 /Published online: 1 February 2017 # The Author(s) 2017. This article is published with open access at Springerlink.com Abstract Primary open angle glaucoma (POAG) is a major type of glaucoma characterized by progressive loss of retinal ganglion cells with associated visual field loss without an iden- tifiable secondary cause. Genetic factors are considered to be major contributors to the pathogenesis of glaucoma. The aim of the study was to identify the causative gene in a large family with POAG by applying whole exome sequencing (WES). WES was performed on the DNA of four affected family mem- bers. Rare pathogenic variants shared among the affected indi- viduals were filtered. Polymerase chain reaction and Sanger sequencing were used to analyze variants segregating with the disease in additional family members. WES analysis identified a variant in TP53BP2 (c.109G>A; p.Val37Met) that segregated heterozygously with the disease. In silico analysis of the sub- stitution predicted it to be pathogenic. The variant was absent in public databases and in 180 population-matched controls. A novel genetic variant in the TP53BP2 gene was identified in a family with POAG. Interestingly, it has previously been dem- onstrated that the gene regulates apoptosis in retinal ganglion cells. This supports that the TP53BP2 variant may represent the cause of POAG in this family. Additional screening of the gene in patients with POAG from different populations is required to confirm its involvement in the disease. Keywords Primary open angle glaucoma . Whole exome sequencing . TP53BP2 * Anneke I. den Hollander Anneke.denHollander@radboudumc.nl 1 Department of Ophthalmology, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, the Netherlands 2 Department of Clinical Genetics, Academic Medical Centre, Amsterdam, the Netherlands 3 Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, the Netherlands Introduction Glaucoma is a leading cause of irreversible blindness world- wide, affecting more than 60 million people around the world [1]. Glaucoma comprises a group of heterogeneous optic neu- ropathies, characterized by progressive optic nerve degenera- tion. The diagnosis of glaucoma is usually late, since the loss of vision often starts in the periphery and progression to the loss of central vision is late. Due to this, glaucoma is also called a silent thief of sight, with devastating consequences to the patient’s quality of life. Glaucoma is classified into two main types: primary and secondary glaucoma. Among primary glaucoma subtypes, primary open angle glaucoma (POAG) represents the major type of glaucoma affecting about 35 million people worldwide and is characterized by a juvenile or adult onset. Patients with POAG have characteristic glaucomatous optic nerve damage with corresponding visual field defects and an open anterior chamber angle at gonioscopy, but no other (congenital) anomalies [2, 3]. One of the significant risk factors for POAG is elevation of intraocular pressure (IOP). However, POAG also occurs in patients without elevated IOP, and an elevated IOP does not necessarily lead to POAG [4]. The grad- ual loss of the retinal ganglion cells (RGCs) is a hallmark of the disease along with the increased IOP, but the exact pathophys- iological mechanisms of the disease are not fully understood. Well-studied risk factors associated with POAG include age, family history, gender, ethnicity, central corneal thickness, and myopia. In addition, genetic factors play an important role in the disease etiology. * Anneke I. den Hollander Anneke.denHollander@radboudumc.nl * Anneke I. den Hollander Anneke.denHollander@radboudumc.nl To date, more than 15 loci have been identified for glauco- ma, and the causative gene has been identified for 5 of these Mol Neurobiol (2018) 55:1387–1395 1388 loci: GLC1A (MYOC/TIGR) [5, 6], GLC1E (OPTN) [7, 8], GLC1F (ASB10) [9, 10], GLC1G (WDR36) [11], and GLC1H (EFEMP1) [12, 13]. In addition, mutations in the CYP1B1 [14] gene were identified in primary congenital, ju- venile onset and adult onset POAG [15–17]. Finding the genes that cause glaucoma is the first step in improving early diagnosis and treatment of patients suffering from glaucoma. However, only less than 10% of POAG cases have pathogenic mutations in these disease-causing genes. Introduction It is therefore likely that the hereditary aspect of many of the remaining cases of POAG is either in the unidentified genes or due to the com- bined effects of several single nucleotide polymorphisms (SNPs). In recent years, several genome-wide association studies (GWAS) have identified several SNPs at different loci including CAV1/CAV2 [18], TMCO1 [19], CDKN2B-AS1 [20], CDC7-TGFBR3 [21], SIX1/SIX6 [22], GAS7, ATOH7, TXNRD2, ATXN2, and FOXC1 [23], to be associated with POAG, but they explain only a fraction of the disease herita- bility. In addition, the mechanisms how the associated loci influence the development of disease are often unclear. Therefore, additional genetic studies are required to explain the heritability, to gain a better understanding in the disease etiology, and to define new targets for treatment. the optic disk was carried out by the Heidelberg Retina Tomograph II (HRT II; Heidelberg Engineering, Heidelberg, Germany), as described elsewhere [25]. An ophthalmic pho- tographer masked to the results of the previous tests conducted the examination. The HRT Moorfields regression analysis (MRA) was used for classification of the optic disk [26]. The diagnosis of POAG was made when the following criteria were met: IOP higher than 22 mmHg (as measured by applanation tonometry in both eyes), glaucomatous optic neu- ropathy present in both eyes at funduscopy, visual field loss consistent with assessed optic neuropathy in at least one eye, and an open anterior chamber angle by gonioscopy. Whole Exome Sequencing and Analysis To identify the underlying genetic cause of the disease in this large family with POAG, WES was performed using genomic DNA of four affected individuals (III:1, III:5, III:6, and III:8) (Fig. 1). Enrichment of exonic sequences was achieved by using the SureSelectXT Human All Exon V.2 Kit (50 Mb) (Agilent Technologies, Inc., Santa Clara, CA, USA). Sequencing was performed on a SOLiD 4 sequencing plat- form (Life Technologies, Carlsbad, CA, USA). The hg19 ref- erence genome was aligned with the reads obtained using SOLiD LifeScope software V.2.1 (Life Technologies). The goal of the current study was to identify the genetic cause of POAG in a large multiplex family using whole ex- ome sequencing (WES). To identify the causative variant, only the variants shared by the four affected individuals were included for further anal- ysis. All variants present within intergenic, intronic, and un- translated regions and synonymous substitutions were exclud- ed. Variants present in the public genetic variant databases, including the Exome Variant Server (http://evs.gs. washington.edu/EVS/), dbSNP132 (http://www.ncbi.nlm. nih.gov/projects/SNP/snp_summary.cgi?build_id=132), and 1000 Genomes (http://www.1000genomes.org/) with an allele frequency >0.5%, were excluded. Linkage Analysis grade III) and normal results of corneal thickness evaluation. The mean age at diagnosis was 54.8 years (range 49–60 years), with a mean IOP of 13.2 ± 2.2 mmHg (after use of IOP low- ering medications). All individuals had bilateral glaucoma: they had glaucomatous optic neuropathy on funduscopy with reproducible compatible glaucomatous visual field loss, and all individuals showed abnormal results on Heidelberg Retina Tomograph II testing. A representative color photo of the optic disk of individual III-9 with POAG, with the corresponding superior arcuate scotoma on Humphrey visual field testing is shown in Fig. 2. From the medical chart, we distilled that the mean highest IOP recorded on diurnal testing was 23.6 ± 4.7 mmHg. Microsatellite markers with a genetic heterogeneity >60% were selected from the UCSC database. Microsatellite markers D1S2655, D1S2891, D1S2629, and D1S229 were amplified with M13 tailed primers, followed by a second PCR with fluorescently labeled M13 primers. Fluorescent am- plification products were visualized on the ABI-310 genetic analyzer, and the size of the alleles was determined with 500LIZ (Applied Biosystems, Bleiswijk, the Netherlands) and analyzed with the GeneMapper software version 3.7 (Applied Biosystems, Bleiswijk, the Netherlands). Multipoint linkage analysis was performed for informative markers to determine the logarithm of the odds (LOD) score using the GeneHunter program (version 2.1).11 in the easyLINKAGE Software package (http://nephrologie. uniklinikum-leipzig.de/nephrologie.site,postext,easylinkage,a_ id,797.html). For linkage analysis, autosomal dominant inheritance was assumed, with a disease-allele frequency of 0.0001 and 95% penetrance. Clinical Evaluation 1 Pedigree of a family with individuals affected by POAG. The (c.109G>A; p.Val37Met) variant in the TP53BP2 gene is indicated with M2, the variant (c.305G >A; p.Arg102His) in the MAPKAPK2 gene is indicated with M1, and the wild type allele is indicated with WT for both genes together with microsatellite markers haplotype. All affected individuals carry both variants heterozygously, while the unaffected individuals do not carry the variant Fig. 1 Pedigree of a family with individuals affected by POAG. The (c.109G>A; p.Val37Met) variant in the TP53BP2 gene is indicated with M2, the variant (c.305G >A; p.Arg102His) in the MAPKAPK2 gene is indicated with M1, and the wild type allele is indicated with WT for both Clinical Evaluation A large family with eight individuals affected by POAG and one unaffected individual was ascertained. Affected and unaf- fected individuals were examined by an ophthalmologist at the Radboud University Medical Center in Nijmegen, the Netherlands. The study adhered to the principles of the Declaration of Helsinki and was approved by the Institutional Ethical Review Board of the Radboud University Medical Center in Nijmegen, the Netherlands. Blood samples were drawn from the family members, after obtaining written in- formed consent. DNA was extracted using standard methods. q y To evaluate the pathogenicity of the variants obtained from WES, bioinformatic analysis was performed using the PhyloP (nucleotide conservation in various species) and Grantham scores (difference in physicochemical nature of amino acid substitutions). Functional predictions were performed using publically available tools, i.e., SIFT (http://sift.bii.a-star. edu.sg/ Sorting Intolerant from Tolerant), MutationTaster (http://www.mutationtaster.org/), and PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/ Polymorphism Phenotyping). Confirmation of variants and segregation analysis in all available family members was performed using PCR and Sanger sequencing. Sequencing was performed using the Big Dye Terminator Cycle Sequencing- Ready Reaction Kit (Applied Biosystems) on a 3730 DNA automated sequencer (Applied Biosystems, Foster City, CA, USA) using standard protocols. Segregating variants were an- alyzed in 180 population-matched controls by restriction frag- ment length analysis. Clinical characterization of the affected individuals includ- ed slit-lamp examination for iris diaphany, funduscopy, and IOP measurement with Goldmann applanation tonometry. Assessment of visual field defects was performed with a Humphrey Visual Field Analyzer (Carl Zeiss Humphrey Systems, Dublin, CA, USA). The decisions about glaucomatous damage on visual fields were based on the di- agnostic criteria of the Hodapp et al. classification [24]. Evaluation of the anterior chamber angle was performed by gonioscopy, and corneal thickness was calculated by ultra- sound pachymetry. In addition, a morphometric analysis of 1389 Mol Neurobiol (2018) 55:1387–1395 Fig. 1 Pedigree of a family with individuals affected by POAG. The (c.109G>A; p.Val37Met) variant in the TP53BP2 gene is indicated with M2, the variant (c.305G >A; p.Arg102His) in the MAPKAPK2 gene is indicated with M1, and the wild type allele is indicated with WT for both genes together with microsatellite markers haplotype. All affected individuals carry both variants heterozygously, while the unaffected individuals do not carry the variant genes together with microsatellite markers haplotype. All affected individuals carry both variants heterozygously, while the unaffected individuals do not carry the variant Fig. Mutation Detection In Table 2, the number of variants that passed the various filtering steps per individual for the four affected individuals, as well as the variants shared by all four affected individuals is shown. The mean coverage of the WES data was 100X. Filtering for variants shared between all four affected individ- uals (III:1, III:5, III:6, and III:8) resulted in nine variants for further analysis (Table 3). Segregation analysis was performed for nine variants with a phyloP >2.7 or a Grantham score >80 (Table 3). Two novel heterozygous variants in the TP53BP2 and MAPKAPK2 genes were found to be segregating with the disease in the family (Fig. 1). The variant in the TP53BP2 gene (c.109G>A; p.Val37Met) was predicted to be deleterious by SIFT, probably damaging by PolyPhen-2 and disease- Clinical Evaluation Table 1 provides detailed clinical information of the eight affected family members diagnosed with POAG. All individ- uals had open drainage angles on gonioscopy (at least Shaffer Mol Neurobiol (2018) 55:1387–1395 1390 Table 1 Clinical features of patients with primary open angle glaucoma with the TP53BP2 mutation Patient number Gender Age at participation Age at diagnosis Presenting IOP Laterality Iris diaphany Filtering surgery Visual field loss at HFAa MRA grade at HRTb Presenting MD (dB) on Humphrey perimetry* Presenting CPSD (dB) on Humphrey perimetry* III:1 Female 78 49 13 Bilateral No Yes In both eyes Outside normal limits ODS −30.49 4.05 III:2 Female 66 53 15 Bilateral Yes Yes In both eyes Borderline OD/outside normal limits OS −24.58 11.00 III:3 Female 74 ? 16 Bilateral suspect No No OD Outside normal limits OD/borderline OS −3.90 3.05 III:4 Male 71 54 11 Bilateral No Yes In both eyes Outside normal limits ODS −30.69 4.00 III:5 Female 70 54 9 Bilateral Yes Yes In both eyes Outside normal limits ODS −31.10 3.11 III:6 Female 68 50 14 Bilateral No No In both eyes Borderline OD/outside normal limits OS −15.82 12.87 III:7 Male 76 61 15 Bilateral No Yes In both eyes Outside normal limits ODS −17.44 11.52 III:9 Female 79 63 13 Bilateral No No In both eyes Outside normal limits ODS −9.99 8.02 Mean ± SD 72.7 ± 4.4 54.8 ± 4.8 13.2 ± 2.2 IOP as measured by applanation tonometry in both eyes, presence of typical glaucomatous optic neuropathy with compatible visual field loss, open drainage angels on gonioscopy, and the absence of a secondary cause for glaucomatous optic neuropathy IOP intraocular pressure, Presenting IOP highest IOP of two measurements in both eyes, HFA Humphrey Field Analyzer, MRA Moorfields regression analysis, HRT Heidelberg Retina Tomograph II, SD standard deviation, OD right eye, OS left eye, ODS both eyes *Of worst affected visual field a Visual filed loss on HFA, as defined by the HODAPP classification [24] b MRA has three grades: “within normal limits,” “borderline,” and “outside normal limits” Mol Neurobiol (2018) 55:1387–1395 1391 ype nucleo- d the amino mong verte- ed with the carry the disease haplotype that includes both genetic variants, which indicates that both variants are in the same linkage interval and are in cis configuration. Discussion In the current study, we used WES to identify the genetic defect in a large family with POAG. We identified potentially pathogenic variants in the TP53BP2 (c.109G>A; p.Val37Met) and MAPKAPK2 (c.305G>A; p.Arg102His) genes. A disease haplotype carrying both variants segregates with the disease, with a maximum LOD score of 2.4. Variants in both TP53BP2 and MAPKAPK2 segregate with the disease, since both of them are on the same haplotype. Therefore, segregation of the MAPKAPK2 variant with the disease is a coincidental finding. Since the inheritance pattern was not obvious from the pedigree, we considered both recessive and dominant in- heritance patterns during the variant prioritization process. However, no homozygous or compound heterozygous vari- ants were identified among the putative pathogenic variants that were shared between affected individuals, and this thus does not support recessive inheritance. Since phenotype data and DNA were not available from the deceased parents, we can only speculate that the inheritance pattern may be autoso- mal dominant. Clinical Evaluation carry the disease haplotype that includes both genetic variants, which indicates that both variants are in the same linkage interval and are in cis configuration. causing by Mutation Taster (Table 3). The wild type nucleo- tide was highly conserved (phyloP score 4.08), and the amino acid residue p.Val37 was completely conserved among verte- brates (Fig. 3a). The second variant that segregated with the disease in the family was identified in the MAPKAPK2 gene (c.305G>A; p.Arg102His) (Fig. 1). This variant was also pre- dicted to be deleterious by SIFT, probably damaging by PolyPhen and disease-causing by Mutation Taster (Table 3). The wild type nucleotide was highly conserved (phyloP score 5.69), and the amino acid residue p.Arg102 is completely conserved among vertebrates (Fig. 3b). Both variants were not identified in 180 population-matched controls and were not present in the Exome Variant Server, dbSNP132 and 1000 genomes. causing by Mutation Taster (Table 3). The wild type nucleo- tide was highly conserved (phyloP score 4.08), and the amino acid residue p.Val37 was completely conserved among verte- brates (Fig. 3a). The second variant that segregated with the disease in the family was identified in the MAPKAPK2 gene (c.305G>A; p.Arg102His) (Fig. 1). This variant was also pre- dicted to be deleterious by SIFT, probably damaging by PolyPhen and disease-causing by Mutation Taster (Table 3). The wild type nucleotide was highly conserved (phyloP score 5.69), and the amino acid residue p.Arg102 is completely conserved among vertebrates (Fig. 3b). Both variants were not identified in 180 population-matched controls and were not present in the Exome Variant Server, dbSNP132 and 1000 genomes. Linkage Analysis Twelve microsatellite markers were used for linkage analysis of the genomic region encompassing the MAPKAPK2 and TP53BP2 genes, but only four markers were informative. A multipoint LOD score of 2.48 was obtained for markers D1S2655 and D1S2891, which is suggestive of linkage and is in accordance with the maximum LOD score that can be achieved considering the structure of the pedigree. The disease-associated haplotype encompasses markers D1S2655, the MAPKAPK2 variant, D1S2891, D1S2629, D1S229, and the TP53BP2 variant. All affected individuals Table 2 Number of variants identified per individual and shared between four affected individuals Filtration steps Individual 1 Individual 2 Individual 3 Individual 4 Variants shared by all 4 individuals Total number of variants 45.755 47.697 45.417 41.943 21.447 SNP frequency <0.5 28.789 30.381 28.614 26.252 9.065 In-house database frequency <0.5 2.152 2.529 2.503 2.373 55 Exonic and canonical splice site variants 796 873 961 987 33 Nonsynonmous variants 535 620 682 696 18 Grantham score >80 248 268 302 323 4 Phylop >2.7 22 31 24 22 9 Table 2 Number of variants identified per individual and shared between four affected individuals Mol Neurobiol (2018) 55:1387–1395 1392 Table 3 Rare variants shared by four affected individuals and segregation analysis Gene ID Protein isoform cDNA position Amino acid position phyloP Segregation Grantham score SIFT Mutation Taster PolyPhen-2 TP53BP2 NM_001031685 109C>T p.Val37Met 4.135 Yes 21 Deleterious Disease causing Probably damaging MAPKAPK2 NM_032960 305G>A p.Arg102His 5.691 Yes 29 Deleterious Disease causing Probably damaging TGFBI NM_000358 895G>A p.Asp299Asn 5.884 No 23 Tolerated Disease causing Probably damaging ADSSL1 NM_199165 578C>T p.Ser193Phe 5.657 No 155 Deleterious Disease causing Probably damaging NCEH1 NM_001146276 142C>T p.Ala48Thr 5.305 No 58 Tolerated Disease causing Probably damaging RNF157 NM_052916 1913C>G p.Cys638Ser 5.254 No 112 Tolerated Disease causing Probably damaging PHC3 NM_024947 1840C>T p.Glu614Lys 3.961 No 56 Tolerated Disease causing Possibly damaging SLITRK3 NM_014926 2581G>A p.Arg861Cys 3.886 No 180 Deleterious Disease causing Possibly damaging RYR2 NM_001035 8162 T>C p.Ile2721Thr 3.683 No 89 Deleterious Disease causing Possibly damaging Table 3 Rare variants shared by four affected individuals and segregation analysis Both ASPP1 and ASPP2 are proapoptotic proteins involved in the regulation of the apoptosis and are encoded by the TP53BP2, and iASPP is encoded by PPP1R13B genes, TP53BP2 encodes a member of the ASPP (apoptosis- stimulating protein of p53) family of p53-interacting proteins comprised of three members: ASPP1, ASPP2, and iASPP. Fig. Linkage Analysis Taken together, these studies support the involvement of the TP53BP2 gene in glaucoma and suggest that the genetic variant identified by WES in the large POAG family may be relevant to the disease. The second variant that segregates with the disease in the family was identified in MAPK-activated protein kinase 2 (MAPKAPK2, also known as MK2), which is one of the downstream targets of p38 MAPK. The Ocular Tissue Database (OTDB, http://genome.uiowa.edu/otdb/) demonstrates a minimal expression in the eye for MAPKAPK2 in contrast to TP53BP2. Therefore, TP53BP2 gene seems to be the strongest candidate to be associated with the disease in this particular family. In glaucoma cell death of the post-mitotic neurons, i.e., RGCs, occurs due to an increased rate of apoptosis. The ASPP proteins are involved in the regulation of apoptosis by activating p53. The expression of ASPP2 affects the DNA binding activity of p53 on the Bax promoter or down- stream targets involved in apoptosis [32]. In the current study, we speculate that binding between ASPP2 and p53 may be affected by an amino acid variant (c.109G>A; p.Val37Met) in the ASPP2 protein, which leads to the increased accumula- tion of p53, followed by an increase in cell death of the RGCs, subsequently leading to glaucoma. Previously, it has been reported that normal ASSP2 protein is required for the activation of apoptosis in a controlled manner. It was ob- served that the blockade of the ASPP-p53 pathway is impor- tant for the survival of neurons after axonal injury [33]. The results of Wilson et al. are further supported by a recent study in an in vivo model of acute optic nerve damage, in which it was shown that iASPP is expressed by injured RGCs and short interference RNA (siRNA)-induced iASPP knockdown exacerbates RGC death, while RGC survival was enhanced by adeno-associated virus (AAV)-mediated iASPP expres- sion. Increased expression of iASPP in RGCs downregulates p53 activity and blocks the expression of proapoptotic targets PUMA and Fas/CD95 [34]. Since iASPP is an inhibitor of p53-mediated apoptosis, it is possible that the mutation in the ASPP2 protein influences the expression of iASPP. Subsequently, it would not be able to perform actively in the survival of retinal ganglion cells due to apoptosis. Linkage Analysis 3 a Evolutionary conservation of valine at position 37 is represented by alignment of the human TP53BP2 (ASPP2) protein sequence to orthologous protein sequences of various vertebrate species. b Evolutionary conservation of arginine at position 102 is represented by alignment of the human MAPKAPK2 protein sequence to orthologous protein sequences of various vertebrate species Mol Neurobiol (2018) 55:1387–1395 1393 respectively [27]. ASPP2 is well known for its binding and activation of the apoptotic function of p53, p63, and p73 by selectively enhancing their DNA-binding and transactivation activities on proapoptotic genes such as BAX and PIG3 [27, 28]. Apoptosis is tightly regulated during normal develop- ment. In the case of abnormal regulation, it mediates cell death of neuronal cells in neurodegenerative diseases such as Alzheimer’s disease or Parkinson’s disease or death of RGCs in glaucoma due to overexpression of p53 [29–31]. Recently, the role of ASPP1 and ASPP2 proteins in neuronal apoptosis and their involvement in the regulation of adult RGCs after injury have been investigated. The results indicat- ed that both ASPP1 and ASPP2 are highly expressed in RGCs and contribute to p53-dependent death of RGCs. respectively [27]. ASPP2 is well known for its binding and activation of the apoptotic function of p53, p63, and p73 by selectively enhancing their DNA-binding and transactivation activities on proapoptotic genes such as BAX and PIG3 [27, 28]. Apoptosis is tightly regulated during normal develop- ment. In the case of abnormal regulation, it mediates cell death of neuronal cells in neurodegenerative diseases such as Alzheimer’s disease or Parkinson’s disease or death of RGCs in glaucoma due to overexpression of p53 [29–31]. Recently, the role of ASPP1 and ASPP2 proteins in neuronal apoptosis and their involvement in the regulation of adult RGCs after injury have been investigated. The results indicat- ed that both ASPP1 and ASPP2 are highly expressed in RGCs and contribute to p53-dependent death of RGCs. cytokines and interleukins, which mediate apoptosis of retinal ganglion cells in glaucoma. In another recent study, the neu- roprotective effect of minocycline in rats with glaucoma was evaluated, and downregulation of TP53BP2 was observed up- on treatment [36]. Minocycline is a tetracycline with anti- inflammatory and anti-apoptotic properties. In previous stud- ies, it has been shown that minocycline significantly delays RGC death in models of experimental glaucoma and optic nerve transaction [37]. Linkage Analysis In conclusion, through WES in a large POAG family, we identified a novel genetic variant in the TP53BP2 gene, which is predicted to be pathogenic and affects a highly conserved amino acid residue. Since it has been demonstrated that the gene regulates apoptosis in RGCs and is downregulated upon minocycline treatment in a glaucoma rat model, TP53BP2 may represent a novel gene associated with POAG. Additional screening of the TP53BP2 gene in other familial and sporadic patients with POAG from different populations is required to confirm its involvement in the disease. Acknowledgements This study was supported by the Stichting Blindenhulp, a Shaffer grant from the Glaucoma Research Foundation and the following foundations: Glaucoomfonds, Oogfonds, and Algemene Nederlandse Vereniging ter Voorkoming van Blindheid, which contributed through UitZicht. Compliance with Ethical Standards The study adhered to the princi- ples of the Declaration of Helsinki and was approved by the Institutional Ethical Review Board of the Radboud University Medical Center in Nijmegen, the Netherlands. 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https://openalex.org/W2994314550	https://gfzpublic.gfz-potsdam.de/pubman/item/item_4875892_3/component/file_4875895/4875892.pdf	English	null	Applying Rare Earth Elements, Uranium, and<sup>87</sup>Sr/<sup>86</sup>Sr to Disentangle Structurally Forced Confluence of Regional Groundwater Resources: The Case of the Lower Yarmouk Gorge	Geofluids	2,019	cc-by	14,251	Hindawi Geoﬂuids Volume 2019, Article ID 6727681, 21 pages https://doi.org/10.1155/2019/6727681 Christian Siebert ,1 Peter Möller,2 Fabien Magri ,3,4 Eyal Shalev ,5 Eliahu Rosenthal ,6 Marwan Al-Raggad,7 and Tino Rödiger8 The Inter-Islamic Network on Water Resources Development and Management (NWRDAM), Amman, Jordan Dept. of Computational Hydrosystems, Helmholtz Centre for Environmental Research (UFZ), Leipzig, Germany 7The Inter-Islamic Network on Water Resources Development and Management (NWRDAM), Amman, Jordan 8Dept. of Computational Hydrosystems, Helmholtz Centre for Environmental Research (UFZ), Leipzig, Germany Correspondence should be addressed to Christian Siebert; christian.siebert@ufz.de Received 24 May 2019; Revised 23 September 2019; Accepted 3 October 2019; Published 3 December 2019 Guest Editor: Tanguy Robert Copyright © 2019 Christian Siebert et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The conjoint discussion of tectonic features, correlations of element concentrations, δ18O, δD, and 87Sr/86Sr of groundwater leads to new insight into sources of groundwater, their ﬂow patterns, and salinization in the Yarmouk Basin. The sources of groundwater are precipitation inﬁltrating into basaltic rock or limestone aquifers. Leaching of relic brines and dissolution of gypsum and calcite from the limestone host rocks generate enhanced salinity in groundwater in diﬀerent degrees. High U(VI) suggests leaching of U from phosphorite-rich Upper Cretaceous B2 formation. Both very low U(VI) and speciﬁc rare earth element including yttrium (REY) distribution patterns indicate interaction with ferric oxyhydroxides formed during weathering of widespread alkali olivine basalts in the catchment area. REY patterns of groundwater generated in basaltic aquifers are modiﬁed by interaction with underlying limestones. Repeated sampling over 18 years revealed that the ﬂow paths towards certain wells of groundwater varied as documented by changes in concentrations of dissolved species and REY patterns and U(VI) contents. In the Yarmouk Gorge, groundwater with basaltic REY patterns but high U(VI) and low Sr2+ and intermediate sulfate concentrations mainly ascends in artesian wells tapping a buried ﬂower structure fault system crossing the trend of the gorge. Siebert, C., Möller, P., Magri, F., Shalev, E., Rosenthal, E., Al-Raggad, M., Rödiger, T. (2019): Applying Rare Earth Elements, Uranium, and 87Sr/86Sr to Disentangle Structurally Forced Confluence of Regional Groundwater Resources: The Case of the Lower Yarmouk Gorge. - Geofluids, 2019. DOI: http://doi.org/10.1155/2019/6727681 1. Introduction yttrium (henceforth termed REY), it was ascertained that thermal groundwater discharging through springs in the LYG is inﬁltrated in basaltic regions of the Hauran plateau, Syria [1]. Parts of these waters are mixed in various propor- tions with limestone water from Ajloun. The hot waters of Hamat Gader and Meizar get salinized by either mixing with relic seawater evaporation brines [2, 3] or leaching of evapo- rites. The recent study is based on 18 years of repeated sampling of wells and springs and reveals signiﬁcant varia- tions in REY patterns and element concentrations suggesting Since Roman times, the hot springs of Hamat Gader (HG), Israel, and Ain Himma, Jordan, in the Lower Yarmouk Gorge (LYG) were used for health care (Figure 1). At present, only Ein Balsam at HG is publically in use. Hydrogeological and hydrochemical studies of springs and well waters in the gorge reveal that groundwater of widely diﬀerent composition dis- charges at short distances [1]. By major and minor elements and distribution patterns of rare earth elements including Geoﬂuids 2 Sampling location Settlement/city Drainage Lake/dam Border Basalt (Cenozoic) Quaternary WC (Miocene) WSC (B5) URC (B4) MCM (B3) AHP (B2b) ASL (B2a) WUG (B1) WSL (A7) F/H/S undiff. (A3-A6) L-Cretac./Kurnub (K) Tayyasir basalt Intrusions Mesozoic J3-J7 J2 J1 Sampling location Sampling location X (UTM) Mukheibeh 1 753119 753241 753212 747753 753018 754257 755495 756312 753267 753845 754268 752652 752706 752725 749705 749859 750014 750190 750429 751661 763884 748804 756255 3622154 M1 3622342 M2 3622331 M4 3618570 M5 3622417 M6 3623142 M7 3624134 M8 3624296 M9 3622856 M10 3622588 M11 3623212 M13 3625884 Me1 3622894 Me2 3622926 Me2 3619324 EB 3619141 EM 3619198 EB 3619926 EB 3619424 EB 3621700 AH 3648639 3653081 3624301 YR Groundwater wells Mukheibeh 2 Mukheibeh 4 Mukheibeh 5 Mukheibeh 6 Mukheibeh 7 Mukheibeh 8 Mukheibeh 9 Mukheibeh 10 Mukheibeh 11 Mukheibeh 13 Meizer 1 Meizer 2 Meizer 3 Ein Balsam Ein Makla Ein Reach Ein Sahina Ain Saraya Spring (Himma) Ain Himma Umm Abu ad Danar Amphy spring River Yarmouk River Spring (Golan Hieghts) Spring (Hamat Gader) Y (UTM) Short Settlement/city Drainage Lake/dam Border Basalt (Cenozoic) Quaternary WC (Miocene) WSC (B5) URC (B4) MCM (B3) AHP (B2b) ASL (B2a) WUG (B1) WSL (A7) F/H/S undiff. Figure 1: Overview of the study area, showing geological background (a) and sampling locations (b) including structural features recently introduced by Inbar et al. [15] and Sneh (unpublished). 1. Introduction (A3-A6) L-Cretac./Kurnub (K) Tayyasir basalt Intrusions Mesozoic J3-J7 J2 J1 1: Overview of the study area, showing geological background (a) and sampling locations (b) including structural features ed by Inbar et al. [15] and Sneh (unpublished). Sampling location X (UTM) Mukheibeh 1 753119 753241 753212 747753 753018 754257 755495 756312 753267 753845 754268 752652 752706 752725 749705 749859 750014 750190 750429 751661 763884 748804 756255 3622154 M1 3622342 M2 3622331 M4 3618570 M5 3622417 M6 3623142 M7 3624134 M8 3624296 M9 3622856 M10 3622588 M11 3623212 M13 3625884 Me1 3622894 Me2 3622926 Me2 3619324 EB 3619141 EM 3619198 EB 3619926 EB 3619424 EB 3621700 AH 3648639 3653081 3624301 YR Groundwater wells Mukheibeh 2 Mukheibeh 4 Mukheibeh 5 Mukheibeh 6 Mukheibeh 7 Mukheibeh 8 Mukheibeh 9 Mukheibeh 10 Mukheibeh 11 Mukheibeh 13 Meizer 1 Meizer 2 Meizer 3 Ein Balsam Ein Makla Ein Reach Ein Sahina Ain Saraya Spring (Himma) Ain Himma Umm Abu ad Danar Amphy spring River Yarmouk River Spring (Golan Hieghts) Spring (Hamat Gader) Y (UTM) Short Figure 1: Overview of the study area, showing geological background (a) and sampling locations (b) including structural features recently introduced by Inbar et al. [15] and Sneh (unpublished). 3 Geoﬂuids variation of ﬂow paths and associated interactions with host k d l hi id l b i d it [4–7]. The gorge seemingly acts as a complex conduit- b i t l hi h d t f th G l i (a) (b) Deep seated brine Intrusive bodies along LYG Cenozoic graben fill w/r-interaction with intrusiva Conductive fault Low-permeable zone Groundwater flow Valley floor (ground) Drilling Spring Figure 2: Schematic geological cross sections (not to scale). Section (a) starts in the Lower Jordan Valley, continues through the LYG, and branches into the Hauran NE-ward, while section (b) cuts from the Ajloun northward across the LYG and the Golan Heights into Mt. Hermon. The map shows the location of cross sections in red. (a) Deep seated brine Intrusive bodies along LYG Cenozoic graben fill w/r-interaction with intrusiva Conductive fault Low-permeable zone Groundwater flow Valley floor (ground) Drilling Spring (a) (b) (b) Figure 2: Schematic geological cross sections (not to scale). Section (a) starts in the Lower Jordan Valley, continues through the LYG, and branches into the Hauran NE-ward, while section (b) cuts from the Ajloun northward across the LYG and the Golan Heights into Mt. Hermon. 2. Hydrogeological Setting The groundwater in the phreatic and shallow basaltic aquifer mainly follows the morphology. Within the Golan Heights, it ﬂows W- and SW-ward towards the Hula Valley, the Lake Tiberias, and the LYG [28, 33]. In the east, a subter- ranean meridional ridge forms a water divide against the Hauran [19] (Figure 2(a)). The thin lava ﬂows east of the water divide, hosts only modest amounts of groundwater, and discharges locally into incised wadis, e.g., the Raqqad. The basaltic cover of the Hauran plateau is mainly recharged at the elevated southeastern ﬂanks of the Hermon Massif and western piedmont of Jebel Druze, from where the groundwa- ter ﬂows SE- and W-ward, respectively. The groundwater most probably converges in the central part of the Hauran and ﬂows from there SW-ward towards the LYG. There, the observed groundwater of this study discharges either nat- urally at the valley ﬂoor through springs in Hamat Gader, Suraya, and Himma or artiﬁcially through the (mostly) arte- sian wells of Mukheibeh and Meizar, located in the ﬂanks of the gorge, either north (Meizar wells) or south (Mukheibeh wells) (Figure 1(b)). Geographically, the Yarmouk drainage basin comprises (i) the volcanic Hauran plateau and the western ﬂank of the Jebel Druze volcano [18], (ii) the southern and southeastern slopes of Hermon, (iii) the Golan Heights with numerous volcanic cones, (iv) the northern plunges of the Ajloun anti- cline, and (v) the Azraq-Dhuleil basin ENE of Ajloun (Figure 1(a)). The Mediterranean climate in the Yarmouk basin causes rainy and cool winters and hot and dry summers. The distinct diﬀerences in altitude and the distance from the Mediterranean force strong gradients in annual precipita- tion. Highest values (up to 1300 mm/a) fall in the Hermon Massif and the highest parts of Jebel Druze; medium elevated regions such as the Hauran and Ajloun plateaus and the Golan Heights receive 600-800 mm/a, while the low-lying LYG and the xeric region SE of the surface drainage basin receive <500 mm/a only (e.g., [18–21]). The resulting recharge fractions are calculated to range from 0.06 to 0.1 [20, 22–24]. In the south of the Yarmouk River, geological formations dip NW-ward (Figure 2). Here, the oldest hydrogeological relevant formations comprise the highly karstiﬁed lime- and dolostones of the Upper Cretaceous A7 aquifer and the over- laying heavily fractured siliciﬁed limestones of the Eocene B2 aquifer, altogether forming the 160 m thick regional A7/B2 aquifer system (Figure 3). 1. Introduction The map shows the location of cross sections in red. variation of ﬂow paths and associated interactions with host rocks and leaching residual brines and evaporites. [4–7]. The gorge seemingly acts as a complex conduit- barrier system, along which groundwater from the Golan in the north and Ajloun in the south converges and drains towards the Lower Jordan Valley (Figure 2). Flow paths in the underground of the gorge possibly occur along faults ori- ented perpendicular to the major axis of the gorge [5, 6, 8]. The chemical and isotopic composition of the large amounts of fresh artesian groundwater produced in the Jor- danian Mukheibeh well ﬁeld contrasts with that of the saline groundwater in the Meizar wells and the springs of Hamat Gader. This gave rise to the conceptual model that the LYG is the surface expression of a fault zone, preventing trans- boundary ﬂow [1]. 2D and 3D modelling supported that con- cept of continuous groundwater aquifers with the absence of transboundary groundwater exchange due to a zone of high hydraulic anisotropy underneath the gorge’s centerline Based on stratigraphic data [9, 10], topographic data, deep seismic survey data [11, 12], shallow fault mapping [13], and thickness irregularities of the Turonian and Seno- nian sequences in the study area [14–17] support the occur- rence of strike-slip ﬂower structure faults along and across the gorge creating a series of structural fault blocks and 4 Geoﬂuids numerous buried faults at close proximity to the Dead Sea Transform Fault (DSTF) (Figure 1). Hermon, and eastward by Wadi Raqqad. The entire Golan Heights is unconformably overlain by Plio-Pleistocene cover basalts, which continue E- and SE-ward into the Hauran pla- teau, Jebel Druze, and Azraq-Dhuleil Basin and form the uppermost supraregional aquifer in the area [26, 27]. Within the Golan Heights, the thickness of the basalts varies with more than 750 m in the central part and less than 50 m along the LYG (Figure 2(b)) and B3 layers form the impervious base of the basaltic aquifer [28–30]. However, the basaltic aquifer is connected to underlying aquifers at certain loca- tions [31], either where B3 was already eroded or where structurally prominent features of post-Pliocene age cut the formations [12, 28, 32, 33]. 2. Hydrogeological Setting This system becomes eﬃciently conﬁned due to its descent and the appearance of the covering B3 aquiclude. On top of the southern ﬂank of the LYG, remnants of the B4 sequence form a local limestone aquifer. 1. Introduction An aeromagnetic survey in N Jordan revealed a SW-NE lineament branching from the DSTF towards Hamat Gader in the LYG [34], which was later proven to be a fault by geological mapping (Sneh et al., unpublished) (Figure 1(b)). Applying REY distribution patterns, U(VI), 87Sr/86Sr, and water isotopes in a new, complete, and synchronous set of sampled spring and well waters in 2016, we aim for joint discussion of hydrochemical and geological features to improve the knowledge of the sources of groundwater and of their ﬂow paths. After the introduction (Section 1), we will present the hydrogeological setting of the studied area (Section 2), the sample acquisition and the techniques used to analyze them (Section 3), the results on major and minor element, particularly on REY and U(VI), and Sr isotopes (Section 4), and a detailed discussion (Section 5). Section 6 concludes this study. 3. Analytical Procedures The elements Ca2+, Mg2+, U(VI), and REY are determined by ICP-MS (Elan DRC-e). K+ and Na+ were analyzed by ICP-AES (Spectro Arcos) using matrix-adjusted standard solution for calibration. Cl-, Br-, and SO4 2- are determined with Dionex ICS (AS18 column). The alkalinity is titrated to pH 4.3 with H2SO4 and given as HCO3 -. p 2 4 g 3 To determine REY and U(VI), preconcentration is required. Therefore, about 4 l of sample is ﬁltered in the ﬁeld by using a peristaltic pump coupled to 0.2 μm ﬁlters (Sartorius, Germany). The samples are acidiﬁed by subboiled (index sbb) HCl, and 1 ml of Tm spike solution is added. At the same day, the samples are adjusted to pH = 2 using HClsbb and subsequently passed through preconditioned C18 Sep-Pak cartridges (Waters, USA), loaded with an ethyl- hexyl phosphate (Merck, Germany) liquid ion exchanger, at a rate of 1 l/h. Thereafter, each cartridge is washed with 50 ml of 0.01 M HClsbb and subsequently eluted with 40 ml of 6 M HClsbb at a rate of 3 ml/min. The eluates are evaporated to incipient dryness, and the residues are dissolved in 1 ml of All formations older than B4 continue in the under- ground of the Golan Heights syncline before they partly resurface in the foothills of the Hermon anticline [25]. Underneath the Golan Heights, Jurassic limestones form the base of the formations before they become uplifted in the Hermon anticline in the north (Figures 1(a) and 2(b)). Since the drainage basin extends into three nations with dif- ferent geological terminologies. Figure 3 compiles the rele- vant parts of the stratigraphic columns for the entire region. Morphologically, the Golan Heights is restricted south- ward by the LYG, westward by the Hula Valley and Lake Tiberias, northward by Wadi Sa’ar at the foothills of Mt. 5 Geoﬂuids System Age Period GH/Mt.H Ajloun DB/H GH/Mt.H Ajloun DB/H Golan/Mt. Hermon Ajloun Quaternary Quaternary Yarmouk Basalt Alkaline-olivine basalt Gravel, gipseous marl, siltite, clay Cover basalt Sandstone, siltite Bira/Gesher Silicified limest., dolomite Alternation congl., marl, limest. 3. Analytical Procedures Hordos Waqqas conglomerate (WC) - Jordan Valley Conglomerates in siltand clay matrice Marl, sand Limestone Jeribe Chilou Lower basalt Alkali olivine basalt Conglomerates in limy matrix Oligocene Susita/Fiq n1 Marlst., sandy dolomite Marl, clay, conglomerates Jaddala Wadi Shallalah chalk (WSC) (B5) e5 Chalk, bituminous Thick banked limestone, upper part chalky, marly Avedat Maresha/ Adulam Umm Rijam (URC) (B4) e4 Marl, chalk, limestone chalky limest., chert beds Massive limestone Upper Maastricht- Paleocene Kermev Bardeh Taqiye Muwaqqar chalk (MCM) (B3) e1-e3 Micritic limest., bituminous (oil shale) Alternating chak, marl Masstrichtian Ghareb Al Hasa phosphorite (AHP) (B2-b) m1, m2 Calcareous, phosphorite beds, limest., chalk, marl Alternating chalk, marl, limest., cherts Campanian Mishash Amman silicified limestone (ASL) (B2a) Limestone., dolomite, chalky, phosphate, chert Santonian Menuha Wadi Umm Ghudran (WUG) (B1) Massive chalk, limest., phosphatic sandst., chert Turonian Wadi Es Sir Limest. (WSL) (A7) t2 Dolomitic limest., sandst., cherts Banked limestone F/H/S-undifferentiated Marl and gypsum Shueib (S) (A5-6) Siltstone, marly limest., dolomite Dolomitic limestone Dolomitic limestone, karstic limestone Karstic limest., chert Fuheis (F) (A3) Chalk beds, thin dolost. Calcareous siltst., marly lime-/ dolost., chalk beds Glauconitic sandstone Limest., dolomite Albian Kurnub Yagur ab Dolomite, limestone Sandstone Marly limestone Yakhini Sandstone Banias, Tayassir basalt Basalts Clayey sandstone, limestone intercalated Barremian Hauterivian- Berriasian Aarda (K1) J6-J7 (Nahal Saar) Limestone Sandstone, siltstone, limestones Limestone J5 (Kidod) Marl, shale Marl Dogger J4 (Hermon/ Zohar) j2-j4 Limestone Dolomitic limestone at base, limestone on top Lias J1-J3 j1 Dolomite, limestone Marl lignite, dolomite, sand- and limestone GH = Golan Heights, Mt.H = Mount Hermon, DB = Damascus Basin, H = Hauran; Syrian geology taken from Brew et al. [26] and Wolfart [27] Alkaline-olivine basalt Jurassic Cretaceous Paleogene Neogene Formation Pliocene Miocene Mt. Scopus Judea Bina Aptian Malm Arad Zarqa Azab Hydrogeological properties Group basalts Banked limestone, partly dolomite Chalk, marl Plateau Basalts Damascus Basin/Hauran a1-a3 j5-j7 c S n3 n2 t1 Kefar Giladi Limestone, dolomite, marly limest. Ajloun Marly limestone, bituminous, chert, phosphorite Eocene Belqa Cenomanian- Turonian Cenomanian Saknin Hummar (H) (A4) Kurnub Deir Hanna Na’ur (NL) (A1-2) Subeihi (K2) Judea Soukhne Figure 3: Stratigraphic table of the hydrogeological formation in the Yarmouk Basin. Figure 3: Stratigraphic table of the hydrogeological formation in the Yarmouk Basin. 4. Results 5 M HNO3 sbb (Merck, Germany) and transferred into 10ml volumetric ﬂasks. 1 ml of spike solution is added which is used, if necessary, for drift corrections of the response factors during the ICP-MS measurements. Depending on the sampling location, the results are classiﬁed in the following way: Mukheibeh well ﬁeld (M1-M13), Ain Himma (AH), Hamat Gader springs (HG), Meizar wells (Me1-Me3), and the Yarmouk River (YR). Sampling loca- tions, which have been repeatedly sampled, are indicated by the year of sampling given in parentheses. The Hebrew and Arabic term of springs is transliterated as Ein and Ain, respectively. Stable isotopes of oxygen and hydrogen are determined in separate ﬁltered samples (0.2 μm) using laser cavity ring- down spectroscopy (Picarro L2120-i, USA) without further treatment of the water samples. The respective analytical pre- cision is ±0.1‰ and ±0.8‰ for δ18O and δD, respectively. The results are reported relative to Vienna Standard Mean Ocean Water (VSMOW). Analyses of 87Sr/86Sr in water samples were performed at TUBAF, Freiberg, Germany. Samples were prepared and analyzed after Tichomirova et al. [35] by applying TIMS (Finnigan MAT 262) with an acceptable relative error of ±0.005% for 87Sr/86Sr. Sr-isotope ratios are given in respect to NBS-987. To analyze Sr2+ in basaltic rock samples, rocks have been powdered to <150 μm, pressed to pellets, and ana- lyzed applying energy-dispersive X-ray ﬂuorescence (EDXRF) (Spectro XEPOS HE 2000). Chemical and isotopic analyses are given in Tables 1–3. 4.1. Major and Minor Element Correlations with Cl-. From the low-salinity Mukheibeh clusters, two (Figures 4(a)–4(f)) or one (Figures 4(g)–4(j)) mixing lines evolve with high- salinity end members. They indicate that two end member brines occur in the study area: one is salinizing the Meizar wells and Ain Himma and the other the springs of Hamat Gader (Ein Maqla, Ein Reach, Ein Balsam, and Ain Sarayah). The Ca2+ concentration in Ain Himma switches between the two trends, probably because the access point to sample the spring water within the increasingly ruined Himma resort 6 Geoﬂuids Table 1: Compilation of groundwater analyses from the Lower Yarmouk Gorge and surrounding areas. ID Source Abbr. 4. Results X Y Sampling pH Eh Temp EC Alk Ca Mg K Sr Na Cl SO4 Br HCO3 U δ18O δ2H Date mV °C μS/cm mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l nmol/l VSMOW Groundwater wells 01-128 Mukheibeh 1 M1 (01) 753119 3622154 ####### 7 67 29.1 797 5.93 91.3 30 3.1 0.69 39 57.2 54.3 0.20 358 105.2 -5.65 -27.40 13/803 Mukheibeh 2 M2 (13) 753243 3622340 ####### 7.12 65 28.14 809 5.18 96 30 3.02 0.54 38.7 58.4 57.2 0.09 312 88.2 -5.64 -26.40 16/08 Mukheibeh 2 M2 (16) 753241 3622342 ####### 6.13 18 28.9 830 4.56 78 26 2.9 0.51 39 56.4 56.1 0.1 274 88.2 -5.38 -25.51 13/802 Mukheibeh 4 M4 (13) 753209 3622333 ####### 7.1 63 28.8 807 5.18 96 29 3 0.51 38.5 55.4 54.7 0 312 96.6 -5.26 -25.40 16/09 Mukheibeh 4 M4 (16) 753212 3622331 ####### 7.04 273 29.1 827 4.76 84 28 2.9 0.5 40 56.5 57.1 0.09 286 92.4 -5.50 -25.47 16/12 Mukheibeh 5 M5 (16) 747753 3618570 ####### 7.32 162 40.9 876 5.08 71 24 3.8 0.94 47 57.2 36.3 0.11 305 4.6 -5.16 -24.54 16/10 Mukheibeh 6 M6 (16) 753018 3622417 ####### 7.06 -40 31 667 5.08 86 29 3.4 0.66 49 73.3 59.8 0.26 306 58.8 -5.43 -26.28 16/14 Mukheibeh 7 M7 (16) 754257 3623142 ####### 7.17 -17 38.5 774 4.8 84 29 2.9 0.54 40 60.5 35.5 288 63.0 -5.58 -27.04 13/804 Mukheibeh 8 M8 (13) 755490 3624127 ####### 7.45 -123 44.9 701 4.72 68 20 5.25 1.4 53.3 64.5 27.8 0.12 283 0.0462 -6.28 -30.99 16/16 Mukheibeh 8 M8 (16) 755495 3624134 ####### 7.16 -62 44.9 723 4.1 59 20 5.1 1.3 56 66.6 12.7 5.55 246 -6.09 -30.01 16/17 Mukheibeh 9 M9 (16) 756312 3624296 ####### 7.2 -67 28.9 1157 7.6 54 29 16 5.3 120 82.6 21 0.19 459 -6.54 -32.16 16/11 Mukheibeh 10 M10 (16) 753267 3622856 ####### 6.96 -117 39 710 4.34 72 25 3.7 0.96 47 59.9 39.5 261 5.0 -5.67 -27.37 16/13 Mukheibeh 11 M11 (16) 753845 3622588 ####### 6.92 200 31.9 821 4.8 76 39 2.8 0.87 44 56.4 53.3 0.09 289 -5.34 -25.75 13/805 Mukheibeh 13 M13 (13) 754280 3623202 ####### 7.38 -76 38.5 752 5.12 81 25 3.74 0.94 43.6 59.1 24.5 0.1 307 3.7 -5.69 -27.78 16/15 Mukheibeh 13 M13 (16) 754268 3623212 ####### 7.12 -47 38.9 778 4.4 72 25 3.7 0.88 46 61.7 34 264 8.0 -5.65 -27.04 16/07 Meizar 1 Me1 752652 3625884 ####### 7.38 -79 35.2 1630 3.64 52 17 13 2.1 230 386 12.7 2.39 217 -5.98 -29.13 01-166 Meizar 2 Me2 (01) 752700 3622914 ####### 6.63 -102 60 1650 4.06 142 34.8 21.8 5.16 178 317 278 3.90 244 0.059 -5.90 -29.40 08/753 Meizar 2 Me2 (08) 752715 3622884 ####### 6.75 -106 57.1 2060 3.76 178 42.1 24.6 5.8 201 373 328 4.44 225 0.063 -6.87 -36.61 16/02 Meizar 2 Me2 (16) 752706 3622894 ####### 6.4 -77 60.6 2080 3.68 170 37 24 5.5 210 392 338 3.6 221 -6.89 -35.25 01-167 Meizar 3 Me3 (01) 752707 3622923 ####### 7.09 -129 41.8 664 5.24 65.3 20.9 4.7 1.25 48.5 61.6 4 0.20 315 0.045 -6.84 -33.00 08/752 Meizar 3 Me3 (08) 752721 3622900 ####### 6.6 -117 57.4 2090 3.72 177 42.2 24.2 5.8 200 365 306 3.97 223 0.078 -6.87 -36.61 16/01 Meizar 3 Me3 (16) 752725 3622926 ####### 6.81 -120 42.2 810 5.48 71 25 3.9 1.1 53 78.7 19.3 0.35 330 -5.85 -28.29 Springs (Hamat Gader fresh) 16/03 Ein Sahina ES 750190 3619926 ####### 7.04 454 28 844 5 87 28 3.3 0.56 44 66.5 48.9 0.2 301 19.3 -5.57 -26.13 Springs (Hamat Gader thermohaline, incl. 4. Results Ain Saraya) 16/04 Ein Balsam EB 749705 3619324 ####### 6.76 11 41.9 1600 4.8 120 34 12 3.5 130 325 133 2.92 289 4.6 -5.84 -28.53 00-107 Ein Reach ER (00) 750349 3619399 ####### 6.85 -96 38 1728 6 142 39.5 12.2 2.94 143 309 115 3.82 362 9.1 -5.84 -29.90 04-585 Ein Reach ER (04) 749985 3618816 ####### 6.81 -147 41.5 1759 5.86 134 39.5 12.6 3.44 156 345 119 4.25 353 7.9 -5.99 -29.90 16/05 Ein Reach ER (16) 750014 3619198 ####### 6.69 -76 43.4 1860 4.84 130 37 14 4.2 160 376 128 3.37 291 7.6 -5.93 -28.59 00-108 Ein Makla EM (00) 749909 3619091 ####### 6.64 -131 47.3 2190 5.64 178 44.2 18 5.21 212 488 158 6.55 340 5.8 -6.00 -31.60 04-586 Ein Makla EM (04) 749811 3618793 ####### 6.73 -167 49.6 2160 5.42 152 42 17.2 5.19 209 465 151 5.98 327 5.7 -6.15 -31.30 16/06 Ein Makla EM (16) 749859 3619141 ####### 6.59 -116 49.5 2160 3.64 150 39 17 5.5 210 475 150 4.51 218 5.9 -6.08 -29.59 16/20 Ain Saraya AS 750429 3619424 ####### 6.83 -16 38.3 1655 4.54 130 38 12 3.8 160 342 120 3.02 273 8.0 -5.85 -28.56 7 Geoﬂuids 7 Table 1: Continued. ID Source Abbr. X Y Sampling pH Eh Temp EC Alk Ca Mg K Sr Na Cl SO4 Br HCO3 U δ18O δ2H Date mV °C μS/cm mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l nmol/l VSMOW Springs (Himma thermohaline) 01-12 Ein Himma AH (01) 751665 3621722 ####### 7.06 -145 41.5 1418 5.59 108 34.9 13.9 2.88 124 208 148 2.25 337 4.1 -6.08 -32.0 07/624 Ein Himma AH (07) 751665 3621722 ####### 7.02 -28 40 1433 113 34 13.8 3.7 122 244 212 2.9 330 2.3 -6.16 -31.1 11/140 Ein Himma AH (11) 751568 3621767 ####### 7.11 492 27.5 1071 5.56 105 34.5 8.7 82.3 143 124 0.97 335 9.3 -5.29 -23.8 13/807 Ein Himma AH (13) 751600 3621710 ####### 7.1 72 37.7 1130 4.84 118 32 8.89 1.9 84.4 150 125 1.06 291 8.4 -6.16 -31.1 16/19 Ein Himma AH (16) 751661 3621700 ####### 7.03 85 40 499 4.06 110 30 9.5 2 94 161 129 1.32 244 6.7 -6.13 -28.9 Springs (Golan Heights, cover basalt, fresh) 18/920 Umm Abu ad Dananir 763884 3648639 ####### 6.68 468 17.8 349 22.4 13.7 5.69 0.25 23.8 25.7 11.8 128 0.06 -6.11 -27. 4. Results 18/921 Ayit fall 757703 3649486 ####### 7.61 433 18 1010 85 43 4.47 0.83 64.5 108 24.9 408 0.02 -4.94 -21. 18/922 Amphy spring 748804 3653081 ####### 7.69 460 18.6 363 26.1 15.4 4.31 0.21 25.5 17.3 5.7 168 0.05 -6.97 -30. Springs (Mt. Hermon, limestone, fresh) 08/786 Ein Dan 751017 3682076 ####### 7.39 445 15.9 472 3.44 65.9 5.44 0.65 0.09 4.34 9.77 8.99 211 1.01 -7.36 -38.3 08/787 Ein Banyas 746939 3682079 ####### 7.49 497 18.4 358 3.52 72.3 12.4 1.46 0.30 9.87 12.9 57.3 206 1.00 -7.29 -37.5 Average 69.1 8.92 1.06 7.11 11.3 33.1 River 16/18 Yarmouk River YR 756255 3624301 ####### 8.4 191 23.5 961 3.18 55 30 5.7 0.57 97 110 147 0.2 184 5.9 -4.32 -21.3 Geoﬂuids 8 Table 2: REY composition of the discussed groundwater from the Lower Yarmouk Gorge. 4. Results ID Location Short La Ce Pr Nd Pm Sm Eu Gd Tb Dy Y Ho Er Tm Yb Lu pmol/l pmol/l pmol/l pmol/l pmol/l pmol/l pmol/l pmol/l pmol/l pmol/l pmol/l pmol/l pmol/l pmol/l 01-128 Mukheibeh 1 2001 M1 (01) 2.35 4.31 0.58 2.33 0.48 0.17 0.54 0.09 0.70 17.71 0.17 0.72 1.01 0.20 13/803 Mukheibeh 2 2013 M2 (13) 1.40 2.11 0.28 1.14 0.22 0.08 0.35 0.06 0.54 14.84 0.14 0.58 0.86 0.17 16-08 Mukheibeh 2 2016 M2 (16) 2.21 3.75 0.43 1.98 0.74 0.08 0.42 0.06 0.65 18.59 0.17 0.67 0.89 0.17 13/802 Mukheibeh 4 2013 M4 (13) 1.51 2.47 0.31 1.42 0.26 0.08 0.29 0.06 0.44 11.57 0.11 0.48 0.72 0.13 16-09 Mukheibeh 4 2016 M4 (16) 22.21 41.72 5.15 21.76 4.34 1.02 3.99 0.54 3.43 52.06 0.70 2.10 1.89 0.31 16-12 Mukheibeh 5 2016 M5 (16) 1.18 1.84 0.18 0.81 0.23 0.09 0.11 2.90 0.16 16-10 Mukheibeh 6 2016 M6 (16) 1.54 2.57 0.23 1.03 0.28 0.21 0.05 0.42 14.17 0.12 0.49 0.77 0.15 16-14 Mukheibeh 7 2016 M7 (16) 4.62 9.02 0.90 3.47 0.63 0.17 0.69 0.10 0.79 17.05 0.18 0.66 0.79 0.13 13/804 Mukheibeh 8 2013 M8 (13) 5.14 8.87 1.10 4.53 0.89 0.23 0.91 0.15 0.90 12.69 0.19 0.56 0.46 0.08 16-16 Mukheibeh 8 2016 M8 (16) 4.66 6.57 0.79 3.02 0.49 0.12 0.53 0.06 0.54 9.73 0.11 0.36 0.34 0.05 16-17 Mukheibeh 9 2016 M9 (16) 14.58 29.29 2.84 8.76 0.51 0.11 0.92 0.07 0.47 8.02 0.09 0.32 0.49 0.12 16-11 Mukheibeh 10 2016 M10 (16) 3.63 7.27 0.79 3.40 0.81 0.18 0.71 0.09 0.62 8.73 0.12 0.38 0.42 0.08 16-13 Mukheibeh 11 2016 M11 (16) 4.22 7.74 0.84 3.60 0.82 0.21 0.89 0.14 1.22 27.90 0.29 0.96 0.99 0.16 13/805 Mukheibeh 13 2013 M13 (13) 7.48 11.31 1.19 4.65 0.77 0.23 0.86 0.10 0.75 11.11 0.16 0.50 0.54 0.09 16-15 Mukheibeh 13 2016 M13 (16) 2.97 6.20 0.62 2.67 0.62 0.16 0.59 0.09 0.69 12.51 0.16 0.49 0.50 0.08 16-07 Meizar 1 Me1 1.40 2.21 0.18 0.96 0.42 0.28 0.53 0.18 3.23 0.14 01-166 Meizar 2 2001 Me2 (01) 4.38 10.32 1.33 4.80 1.03 0.23 1.05 0.14 0.88 15.75 0.17 0.49 0.40 0.06 08/753 Meizar 2 2008 Me2 (08) 7.17 18.48 3.23 15.86 5.48 1.40 6.32 0.84 4.18 34.51 0.75 1.91 1.21 0.15 16-02 Meizar 2 2016 Me2 (16) 6.14 10.28 1.14 4.51 0.85 0.22 1.09 0.16 1.15 24.60 0.25 0.72 0.51 0.07 01-167 Meizar 3 2001 Me3 (01) 9.98 18.14 2.48 10.12 2.24 0.56 2.25 0.31 1.81 23.87 0.35 1.02 0.86 0.14 08/752 Meizar 3 2008 Me3 (08) 30.39 86.80 17.80 96.20 31.56 7.83 36.36 4.79 23.55 140.96 3.97 8.98 5.48 0.77 16-01 Meizar 3 2016 Me3 (16) 1.33 2.39 0.25 1.03 0.20 0.20 0.04 0.22 4.63 0.05 0.17 0.25 0.05 16-03 Ein Sahina ES 1.84 3.24 0.32 1.43 0.28 0.33 0.35 9.18 0.09 0.30 0.30 0.04 16-04 Ein Balsam 2016 EB 4.68 9.13 0.98 4.20 0.75 0.20 0.80 0.10 0.76 17.16 0.16 0.53 0.61 0.10 00- 108 Ein Makla 2000 ER (00) 12.49 25.59 1.87 11.20 1.81 0.49 2.16 0.33 2.26 50.15 0.55 1.91 2.03 0.35 04-586 Ein Makla 2004 ER (04) 10.31 14.67 1.81 7.43 1.28 0.38 1.83 0.31 1.99 47.02 0.48 1.84 1.92 0.33 16-06 Ein Makla 2016 ER (16) 8.81 11.05 1.44 6.23 1.26 0.38 1.70 0.27 2.23 50.26 0.52 1.84 2.03 0.36 00- 107 Ein Reach 2000 EM (00) 19.71 1.46 9.53 1.35 0.38 1.50 0.24 1.50 27.60 0.31 1.17 1.25 0.22 04-585 Ein Reach 2004 EM (04) 4.75 7.70 0.98 3.61 0.62 0.21 0.84 0.15 1.04 19.69 0.22 0.73 0.92 0.17 16-05 Ein Reach 2016 EM (16) 14.61 25.69 2.88 12.01 2.16 0.60 2.41 0.33 2.40 43.53 0.53 1.75 1.72 0.30 16-20 Ain Saraya AS 12.27 18.80 2.33 7.34 0.49 0.11 0.84 0.08 0.64 15.98 0.16 0.62 0.85 0.17 01-12 Ain Himma 2001 AH (01) 4.31 23.39 3.48 13.06 2.98 0.78 3.38 0.52 3.33 51.76 0.73 2.21 2.02 0.34 07/624 Ain Himma 2007 AH (07) 16.34 65.43 3.42 14.15 3.38 0.92 4.19 0.68 4.29 62.85 0.93 2.79 2.57 0.42 13/807 Ain Himma 2013 AH (13) 4.06 7.58 0.74 2.91 0.53 0.13 0.53 0.09 0.61 13.83 0.16 0.52 0.66 0.11 16-19 Ain Himma 2016 AH (16) 43.50 61.06 8.54 25.95 0.76 0.19 2.11 0.12 0.92 19.85 0.21 0.80 1.05 0.23 9 Geoﬂuids 9 Geoﬂuids 10 Table 3: Sr2+ concentration and 87Sr/86Sr isotope signatures of groundwater and rocks from the Lower Yarmouk Gorge and surrounding areas. 4. Results ID Well/spring Sr (mg/l) 87Sr/86Sr 16/08 Mukheibeh 2 0.51 0.70769 16/09 Mukheibeh 4 0.5 0.70764 16/12 Mukheibeh 5 0.94 0.70770 16/10 Mukheibeh 6 0.66 0.70767 16/14 Mukheibeh 7 0.54 0.70757 16/16 Mukheibeh 8 1.3 0.70748 16/17 Mukheibeh 9 5.3 0.70778 16/11 Mukheibeh 10 0.96 0.70757 16/13 Mukheibeh 11 0.87 0.70767 16/15 Mukheibeh 13 0.88 0.70757 16/07 Meizar 1 2.1 0.70754 16/02 Meizar 2 5.5 0.70760 16/01 Meizar 3 1.1 0.70755 16/03 Ein Sahina 0.56 0.70763 16/04 Ein Balsam 3.5 0.70765 16/06 Ein Makla 5.5 0.70767 16/05 Ein Reach 4.2 0.70776 16/20 Ain Saraya 3.8 0.70771 16/19 Ain Himma 2 0.70759 18/920 Umm Abu ad Dananir 0.252 0.70458 18/922 Amphy spring 0.21 0.70457 16/18 Yarmouk 0.57 0.70708 ID Rock sample Sr (mg/kg) 87Sr/86Sr 18/A Golan Heights 1 1934 0.70330 18/B Golan Heights 2 1040 0.70350 ID Well/spring Sr (mg/l) 87Sr/86Sr 16/08 Mukheibeh 2 0.51 0.70769 16/09 Mukheibeh 4 0.5 0.70764 16/12 Mukheibeh 5 0.94 0.70770 16/10 Mukheibeh 6 0.66 0.70767 16/14 Mukheibeh 7 0.54 0.70757 16/16 Mukheibeh 8 1.3 0.70748 16/17 Mukheibeh 9 5.3 0.70778 16/11 Mukheibeh 10 0.96 0.70757 16/13 Mukheibeh 11 0.87 0.70767 16/15 Mukheibeh 13 0.88 0.70757 16/07 Meizar 1 2.1 0.70754 16/02 Meizar 2 5.5 0.70760 16/01 Meizar 3 1.1 0.70755 16/03 Ein Sahina 0.56 0.70763 16/04 Ein Balsam 3.5 0.70765 16/06 Ein Makla 5.5 0.70767 16/05 Ein Reach 4.2 0.70776 16/20 Ain Saraya 3.8 0.70771 16/19 Ain Himma 2 0.70759 18/920 Umm Abu ad Dananir 0.252 0.70458 18/922 Amphy spring 0.21 0.70457 16/18 Yarmouk 0.57 0.70708 ID Rock sample Sr (mg/kg) 87Sr/86Sr 18/A Golan Heights 1 1934 0.70330 18/B Golan Heights 2 1040 0.70350 All of the above patterns show positive Y anomalies. The dissolution of REY-enriched HFO yields convex patterns of type t5 with enhanced abundances of medium REE com- pared to light and heavy REE and negative Y anomalies (Figure 6(f)). These Y anomalies develop because Y prefers to stay in the aqueous phase during the stage of REY adsorp- tion by HFO [36]. The water from Ain Himma in 2001 and 2007 and well Mukheibeh 4(16) shows REY patterns, typical of water from limestone aquifers such as those of Ein Dan and Ein Banyas in the Mt. Hermon Massif but without the negative Ce anom- alies typical of spring waters from karstic limestones (Figure 6(g)) or from Cretaceous limestones along the rift valley [37]. 4. Results Note that the REY abundance in waters from Mukheibeh 4(16) and Ain Himma from years 2000 and 2007 is lower than that in the spring waters of Dan and Banyas, which may be a result of interaction with HFO. 4.4. 87Sr/86Sr. Although the waters show a wide spread in Sr2+, their 87Sr/86Sr isotope ratios vary only between 0.7070 and 0.7077 (Figure 7). This corresponds with the range of 87Sr/86Sr in Cretaceous limestones of Israel, which is about 0.7070-07086 (Wilske et al., unpublished data). Only the Yarmouk River with 0.70710 points to mixing with basaltic rock drainage water which shows an 87Sr/86Sr value of 0.70455, slightly above Phanerozoic upper mantel alkali olivine basalts from Israel with 87Sr/86Sr of 0.7033-07035 (Table 3). varied over the years. Hamat Gader brines are lower in SO4 2- but higher in Cl- and Br- than Meizar brines. Waters from wells such as Meizar 1 and Mukheibeh 8, 9, and 11 sometimes deviate from the indicated trend lines. The Yarmouk River water is mostly comparable to Mukheibeh water, but not in diagramms with Na+, Cl- and SO4 2-. varied over the years. Hamat Gader brines are lower in SO4 2- but higher in Cl- and Br- than Meizar brines. Waters from wells such as Meizar 1 and Mukheibeh 8, 9, and 11 sometimes deviate from the indicated trend lines. The Yarmouk River water is mostly comparable to Mukheibeh water, but not in diagramms with Na+, Cl- and SO4 2-. In the plot of Sr2+ vs. 87Sr/86Sr, Mukheibeh ﬁeld groundwater clusters at low Sr2+, whereas the samples from Hamat Gader, Meizar 2, and Himma show a wide spread. Mukheibeh 8 water ﬁts into the Hamat Gader- Meizar 2-Himma trend, whereas Meizar 3 approaches the Mukheibeh ﬁeld. 4.2. Uranium. U(VI) is correlated neither with any other ele- ment mentioned before nor with Eh varying between -200 and +200 mV (Table 1). The Mukheibeh ﬁeld splits into three subgroups (Figure 5). U(VI) with 80-105 nmol/l has the highest values in Mukheibeh groundwater. Groundwater from wells Mukheibeh 5 and 11, Ain Himma, Hamat Gader shows values between 3 and 10 nmol/l. The groundwa- ter with <0.1 nmol/l and that with U(VI) below the detection limit comprise all well waters from Meizar 2 and 3 and Mukheibeh 1, 8, and 9. The lowest U(VI) values are either in the lowest or in the highest sulfate groundwater (Figure 5(b)). 4.5. δ18O vs. δD. 4. Results under oxidizing conditions. These colloids later aggregate to gels on all solid surfaces along the pathways within and below the basaltic layer. In aqueous systems, however, HREE and Y are slightly fractionated. The REY patterns of samples in this study are subdivided into 6 types. The ﬁrst group (t1) typiﬁes groundwater derived from weathered alkali olivine basalts. The patterns t2 and t3 show the results of increasing mixing with limestone water (t4) (Figures 6(a)–6(d)). In Figure 6(e), three REY patterns of type t2∗are compiled which show very high LREE contents but low HREE and Y. Otherwise, they resemble type t2. Another diﬀerent feature of t2∗is that positive Gd anomalies exceed those of Y. varied over the years. Hamat Gader brines are lower in SO4 2- but higher in Cl- and Br- than Meizar brines. Waters from wells such as Meizar 1 and Mukheibeh 8, 9, and 11 sometimes deviate from the indicated trend lines. The Yarmouk River water is mostly comparable to Mukheibeh water, but not in diagramms with Na+, Cl- and SO4 2-. 4.2. Uranium. U(VI) is correlated neither with any other ele- ment mentioned before nor with Eh varying between -200 and +200 mV (Table 1). The Mukheibeh ﬁeld splits into three subgroups (Figure 5). U(VI) with 80-105 nmol/l has the highest values in Mukheibeh groundwater. Groundwater from wells Mukheibeh 5 and 11, Ain Himma, Hamat Gader shows values between 3 and 10 nmol/l. The groundwa- ter with <0.1 nmol/l and that with U(VI) below the detection limit comprise all well waters from Meizar 2 and 3 and Mukheibeh 1, 8, and 9. The lowest U(VI) values are either in the lowest or in the highest sulfate groundwater (Figure 5(b)). 4.3. Rare Earths and Yttrium. Weathering of omnipresent Table 3: Sr concentration and Sr/ Sr isotope signatures of groundwater and rocks from the Lower Yarmouk Gorge and surrounding areas. 4. Results The stable water isotopes in the LYG range from low values of Meizar 2 in the southern Golan Heights and springs and wells on the eastern plunge of the Mt. Hermon Massif towards the Hauran plateau to high values in water of the Yarmouk River (Figure 8). All data from the LYG are plotted between the Syrian and Mt. Hermon meteoric water lines (MWL). The Mukheibeh waters like the groundwater from the Hauran plateau nearly cover the whole array, whereas the samples of Meizar, Hamat Gader, and Himma cluster. Ein Sahina, located uphill of the Hamat Gader group, is plotted among the Mukheibeh ﬁeld. Ain Sarayah, located close to Ein Reach of the Hamat Gader 4.3. Rare Earths and Yttrium. Weathering of omnipresent alkali olivine basalts in the Yarmouk basin releases Fe(II) which precipitates as colloidal ferric oxyhydroxides (HFO) 11 11 Geoﬂuids 0 100 200 300 400 500 Cl (mg/l) 0 50 100 150 200 250 Na (mg/l) Me1 (16) M9 (16) Hamat Gader HG Ein Sahina Ain Himma Meizar wells Mukheibeh wells Yarmouk River (a) Cl (mg/l) Ca (mg/l) 0 100 200 300 400 500 50 100 150 200 Me1 (16) M9 (16) Group: 1 Group: 2 Group: 3 Group: 4 Group: 5 Group: 6 (b) Cl (mg/l) K (mg/l) 0 100 200 300 400 500 0 5 10 15 20 25 Me1 (16) M9 (16) Group: 1 Group: 2 Group: 3 Group: 4 Group: 5 Group: 6 (c) Cl (mg/l) Sr (mg/l) 0 100 200 300 400 500 0 1 2 3 4 5 6 Me1 (16) M9 (16) Group: 1 Group: 2 Group: 3 Group: 4 Group: 5 Group: 6 (d) Cl (mg/l) SO4 (mg/l) 0 100 200 300 400 500 0 50 100 150 200 250 300 350 Me1 (16) M9 (16) Group: 1 Group: 2 Group: 3 Group: 4 Group: 5 Group: 6 (e) Br (mg/l) Rb (𝜇g/l) 0 1 2 3 4 5 6 7 0 5 10 15 20 25 30 35 Me1 (16) Me2 (16) M2 (16) M8 (16) Group: 1 Group: 2 Group: 3 Group: 4 Group: 5 Group: 6 (f) Figure 4: Continued. 4. Results Cl (mg/l) Ca (mg/l) 0 100 200 300 400 500 50 100 150 200 Me1 (16) M9 (16) Group: 1 Group: 2 Group: 3 Group: 4 Group: 5 Group: 6 (b) 0 100 200 300 400 500 Cl (mg/l) 0 50 100 150 200 250 Na (mg/l) Me1 (16) M9 (16) Ca (mg/l) Cl (mg/l) K (mg/l) 0 100 200 300 400 500 0 5 10 15 20 25 Me1 (16) M9 (16) Cl (mg/l) Sr (mg/l) 0 100 200 300 400 500 0 1 2 3 4 5 6 Me1 (16) M9 (16) Group: 1 Group: 2 Group: 3 Group: 4 Group: 5 Group: 6 (d) Sr (mg/l) K (mg/l) Cl (mg/l) SO4 (mg/l) 0 100 200 300 400 500 0 50 100 150 200 250 300 350 Me1 (16) M9 (16) Group: 1 Group: 2 Group: 3 Group: 4 Group: 5 Group: 6 (e) Br (mg/l) Rb (𝜇g/l) 0 1 2 3 4 5 6 7 0 5 10 15 20 25 30 35 Me1 (16) Me2 (16) M2 (16) M8 (16) Group: 1 Group: 2 Group: 3 Group: 4 Group: 5 Group: 6 (f) Figure 4: Continued. g/l) 300 400 500 Me1 (16) Group: 4 Group: 5 Group: 6 Br (mg/l) Rb (𝜇g/l) 0 1 2 3 4 5 6 7 0 5 10 15 20 25 30 35 Me1 (16) Me2 (16) M2 (16) M8 (16) Group: 1 Group: 2 Group: 3 Group: 4 Group: 5 Group: 6 (f) Figure 4: Continued. Br (mg/l) Rb (𝜇g/l) 0 1 2 3 4 5 6 7 0 5 10 15 20 25 30 35 Me1 (16) Me2 (16) M2 (16) M8 (16) Group: 1 Group: 2 Group: 3 Group: 4 Group: 5 Group: 6 (f) d SO4 (mg/l) Rb (𝜇g/l) Figure 4: Continued. 4. Results Meizar 2 and Meizar 3(08) show the lowest isotope values. cluster, is plotted among the heaviest Mukheibeh waters. Meizar 2 and Meizar 3(08) show the lowest isotope values. 4. Results 12 12 Geoﬂuids Cl (mg/l) Mg (mg/l) Group: 1 Group: 2 Group: 3 Group: 4 Group: 5 Group: 6 0 100 200 300 400 500 15 20 25 30 35 40 45 Me1 (16) Me2 (16) Me3 (16) M8 (16) M11 (16) (g) Sr (mg/l) Rb (𝜇g/l) Group: 1 Group: 2 Group: 3 Group: 4 Group: 5 Group: 6 0 1 2 3 4 5 6 0 5 10 15 20 25 30 35 Me1 (16) Me2 (16) Me3 (16) M2 (16) M9 (16) (h) Cl (mg/l) Br (mg/l) Group: 1 Group: 2 Group: 3 Group: 4 Group: 5 Group: 6 0 100 200 300 400 500 0 1 2 3 4 5 6 7 Me1 (16) Me2 (16) Me3 (16) M8 (16) (i) Cl (mg/l) Rb (𝜇g/l) Group: 1 Group: 2 Group: 3 Group: 4 Group: 5 Group: 6 0 100 200 300 400 500 0 5 10 15 20 25 30 35 Me1 (16) Me2 (16) Me3 (16) M2 (16) (j) Figure 4: Crossplots of elements in the groundwater in the Lower Yarmouk Gorge. For details, refer to text. Sr (mg/l) Rb (𝜇g/l) Group: 1 Group: 2 Group: 3 Group: 4 Group: 5 Group: 6 0 1 2 3 4 5 6 0 5 10 15 20 25 30 35 Me1 (16) Me2 (16) Me3 (16) M2 (16) M9 (16) (h) ( ) Cl (mg/l) Rb (𝜇g/l) Group: 1 Group: 2 Group: 3 Group: 4 Group: 5 Group: 6 0 100 200 300 400 500 0 5 10 15 20 25 30 35 Me1 (16) Me2 (16) Me3 (16) M2 (16) (j) Cl (mg/l) Br (mg/l) Group: 1 Group: 2 Group: 3 Group: 4 Group: 5 Group: 6 0 100 200 300 400 500 0 1 2 3 4 5 6 7 Me1 (16) Me2 (16) Me3 (16) M8 (16) (i) (i) Figure 4: Crossplots of elements in the groundwater in the Lower Yarmouk Gorge. For details, refer to text. The water of Meizar 2(08) and Meizar 3(08) shows simi- lar chemical and isotopic composition and the same type of REY patterns (t5). Although showing similar U(VI) concen- trations, Meizar 2(01) and Meizar 2(16) are dissimilar in REY patterns (t4 and t5). This suggests that these types of ground- water discharge from the same reservoir but the ﬂow path of recharging water diﬀers over the years. cluster, is plotted among the heaviest Mukheibeh waters. 5. Discussion A second mixing line is indicated by Ein Sahina and Mukheibeh wells 1 and 6; both lines only diﬀer in the Mukheibeh end member. water is characterized by Na+/Cl−≫1 (Table 1) and typical REY patterns of type t1 (Figure 6(a)). With increasing leach- ing of halite from sedimentary rocks, the basaltic waters approach the lowest Na+/Cl- value of about 1, whereas mix- ing with evaporated seawater brines yields Na+/Cl−< 1 (Figure 9). Comparison of the Mukheibeh waters with those of basaltic composition reveals that the former waters are enriched in all elements (Table 1). The dissolution of anhy- drite/gypsum by thermal waters of Meizar and Himma leads to enhanced concentrations of Ca2+ and SO4 2- (Figure 4(b)). Ca2+ may also increase by dissolution of calcite at enhanced temperatures and albitization of plagioclases in basalts. Mg2+, Rb+, Br-, and K+ may be gained by ion exchange against Ca2+ in marly layers in the aquifers (Figures 3 and 4). A Br- increase may be gained by contact with the bituminous-rich B2 formation. The correlations of Cl- with SO4 2-, Ca2+, Mg2+, Sr2+, Rb+, and Br- reveal that, with few exceptions, waters from Hamat Gader, Himma, and Meizar are mixtures of basaltic water and remnants of brines from the Triassic-Cretaceous Arabian carbonate platform. The strong correlation of Rb+ and Sr2+ indicates a common source but not necessarily the same mineral (Figure 4(h)). The two trends in the correlation of Rb+ and Br- verify the diﬀerent sources of both elements (Figure 4(j)). 5.2. The Impact of HFO Precipitation on U and REY. U(VI) is highly adsorbed onto the high surface area of HFO [40]. The U content of alkali olivine basalts is in the range of 1 ppb [41]. The inﬁltrating basaltic groundwater with low U(VI) content passes the growing HFO “ﬁlter” within and below the basaltic cover of the Hauran plateau and elsewhere. During the alter- ation of HFO to goethite, lepidocrocite, or hematite, the adsorbed U(VI) is reduced to U(V) which is more resilient to oxidation than uraninite (UO2) or adsorbed U(IV) [42, 43]. Adsorption of U(VI) in the pH range of 6.6-7.3 (Table 1) is not aﬀected by additional adsorption of phosphate [44]. y The high U(VI) contents of 80 to 105 nmol/l in the groundwater of Mukheibeh artesian wells 1, 2, 4, 6, and 7 are most probably supplied later from the phosphorite-rich B2 aquifer. 5. Discussion 5.1. Sources of Groundwater. The stable isotopes of water and the element correlations reveal diﬀerent origins of fresh and saline contributions to the groundwater in the LYG. Distinct groups of stable isotopes suggest regional inﬁltration areas at diﬀerent elevations. The Meizar 2 groundwater from 2001 to 2016 with (i) light δ2H and δ18O signatures and (ii) REY pat- terns of nearly limestone water shape and least aﬀected by HFO (t4 in Figure 6(d)) suggests inﬁltration of precipitation on the outcropping Triassic to Cretaceous limestones of the foothills of the Mt. Hermon Massif. The increase in Cl- is higher than that in Na+ probably pointing to mobilization of highly evaporated seawater brines and admix of these brines to the limestone water. g g y The groundwater with δ18O and δD of about -6‰ and -30‰, respectively, typiﬁes the groundwater from Hamat Gader, Himma, Meizar 3 in 2001 and 2016, and the Mukhei- beh ﬁeld. Most of the Mukheibeh and Hauran groundwater shows a trend of increasingly heavy stable isotopes of water, suggesting evaporation of recharge prior to inﬁltration (Figure 8). The eﬀect of evaporation on stable isotope enrich- ment is shown by heaviest δD and δ18O signatures in the Yarmouk River. High molar values of Na+/Cl- and Ca2+/SO4 2- but low Br-/Cl- and low concentrations of Na+, Cl-, Ca2+, Mg2+, K+, Sr2+, and Br- typify the basaltic waters [31]. Pure basaltic 13 Geoﬂuids .5 1.0 1.5 2.0 2.5 Na/Cl (mol/mol) .01 .1 1 10 100 U (mmol/l) Me1 (16) M8 (16) LOD Group: 1 Group: 2 Group: 3 Group: 4 Group: 5 Group: 6 (a) U (mmol/l) Group: 1 Group: 2 Group: 3 Group: 4 Group: 5 Group: 6 0 50 100 150 200 250 300 350 SO4 (mg/l) .01 .1 1 10 100 Me1 (16) Me2 (08) Me3 (08) LOD (b) Figure 5: Crossplots of U(VI) and Na/Cl values in the groundwater of the Lower Yarmouk Gorge. U (mmol/l) Group: 1 Group: 2 Group: 3 Group: 4 Group: 5 Group: 6 0 50 100 150 200 250 300 350 SO4 (mg/l) .01 .1 1 10 100 Me1 (16) Me2 (08) Me3 (08) LOD (b) i th d t f th L Y k G U (mmol/l) U (mmol/l) (b) Figure 5: Crossplots of U(VI) and Na/Cl values in the groundwater of the Lower Yarmouk Gorge. ing at SE shoreline of the Lake Tiberias [2, 38, 39]. 5. Discussion The phosphorites from the B2 formation in Syria, Jordan, and Israel contain about 100 ppm U [45]. Assuming that U(VI) is mobilized by phosphate as UO2(HPO4)2 2+ [46], the phosphate concentration should be in the range of 0.2 μmol/l or 6 μg/l which was much below our routine detection limit of phosphate of 1 mg/l. g j The molar 1000Br-/Cl- vs. Na+/Cl- values show several trends for groundwater in the Yarmouk basin and the trend of evaporated seawater in salt pans (Figure 9). In this plot, the springs of Hamat Gader, Himma, and well Meizar 2 deﬁne vertical trends which are only explainable by leaching of Br- from the organic-rich B2 formation (Figure 3). Meizar 3 in 2001 and 2016 and all the groundwater with the lowest Br-/Cl- values in the vertical groups suggest mixing between Mukheibeh groundwater and seawater brine characterized by Na+/Cl- and 1000Br-/Cl- of about 0.5 and 5.3, respectively. Such ratios resemble those of the Ha’On type of brine, emerg- Meizar 2 water has its source in the ﬂanks of the Mt. Hermon Massif and in the western elevations of the Hauran plateau, which agrees with light stable isotopes of water. Although limestone waters contain 2-20 nmol/l U(IV) from elsewhere in Israel (Siebert unpublished), Meizar 2 and Mukheibeh 8, 9, and 11 waters show less than 0.1 nmol/l U(VI) suggesting that these waters must have had contact with HFO but did not interact with the B2 for- mation. 5. Discussion Though having similar low U, considerably heavier stable isotope signatures in Mukheibeh 8 and 9, the most 14 Geoﬂuids –7 –6 –5 –4 Log (REY, C1-normalized) M1 (01) M2 (13) M2 (16) M4 (13) M6 (16) M11 (16) Me3 (16) AH (13) ER (16) La Ce Pr Nd Pm Sm Eu Gd Tb Dy Y Ho Er Tm Yb Lu (a) –7 –6 –5 –4 EM (00) EM (04) ER (00) ER (04) EM (16) M7 (16) M10 (16) EB La Ce Pr Nd Pm Sm Eu Gd Tb Dy Y Ho Er Tm Yb Lu (b) –7 –6 –5 –4 Log (REY, C1-normalized) M8 (16) M13 (13) M13 (16) ES YR La Ce Pr Nd Pm Sm Eu Gd Tb Dy Y Ho Er Tm Yb Lu (c) –7 –6 –5 –4 AH (01) AH (07) M4 (16) M8 (13) Me2 (01) Me2 (16) Me3 (01) La Ce Pr Nd Pm Sm Eu Gd Tb Dy Y Ho Er Tm Yb Lu (d) –7 –6 –5 –4 Log (REY, C1-normalized) M9 (16) AH (16) AS La Ce Pr Nd Pm Sm Eu Gd Tb Dy Y Ho Er Tm Yb Lu (e) –7 –6 –5 –4 La Ce Pr Nd Pm Sm Eu Gd Tb Dy Y Ho Er Tm Yb Lu Me2 (08) Me3 (08) (f) –7 –6 –5 –4 Log (REY, C1-normalized) M1 (01) M2 (13) M2 (16) M4 (13) M6 (16) M11 (16) Me3 (16) AH (13) ER (16) La Ce Pr Nd Pm Sm Eu Gd Tb Dy Y Ho Er Tm Yb Lu (a) –7 –6 –5 –4 EM (00) EM (04) ER (00) ER (04) EM (16) M7 (16) M10 (16) EB La Ce Pr Nd Pm Sm Eu Gd Tb Dy Y Ho Er Tm Yb Lu (b) (b) –7 –6 –5 –4 Log (REY, C1-normalized) M8 (16) M13 (13) M13 (16) ES YR La Ce Pr Nd Pm Sm Eu Gd Tb Dy Y Ho Er Tm Yb Lu (c) –7 –6 –5 –4 Log (REY, C1-normalized) –7 –6 –5 –4 AH (01) AH (07) M4 (16) M8 (13) Me2 (01) Me2 (16) Me3 (01) La Ce Pr Nd Pm Sm Eu Gd Tb Dy Y Ho Er Tm Yb Lu (d) 7 –6 –5 –4 Log (REY, C1-normalized) –7 –6 –5 –4 Log (REY, C1-normalized) La Ce Pr Nd Pm Sm Eu Gd Tb Dy Y Ho Er Tm Yb Lu –7 –6 –5 –4 L C P Nd P S E Gd Tb D Y H E T Yb L Log (REY, C1-normalized) –7 La Ce Pr Nd Pm Sm Eu Gd Tb Dy Y Ho Er Tm Yb Lu La Ce Pr Nd Pm Sm Eu Gd Tb Dy Y Ho Er Tm Yb Lu (f) Figure 6: Continued. 5. Discussion Figure 6: Continued. 15 15 Geoﬂuids –7 –6 –5 –4 La Ce Pr Nd Pm Sm Eu Gd Tb Dy Y Ho Er Tm Yb Lu Log (REY, C1-normalized) AH (01) AH (07) EBY ED (g) Figure 6: REY distribution patterns of groundwater in the Lower Yarmouk Gorge. The visual grouping of patterns shows their high variability. –7 –6 –5 –4 La Ce Pr Nd Pm Sm Eu Gd Tb Dy Y Ho Er Tm Yb Lu Log (REY, C1-normalized) AH (01) AH (07) EBY ED (g) n patterns of groundwater in the Lower Yarmouk Gorge. The visual grouping of patterns shows their hi –7 –6 –5 –4 La Ce Pr Nd Pm Sm Eu Gd Tb Dy Y Ho Er Tm Yb Lu Log (REY, C1-normalized) AH (01) AH (07) EBY ED (g) e 6: REY distribution patterns of groundwater in the Lower Yarmouk Gorge. The visual grouping –7 –6 –5 –4 La Ce Pr Nd Pm Sm Eu Gd Tb Dy Y Ho Er Tm Yb Lu Log (REY, C1-normalized) AH (01) AH (07) EBY ED (g) Figure 6: REY distribution patterns of groundwater in the Lower Yarmouk Gorge. The visual grouping of patterns shows their high variability. Hamat Gader HG Ein Sahina Ain Himma Meizar wells Mukheibeh wells Yarmouk River Springs cover basalt (GH) Model curve: SrBW = 0.05 mg/l; 87Sr/86SrBW = 0.70455 Srbrine = 79 mg/l; 87Sr/86Srbrine = 0.7078 Srbrine = 79 mg/l; 87Sr/86Srbrine = 0.7078 Model curve: SrBW = 0.21 mg/l; 87Sr/86SrBW = 0.70455 0 1 2 3 4 5 6 Sr (mg/l) .7066 .7068 .7070 .7074 .7076 .7078 .7080 .7064 .7072 .7046 .7044 87Sr/86Sr Me3 (16) M9 (16) M8 (16) (a) 0 1 2 3 4 5 6 Sr (mg/l) .7045 .7050 .7070 .7075 .7080 Me3 (16) .7085 .7090 M8 (16) M9 (16) 87Sr/86Sr Hamat Gader HG Ein Sahina Ain Himma Meizar wells Mukheibeh wells Yarmouk River Springs cover basalt (GH) Model curve: SrBW = 0.21 mg/l; 87Sr/86SrBW = 0.70455 Srbrine = 300 mg/l; 87Sr/86Srbrine = 0.7089 Srbrine = 300 mg/l; 87Sr/86Srbrine = 0.7078 Model curve: SrBW = 0.21 mg/l; 87Sr/86SrBW = 0.70455 (b) Figure 7: Crossplots of Sr isotope ratios and Sr2+ in the groundwater from the Lower Yarmouk Gorge. 5. Discussion 0 1 2 3 4 5 6 S ( /l) .7045 .7050 .7070 .7075 .7080 Me3 (16) .7085 .7090 M8 (16) M9 (16) 87Sr/86Sr Hamat Gader HG Ein Sahina Ain Himma Meizar wells Mukheibeh wells Yarmouk River Springs cover basalt (GH) Model curve: SrBW = 0.05 mg/l; 87Sr/86SrBW = 0.70455 Srbrine = 79 mg/l; 87Sr/86Srbrine = 0.7078 Srbrine = 79 mg/l; 87Sr/86Srbrine = 0.7078 Model curve: SrBW = 0.21 mg/l; 87Sr/86SrBW = 0.70455 0 1 2 3 4 5 6 Sr (mg/l) .7066 .7068 .7070 .7074 .7076 .7078 .7080 .7064 .7072 .7046 .7044 87Sr/86Sr Me3 (16) M9 (16) M8 (16) (a) 2 0 1 2 3 4 5 6 Sr (mg/l) .7066 .7068 .7070 .7074 .7076 .7078 .7080 .7064 .7072 .7046 .7044 87Sr/86Sr Me3 (16) M9 (16) M8 (16) (b) (a) Figure 7: Crossplots of Sr isotope ratios and Sr2+ in the groundwater from the Lower Yarmouk Gorge. northeastern samples in the LYG, refer to a recharge area dif- fering from Meizar 2. phosphate and REY resulting in type t1 patterns. This seems to be indicated in type t2∗, which is possibly due to Y-phosphate precipitation (possibly churchite, Y, and HREEPO4) due to which the light REE are released [47]. g HFO scavenges not only U(IV) but also REY and HPO4 2-. There may be some synergetic interaction between 16 Geoﬂuids Hamat Gader HG Ein Sahina Meizar wells Ain Himma Mukheibeh wells Yarmouk River GW Hauran plateau MWL Mt. Hermon –20 –25 –30 𝛿2H (VSMOW) –35 –40 –7.5 –7.0 –6.5 –6.0 –5.5 𝛿18O (VSMOW) –5.0 –4.5 –4.0 MWL SW Syria Figure 8: δD vs. δ18O in groundwater of the Lower Yarmouk Gorge ([1]; this study) and the Hauran plateau [18]. MWL for Mt. Hermon and SW Syria are taken from Brielmann [52] and Al Charideh and Zakhem [53], respectively. –20 –25 –30 𝛿2H (VSMOW) –35 –40 –7.5 –7.0 –6.5 –6.0 –5.5 𝛿18O (VSMOW) –5.0 –4.5 –4.0 𝛿2H (VSMOW) Yarmouk River GW Hauran plateau MWL Mt. Hermon MWL SW Syria Figure 8: δD vs. δ18O in groundwater of the Lower Yarmouk Gorge ([1]; this study) and the Hauran plateau [18]. MWL for Mt. Hermon and SW Syria are taken from Brielmann [52] and Al Charideh and Zakhem [53], respectively. 5. Discussion .0 .5 1.0 1.5 2.0 2.5 Na/Cl 0 1 2 3 4 5 6 1000Br/Cl Me1 (16) M1 (01) M9 (16) Hamat Gader HG Ein Sahina Ain Himma Meizar Wells Mukheibeh Wells Yarmouk River MacCaffrey et al. (1995) Ha’On (Moller et al. 2012) Figure 9: Crossplots of 1000Br/Cl and Na/Cl of the groundwater from the Lower Yarmouk Gorge. .0 .5 1.0 1.5 2.0 2.5 0 1 2 3 4 5 6 1000Br/Cl Me1 (16) M1 (01) M9 (16) 1000Br/Cl 1000Br/Cl Me1 (16) Figure 9: Crossplots of 1000Br/Cl and Na/Cl of the groundwater from the Lower Yarmouk Gorge. All groundwater in the gorge is produced from limestone aquifers. When the REY poor basaltic water passes the lime- stones at enhanced temperatures, some calcite dissolves and thereby its aliquot of REY is released and mixed with REY load of the groundwater. More than 99% of the REY is immediately adsorbed onto calcite surfaces [48]. This way, the REY patterns of groundwater change from type t1 to t2, t3, and ﬁnally t4 (Figures 6 and 10). At enhanced 17 Geoﬂuids 17 –7 –6 –5 –4 La Ce Pr Nd Pm Sm Eu Gd Tb Dy Y Ho Er Tm Yb Lu Log (REY, C1-normalized) Me3 (01) 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 M4 (13) Figure 10: Mixing of basaltic and limestone groundwater showing the change in rare earth distribution patterns. gure 10: Mixing of basaltic and limestone groundwater showing the change in rare earth distribution patterns. Figure 10: Mixing of basaltic and limestone groundwater showing the change in rare earth distribution patter 5.3. Tracing Mixing by Sr2+ and 87Sr/86Sr. The above dis- cussed ﬁndings, which trace back the genesis of the ground- water in the LYG by variable interactions of basaltic water with late Tertiary brines of Ha’On type and with calcite and limestone of the discharging Cretaceous/Paleogene aquifers, can be fortiﬁed by model calculations, which try to resemble the measured 87Sr/86Sr values in the ground- water of the LYG by at least interaction of basaltic water and brine (Figure 7). temperatures, release of LREE is faster than that of HREE and Y because their Coulomb binding forces are less for the for- mer bigger than the latter smaller ions. This may qualitatively explain the change in REY patterns of groundwater in the Yarmouk basin. 5. Discussion Although the groundwater of the Yarmouk Gorge is produced from limestone aquifers, their REY patterns still indicate that the groundwater originates from basaltic catch- ment regions or, more precisely, has passed HFO layers. Although the patterns are similar in shape, the spring waters of Dan and Banyas from limestones of the Mt. Hermon Massif without contact with HFO show higher abundances than the limestone-like waters from the Yarmouk Gorge such as in Himma spring and Mukheibeh well 4(16) (Figure 6(g)). Types t1 to t4 in Figure 5 represent the con- tinuous change of REY patterns due to the interaction of basaltic groundwater after passing the HFO ﬁlter t1 and limestones resulting in changes according to t2-t4. These types of patterns result from mixing limestone and basaltic rock waters. It could well be that not the whole volume of water changes due to the interaction but only parts of it and mixing of various types yields the ﬁnal patterns as shown in Figure 10. Using the fraction εbrine of brine in the mixture of brine and basaltic water, the mix of Sr2+ (Equation (1)) and the mix of the Sr2+ isotope ratios (Equation (2)) are estimated. ð1Þ Srmix = εbrine × Srbrine + 1 −εbrine ð Þ × SrBW, ð1Þ 87Sr/86Srmix = 87Sr/86Srbrine × εbrine × Srbrine/Srmix + 87Sr/86SrBW × 1 −εbrine ð Þ × SrBW/Srmix, Srmix = εbrine × Srbrine + 1 −εbrine ð Þ × SrBW, ð1Þ 87Sr/86Srmix = 87Sr/86Srbrine × εbrine × Srbrine/Srmix + 87Sr/86SrBW × 1 −εbrine ð Þ × SrBW/Srmix, ð2Þ ð2Þ where index BW is the basaltic water. where index BW is the basaltic water. 2 Considering the analytical data on Sr2+ concentration of groundwater in Table 3, brine, basaltic water, and dissolved calcite and gypsum and their corresponding 87Sr/86Sr values and the Sr2+ concentration of basaltic water must be below 0.5 mg/l, the lowest value in Mukheibeh water. Indeed, pure basaltic water sampled from 2 springs in the cover basalt of the Golan Heights shows Sr2+ = 0:2 mg/l. The Sr2+ con- centration of the brine may be between 79 mg/l as ana- lyzed in Ha’On brine [2] and 300 mg/l, depending on the amount of dissolution of calcite from limestone with assumed average Sr2+ concentrations of 100 mg/mol calcite and about 25mg/mol gypsum from evaporites [49]. 5. Discussion The 87Sr/86Sr value of basaltic water is 0.70455 to 0.70457, and that of the brine is assumed to be 0.7078, matching the spread of data in Figure 7. The 87Sr/86Sr value of 0.7078 may result from mixing of Late Tertiary Tethys Type t5 (Figure 6(e)) is not showing dissolution of phosphate minerals such as apatite but leachates of altering HFO that loses REY at high levels. The diﬀerence between the latter two is that the former should show a positive Eu anomaly [36], whereas the latter is characterized by a negative one. How does it come that these types of groundwater still show REY patterns typical after inﬁltration in basaltic catch- ment areas? The reason is that the REY in calcite surfaces along the pathways in limestones equilibrate with the low REY abundance from the basaltic catchment. Under steady- state conditions, the groundwater from limestones shows patterns achieved by the interaction with groundwater that has passed HFO layers [31]. 18 Geoﬂuids 3625000 3622000 3620000 3625000 3622000 3620000 3625000 3622000 3620000 3625000 3622000 3620000 747500 750000 752500 755000 757500 747500 750000 752500 755000 757500 747500 750000 752500 (a) (b) (c) (d) 755000 757500 747500 750000 752500 755000 757500 (a) (b) Figure 11: Regional distribution of U(VI) (a), Sr2+ (b), rare earth distribution patterns (c), and sulfate (d) in the Lower Yarmouk Gorge. Legends given in (a) and (c) are representative for the entire Figure 11. 3625000 3622000 3620000 747500 750000 752500 755000 757500 3625000 3622000 3620000 747500 750000 752500 (b) (d) 755000 757500 (b) Figure 11: Regional distribution of U(VI) (a), Sr2+ (b), rare earth distribution patterns (c), and sulfate (d) in the Lower Yarmouk Gorge. Legends given in (a) and (c) are representative for the entire Figure 11. (4) If 87Sr/86Sr values of brine are larger than 0.7078, nei- ther the group of groundwater from Mukheibeh wells and Meizar 3 nor the group of Hamat Gader, Meizar 2, and Ain Himma can be represented (Figure 7(b)) seawater of 0.7089 [50] and dissolved average Upper Cretaceous limestone in Israel ranging between 0.7076 and 0.7078 (Wilske et al., unpublished data). 2 seawater of 0.7089 [50] and dissolved average Upper Cretaceous limestone in Israel ranging between 0.7076 and 0.7078 (Wilske et al., unpublished data). 2 seawater of 0.7089 [50] and dissolved average Upper Cretaceous limestone in Israel ranging between 0.7076 and 0.7078 (Wilske et al., unpublished data). 5. Discussion 2 The model curves in Figure 6 are ﬁtted by varying Sr2+ in basaltic water and in brine as well as the 87Sr/86Sr value of the brine. Several information can be derived by the following procedure. In summary, the 87Sr/86Sr of the groundwater in the LYG is the result of relic brine, which is diluted by basaltic water and subsequently dissolves calcite and gypsum and experi- ences some exchange of Ca2+ against Mg2+, Na+, and K+ in marly layers of the aquifers (Figure 3). Only Meizar 3 is mainly limestone water. (1) The observed groundwater cannot be ﬁtted by one curve, and the results are sensitive to assumed values of 87Sr/86Sr and the resulting εbrine (2) To ﬁt most Mukheibeh groundwater and that of Me3(16), the requested Sr2+ concentrations must be 0.05 mg/l, much lower than the observed 0.21mg/l (Figure 7(a)). Hence, the positive shift of these types of groundwater along the ordinate is assumed to result from the interaction of the proposed ﬂuid mix with calcite and gypsum in the discharging lime- stone aquifers, which show 87Sr/86Sr values as high as 0.7078 (Wilske et al., unpublished data) 5.4. Regional Distribution of Dissolved Species. The regional distribution of U(VI), Sr2+, and REY shows comparable structures, whereas SO4 2- behaves diﬀerently. High and low U(VI) concentrations are present in the NE of the Lower Yar- mouk Gorge (Figure 11(a)). The high values of 80-105 nmol/l U mark the area in which Mukheibeh wells 1, 2, 4, 5, 6, and 7 produce artesian water from the phosphorite-rich B2 aquifer. These high U(VI) concentrations decrease to 20 nmol/l SW-ward, downstream the Yarmouk River and to both sides of the gorge. North of and NE-ward in the gorge groundwater contain U(VI) below 1 nmol/l. Such low values can only be established by adsorption of U(VI). In the case of 5.4. Regional Distribution of Dissolved Species. The regional distribution of U(VI), Sr2+, and REY shows comparable structures, whereas SO4 2- behaves diﬀerently. High and low U(VI) concentrations are present in the NE of the Lower Yar- mouk Gorge (Figure 11(a)). The high values of 80-105 nmol/l U mark the area in which Mukheibeh wells 1, 2, 4, 5, 6, and 7 produce artesian water from the phosphorite-rich B2 aquifer. These high U(VI) concentrations decrease to 20 nmol/l SW-ward, downstream the Yarmouk River and to both sides of the gorge. 6. Conclusions Mukheibeh 8 and 9, this could be HFO in the Hauran pla- teau; in the case of Meizar, saline groundwater contact with dissolving HFO is documented in Figure 7(f) in the year 2008. According to Shimron [51], basaltic intrusions are present in the Mt. Hermon anticline, being probably responsible for the low U(VI). Additionally, the long path- way through the limestone aquifers from Mt. Hermon to the LYG altered the REY patterns in groundwater to type t4. In 2016, Meizar 3 shows the REY pattern of type t1. However, in 2001, it resembled type t4 of Meizar 2 in 2001 and 2016. The conjoint study of major, minor, and trace elements, δ18O, δD, and 87Sr/86Sr in the groundwater of the LYG reveals the following: (i) Mixtures of water from basaltic rocks and limestones are almost omnipresent in the LYG. A clear excep- tion is Meizar 2 that produces groundwater that was inﬁltrated at the ﬂanks of Mt. Hermon Massif. The mixtures vary from nearly pure basaltic water to nearly pure limestone water. In addition, leaching of residual brines and evaporites enhances the salin- ity of the various types of groundwater In the central part of the LYG, Sr2+ is about 0.55 mg/l (Figure 11(b)), while it increases to 1 mg/l NE-ward, to 3 mg/l in Himma, to 4 mg/l in Hamat Gader, and to 5 mg/l in both Meizar wells 2 and 3. (ii) The sources of salinization in limestone aquifers are given by relic brines, leaching of gypsum, and disso- lution of calcite. The origin of high sulfate concen- trations could be either the Late Triassic gypsum beds occurring at approximate depths of 2000m or evaporites of the Late Tertiary rift brine of the inland sea. For instance, groundwater in Meizar 2 and Hamat Gader has leached diﬀerent amounts of gyp- sum/anhydrite and calcite. Ion exchange of Ca2+ against Mg2+, Na+, and K+ enhanced the concentra- tions of the latter. Meizar 3 in 2008 resembles Meizar 2 in the same year. Their REY patterns show that this groundwater had dissolved HFO on its altered ﬂow path. 6. Conclusions The regional distribution of U(VI), Sr2+, and SO4 2- and REY distribution patterns reveal that there is a zone with strongly conﬁned groundwater and the hydrochemical composition changes systematically sideward and downstream along the gorge (ii) The sources of salinization in limestone aquifers are given by relic brines, leaching of gypsum, and disso- lution of calcite. The origin of high sulfate concen- trations could be either the Late Triassic gypsum beds occurring at approximate depths of 2000m or evaporites of the Late Tertiary rift brine of the inland sea. For instance, groundwater in Meizar 2 and Hamat Gader has leached diﬀerent amounts of gyp- sum/anhydrite and calcite. Ion exchange of Ca2+ against Mg2+, Na+, and K+ enhanced the concentra- tions of the latter. Meizar 3 in 2008 resembles Meizar 2 in the same year. Their REY patterns show that this groundwater had dissolved HFO on its altered ﬂow path. The regional distribution of U(VI), Sr2+, and SO4 2- and REY distribution patterns reveal that there is a zone with strongly conﬁned groundwater and the hydrochemical composition changes systematically sideward and downstream along the gorge A similar shell-like behavior is observable in the REY pat- terns with t1 patterns in the center followed by t2 SW-ward and t3 type SE-ward and patterns of t4 to t6 in the NW (Figure 11(c)). The high-uranium water shows SO4 2- concentrations of 30-50 mg/l (Figure 11(d)). Outside that central part, the groundwater shows either much higher SO4 2- concentra- tions, such as in Hamat Gader (150 mg/l) and Meizar (300 mg/l), or almost no dissolved sulfate as in the NE (0.12 mg/l). The increasing SO4 2- outside the marked center may prove depletion of gypsum in the central region of ascending groundwater. Comparing spatial concentration distribution patterns of Sr2+ and SO4 2- results in similar patterns, though the concentration levels diﬀer signiﬁcantly. Leaching of brines and/or evaporites alters the chemical composition of the initial basaltic water. The light signatures of water isotopes of Meizar 2 support a catchment area at the Mt. Hermon foothills or at elevated places in the Hauran. Meizar 3 water isotopes correspond with those of Hamat Gader and Himma, which may be taken as an indirect proof for its basaltic water. Their variable REY patterns of types t1, t4, and t5 suggest various ﬂow paths of the groundwater including diﬀering contacts with HFO. 6. Conclusions The shortest pathway of groundwater ﬂow is indicated by REY patterns of type t1 (Figure 6), while patterns of type t2 and t3 suggest a longer pathway with more intense REY exchange with calcite in limestones. The longest pathways are typiﬁed by REY pattern type t4. The REY types and the concentrations of U(VI), Sr2+, and SO4 2- characterize complex ﬂow patterns of groundwater towards the gorge. (iii) The regional variation of their chemical composition of groundwater is related to a complex ﬂower- structured fault system crossing the gorge. Ground- water ﬂow in the gorge and the mixing between the diﬀerent water bodies are controlled by these struc- tural features (iii) The regional variation of their chemical composition of groundwater is related to a complex ﬂower- structured fault system crossing the gorge. Ground- water ﬂow in the gorge and the mixing between the diﬀerent water bodies are controlled by these struc- tural features 5. Discussion North of and NE-ward in the gorge groundwater contain U(VI) below 1 nmol/l. Such low values can only be established by adsorption of U(VI). In the case of (3) The ﬁtting curves are invariant in respect to varia- tions of Sr2+ in the brine (compare red curve in Figure 6(a) and blue curve in Figure 7(b)) 19 Geoﬂuids Geoﬂuids Conflicts of Interest The authors declare that they have no conﬂicts of interest. Data Availability All underlying data of the research study are included in the manuscript in the form of Tables 1–3. The most distinct basaltic water is produced from the B1/B2 limestone aquifers fractured by a complex fault system crossing the LYG [15] (Figure 1(b)). This marks the most important ﬂow path of drainage water from the Hauran into the LYG. The springs of Hamat Gader (including Ein Sahina) and Himma are positioned on an uptilted block, whereby both spring ﬁelds are separated from the Meizar ﬁeld. The deep aquifer which is tapped by Meizar 2 also produced water in the shallow well Meizar 3 in 2008. References [17] E. Rosenthal, G. Weinberger, A. Almogi-Labin, and A. Flexer, “Late cretaceous-early tertiary development of depositional basins in Samaria (Israel) as part of the evolution of regional folding systems,” AAPG Bulletin, vol. 84, pp. 997–1042, 1999. [1] C. Siebert, P. Möller, S. Geyer et al., “Thermal waters in the Lower Yarmouk Gorge and their relation to surrounding aqui- fers,” Geochemistry, vol. 74, no. 3, pp. 425–441, 2014. [2] P. Möller, C. Siebert, S. Geyer et al., “Relationship of brines in the Kinnarot Basin, Jordan‐Dead Sea Rift Valley,” Geoﬂuids, vol. 12, no. 2, 181 pages, 2012. [18] Z. 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Acknowledgments The research was partly funded by the German Science Foundation (DFG) (grant MA4450/2) and the DESERVE Virtual Institute (VH-VI-527) funded by the Helmholtz- Association of German Research Centers. The authors thank the Mekorot Co. Ltd. and the IDF and JAF for providing access and security during sampling along the international borders. Although producing from the same aquifer, the hydro- chemical diﬀerences in groundwater from Ein Himma and Meizar 3 disprove any transboundary ﬂow below the Yarmouk River. The conﬁned water from basaltic inﬁltration areas in Syria, however, is present on both sides of the gorge. 20 Geoﬂuids References Magri, “Hydrochemi- cal considerations for identifying water from basaltic aquifers: the Israeli experience,” Journal of Hydrology: Regional Studies, vol. 5, pp. 33–47, 2016. [14] A. Flexer, “Stratigraphy and facies development of Mt. 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References Zak, “The geological history of Pliocene-Pleistocene evaporites in Applied & Hindawi www.hindawi.com Volume 2018 International Journal of Geophysics Environmental and Public Health Journal of Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Volume 2018 Public Health Advances in Hindawi www.hindawi.com Volume 2018 Meteorology Advances in Hindawi Publishing Corporation http://www.hindawi.com Volume 2013 Hindawi www.hindawi.com The Scientific World Journal Volume 2018 Hindawi www.hindawi.com Volume 2018 Chemistry Advances in Scientifca Hindawi www.hindawi.com Volume 2018 Submit your manuscripts at www.hindawi.com Hindawi www.hindawi.com Volume 2018 International Journal of Geophysics Hindawi www.hindawi.com Public Advances Hindawi Publishing Corporation http://www.hindawi.com Volume 2013 Hindawi www.hindawi.com The Scientific World Journal Volume 2018 Hindawi www.hindawi.com Volume 2018 Chemistry Advances in Scientifca Hindawi www.hindawi.com Volume 2018 Scientifca Hindawi www.hindawi.com Volume 2018 Hindawi Publishing Corporation Hindawi The Scientific World Journal Hindawi www.hindawi.com Volume 2018 Journal of Chemistry Archaea Hindawi www.hindawi.com Volume 2018 Marine Biology Journal of Hindawi www.hindawi.com Volume 2018 Biodiversity International Journal of Hindawi www.hindawi.com Volume 2018 Ecology International Journal of Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Applied & Environmental Soil Science Volume 2018 Forestry Research International Journal of Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Volume 2018 Geophysics Environmental and Public Health Journal of Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Volume 2018 International Journal of Microbiology Hindawi www.hindawi.com Volume 2018 Public Health Agriculture Advances in Hindawi www.hindawi.com Volume 2018 Agronomy Hindawi www.hindawi.com Volume 2018 International Journal of Hindawi www.hindawi.com Volume 2018 Meteorology Advances in Hindawi Publishing Corporation http://www.hindawi.com Volume 2013 Hindawi www.hindawi.com World Journal Volume 2018 Hindawi www.hindawi.com Volume 2018 Chemistry Scientifca Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Volume 2018 Geological Research Journal of Analytical Chemistry International Journal of Hindawi www.hindawi.com Volume 2018 Submit your manuscripts at www.hindawi.com Submit your manuscripts at www.hindawi.com Hindawi www.hindawi.com Volume 2018 Geological Research Journal of Submit your manuscripts at www.hindawi.com Hindawi www.hindawi.com Volume 2018 Journal of Chemistry Archaea Hindawi www.hindawi.com Volume 2018 Marine Biology Journal of Hindawi www.hindawi.com Volume 2018 Biodiversity International Journal of Hindawi www.hindawi.com Volume 2018 Ecology International Journal of Hindawi www.hindawi.com Volume 2018 Forestry Research International Journal of Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Volume 2018 International Journal of Microbiology Agriculture Advances in Hindawi www.hindawi.com Volume 2018 Agronomy Hindawi www.hindawi.com Volume 2018 International Journal of Analytical Chemistry International Journal of Hindawi www.hindawi.com Volume 2018 Marine Biology Journal of Hindawi www.hindawi.com Volume 2018
https://openalex.org/W2985276039	https://boa.unimib.it/bitstream/10281/254022/2/10281-254022.pdf	English	null	Age and Sex Influence the Neuro-inflammatory Response to a Peripheral Acute LPS Challenge	Frontiers in aging neuroscience	2,019	cc-by	9,811	ORIGINAL RESEARCH published: 05 November 2019 doi: 10.3389/fnagi.2019.00299 Keywords: aging, neuroinﬂammation, microglia, astrocytes, 18 kDa translocator protein, triggering receptor expressed on myeloid cells 2 Citation: Murtaj V, Belloli S, Di Grigoli G, Pannese M, Ballarini E, Rodriguez-Menendez V, Marmiroli P, Cappelli A, Masiello V, Monterisi C, Bellelli G, Panina-Bordignon P and Moresco RM (2019) Age and Sex Inﬂuence the Neuro-inﬂammatory Response to a Peripheral Acute LPS Challenge. Front. Aging Neurosci. 11:299. doi: 10.3389/fnagi.2019.00299 Age and Sex Inﬂuence the Neuro-inﬂammatory Response to a Peripheral Acute LPS Challenge Valentina Murtaj 1,2, Sara Belloli 2,3, Giuseppe Di Grigoli 2,3, Maria Pannese 4, Elisa Ballarini 5,6, Virginia Rodriguez-Menendez 5,6, Paola Marmiroli 5,6, Andrea Cappelli 7, Valeria Masiello 2, Cristina Monterisi 6, Giuseppe Bellelli 8, Paola Panina-Bordignon 4,9† and Rosa Maria Moresco 2,6† 1PhD Program in Neuroscience, School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy, 2PET and Nuclear Medicine Unit, San Raffaele Scientiﬁc Institute, Milan, Italy, 3Institute of Molecular Bioimaging and Physiology of National Reasearch Council, Segrate, Italy, 4Neuroimmunology Unit, Division of Neuroscience, IRCCS San Raffaele Scientiﬁc Institute, Milan, Italy, 5Milan Center for Neuroscience, School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy, 6Department of Medicine and Surgery, Tecnomed Foundation, University of Milano-Bicocca, Monza, Italy, 7Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Siena, Italy, 8Acute Geriatric Unit, School of Medicine and Surgery, San Gerardo Hospital, University of Milano-Bicocca, Monza, Italy, 9School of Medicine and Surgery, San Raffaele Vita-Salute University, Milan, Italy Reviewed by: Tahir Ali, University of Calgary, Canada Chan Young Shin, Konkuk University, South Korea Bernd L. Fiebich, University of Freiburg, Germany Correspondence: Rosa Maria Moresco moresco.rosamaria@hsr.it Paola Panina-Bordignon panina.paola@hsr.it Correspondence: Rosa Maria Moresco moresco.rosamaria@hsr.it Paola Panina-Bordignon panina.paola@hsr.it †These authors have contributed equally to this work as co-last authors Received: 24 June 2019 Accepted: 17 October 2019 Published: 05 November 2019 Aging is associated with an exaggerated response to peripheral inﬂammatory challenges together with behavioral and cognitive deﬁcits. Studies considering both age and sex remain limited, despite sex dimorphism of astrocytes and microglial cells is largely recognized. To ﬁll this knowledge gap, we investigated the effect of a single intraperitoneal lipopolysaccharide (LPS) administration in adult and aged mice. We assessed the expression of different inﬂammatory mediators, and the microglial response through binding of [18F]-VC701 tracer to translocator protein (TSPO) receptors in the male and female brain. Aged female brain showed a higher pro-inﬂammatory response to LPS compared to adult female and to aged male, as revealed by ex vivo binding to TSPO receptors and pro-inﬂammatory mediator transcript levels. The highest astroglial reaction was observed in the brain of aged females. Differently to the other groups of animals, in aged males LPS challenge did not affect transcription of triggering receptor expressed on myeloid cells 2 (TREM2). In conclusion, our study shows that in the mouse’s brain the neuro-inﬂammatory response to an acute peripheral insult is sex- and age-dependent. Moreover, our results might set the basis for further studies aimed at identifying sex-related targets involved in the modulation of the aberrant neuro- inﬂammatory response that characterizes aging. This knowledge could be relevant for the treatment of conditions such as delirium and dementia. Edited by: Diego Ruano, University of Seville, Spain Reviewed by: Tahir Ali, University of Calgary, Canada Chan Young Shin, Konkuk University, South Korea Bernd L. Fiebich, University of Freiburg, Germany *Correspondence: Rosa Maria Moresco moresco.rosamaria@hsr.it Paola Panina-Bordignon panina.paola@hsr.it Edited by: Diego Ruano, University of Seville, Spain Reviewed by: Tahir Ali, University of Calgary, Canada Chan Young Shin, Konkuk University, South Korea Bernd L. Fiebich, University of Freiburg, Germany Edited by: Diego Ruano, University of Seville, Spain INTRODUCTION However, most evidence on molecular and pathological CNS effects induced by peripheral inflammatory challenges, particularly during aging, derive from pre-clinical studies on rodents (Cunningham and Maclullich, 2013). As shown in the literature, aging is associated with an exaggerated response to peripheral inflammatory challenges together with behavioral and cognitive deficits (Hoogland et al., 2015; Schreuder et al., 2017). Indeed, in neurodegenerative disorders as well as in normal aging, microglia cells lose their supportive role in neuroplasticity and undertake a primed over-reactive phenotype promoting cognitive decline and synaptic dysfunction (Godbout and Johnson, 2006; Maclullich et al., 2008; Teeling and Perry, 2009). A recent gene expression profiling of microglia showed that aging is associated with over-expression of immune-related genes with an intermediate signature between acute and primed microglial genes (Holtman et al., 2015). Translocator protein (TSPO) has been shown to be a reliable biomarker of microglia activation in a large number of studies of neuro-inflammation by pre-clinical imaging (Liu et al., 2014). During brain injury or inflammatory insults, TSPO is overexpressed in activated microglia cells. For this reason, TSPO ligands for PET imaging have been extensively used for the in vivo monitoring of microglia activation/macrophage infiltration in different neuropsychiatric conditions as well as in neurodegenerative and neuro-inflammatory animal models (Liu et al., 2014). Currently, not only is there a lack of studies on the inflammatory responses in males and females but also studies considering both sex and age remain limited. For this reason, the main goal of this study was to test whether sex and age influence the early brain response to an acute peripheral inflammatory challenge. This experimental setting could reproduce the delirium syndrome, a transient and serious neurocognitive disorder characterized by an acute onset and fluctuating course (Inouye et al., 2014) To this aim, we investigated the effect of a single intraperitoneal LPS administration in male and female, adult and aged mice. The association between genes regulating monocytes or microglial response with neurodegenerative disorders also supports the major role that neuroinflammation exerts in cognitive dysfunction. An example of this is the Triggering Receptor Expressed on Myeloid (TREM), a key component of innate and adaptive immunity, which is expressed by a variety of innate cells of the myeloid lineage including neutrophils, monocytes, osteoclasts, macrophages, dendritic cells and microglia. In particular, TREM2 has been shown to bind to poly-anionic ligands such as bacterial LPS and phospholipids (Wang et al., 2015). INTRODUCTION Upon ligand binding, TREM2 signals intracellularly through the adaptor protein DAP12, eventually regulating different cellular functions like phagocytosis, cytokine production, proliferation and survival (Thankam et al., 2016). Genetic studies have identified TREM2 variants that are associated with an increased risk of Alzheimer’s disease (AD; Zheng et al., 2018). Another protein of potential interest is the TREM cells Like 2 (TREML2 also named TLT2). TREML2 is upregulated on B cells, neutrophils and macrophages during inflammation, and recent data suggest a potential modulatory role in pro-inflammatory responses (Thomas et al., 2016). Indeed, a missense variant of TREML2 (rs3747742) has been associated with a reduced susceptibility to develop INTRODUCTION The activation of the peripheral immune system is reflected by a pro-inflammatory milieu in the central nervous system (CNS; Norden and Godbout, 2013). The mechanisms of neuroinflammation are the subject of an intense pre-clinical research effort. However, most studies focus only on male animals, as preferred sex. A recent systematic review showed that only November 2019 \| Volume 11 \| Article 299 Frontiers in Aging Neuroscience \| www.frontiersin.org Age and Sex Inﬂuence Neuroinﬂammation Murtaj et al. 3 out of 51 publications included both male and female mice or rats, and only a few of them focused on aged animals (Hoogland et al., 2015). This issue is relevant since ample evidence support sex-related differences in age-dependent neurodegenerative disorders (Bouman et al., 2005; Marriott and Huet-Hudson, 2006; Azad et al., 2007; Ycaza Herrera and Mather, 2015; Vom Steeg et al., 2016). Precise regulation of the immune responses maintains brain tissue homeostasis, while a chronic inflammatory state may influence the loss of neuronal function and plasticity (Besedovsky and Rey, 2008). In the presence of immune senescence, systemic inflammation represents a major precipitating factor for cognitive disorders (Perry, 2004; Costantini et al., 2018). AD (Benitez et al., 2014; Bhattacharjee et al., 2014; Zhao and Lukiw, 2015). Females have a higher prevalence of AD compared to males, thus sex is included among the risk factors for dementia (McCarthy et al., 2012). Using in vivo imaging, Mosconi et al. (2017) demonstrated the presence of AD endo-phenotypes in the brain of asymptomatic peri-menopausal or menopausal women when compared to age-matched men. Sex dimorphism of astrocytes and microglial cells is largely recognized and has been recently demonstrated by Villa et al. (2018). Adult female microglial cells carry a neuroprotective phenotype even when transplanted into male brain (Amateau and McCarthy, 2002; Hanamsagar et al., 2017; Villa et al., 2018). Interestingly, this protective phenotype seems to be in contrast with what has been observed in aged subjects, as suggested by a whole genome profile showing that old female brains exhibit higher transcription of genes of the complement system when compared to old males. The different neuro-inflammatory signatures may explain the sex-specific susceptibility to cognitive disorders (Mangold et al., 2017). A neuro-inflammatory response induced by peripheral challenge with lipopolysaccharide (LPS) has been recently demonstrated in the brain of adult healthy volunteers using Positron Emission Tomography (PET) and a radio ligand for in vivo imaging of brain inflammation (Sandiego et al., 2015). Animals and LPS Challenge The two-way ANOVA following by Tukey post hoc test was used to evaluate statistical significance between LPS-treated groups while parametric unpaired Student’s t-test were used to compare LPS-treated to control group. Genes and relative primer sets used are listed in Table 2. homogenized in the supplied homogenization buffer and stored at −80◦C. Total RNA was extracted with the Promega Maxwell® 16 LEV simplyRNA kit using the Maxwell® 16 Instrument (Promega). cDNA synthesis was performed using 250 ng of total RNA with the ThermoScript RT-PCR System (Invitrogen) and Random Primers (Promega) in a final volume of 20 µl according to the manufacturer’s instructions. The cDNA from each sample was used to perform RT-qPCR using LightCycler 480 SYBR Green I Master Mix (Roche) on the LightCycler 480 Instrument (Roche). β-actin was used as housekeeping gene for sample normalization. All analyses were performed in triplicate. The 2−∆∆CT method was used to calculate the relative changes in gene expression in LPS-treated relative to vehicle-treated mice. The two-way ANOVA following by Tukey post hoc test was used to evaluate statistical significance between LPS-treated groups while parametric unpaired Student’s t-test were used to compare LPS-treated to control group. Genes and relative primer sets used are listed in Table 2. maintained until reaching the age of 17–18 months (aged) and were further processed. A total number of 76 mice were included in the study. For each experimental task, adult and aged mice of both sexes were randomly assigned to LPS or vehicle (saline) treatment as reported in Table 1 and in the diagram below (Figure 1). Animals were intraperitoneally injected with 0.63 mg/kg of LPS (Lipopolysaccharides of Escherichia coli, serotype 055:B5; Sigma-Aldrich) freshly dissolved in sterile saline prior to injection, or vehicle (2 mg/5 ml saline), and neuro-inflammatory responses were evaluated 6 h later (Biesmans et al., 2013). Ex vivo Binding of [18F]-VC701 to TSPO Protein There were eight mice per group and treatment was dedicated to ex vivo imaging studies. Microglia activation was evaluated using the [18F]-labeled VC701, a TSPO radio ligand developed in our facility (Di Grigoli et al., 2015; Belloli et al., 2018). This radiopharmaceutical binds to 18 kDa TSPO. In this study, we used the novel TSPO tracer [18F]-VC701 that allows to obtain adequate signal to noise ratios for the measurements of TSPO binding in mouse brain (Belloli et al., 2018). Binding of [18F]- VC701 was evaluated ex vivo at 6 h after the intraperitoneal administration of LPS or saline. Animals were injected with 4.55 ± 1.85 MBq (specific activity: 3.17 ± 1.4 Ci/µMole) of [18F]-VC701 through the tail vein and sacrificed under light anesthesia 120 min later. Blood samples were collected and counted in a gamma counter (LKB Compugamma CS1282, Wallac). Brains were rapidly removed and divided into two hemispheres. Cortex, hippocampus and cerebellum were collected and placed in pre-weighed tubes. Radioactivity concentration was counted while the tissue used for RT-PCR was stored at −80◦C for transcript measurement. After counting, tissue and blood radioactivity concentration were corrected for the half-life of [18F] (108.9 min) and expressed as percentage of the injected dose per gram of tissue (%I.D./g). To avoid a potential confounding effect due to differences in blood circulating levels, binding data were also normalized to blood uptake value. Animals and LPS Challenge g Adult (2 months) and aged (17–18 months) non-breeding male and female C57Bl/6J mice were purchased from Charles River. Animals were maintained and handled in compliance with our institutional guidelines for the care and use of experimental animals (IACUC) and the national law for animals used in research (Prot. N. SK552/2012 D.lsg. 116/1992 and N. 722/2016-PR D.lsg. 26/2016). Mice were housed in the San Raffaele Hospital animal facility, maintained in 12/12 h light/dark cycle with access ad libitum to food and water. Following 1 week of acclimation, one group of 2 months old mice (adult) were processed according to the experimental tasks described below, whereas another group of mice was November 2019 \| Volume 11 \| Article 299 Frontiers in Aging Neuroscience \| www.frontiersin.org 2 Age and Sex Inﬂuence Neuroinﬂammation Murtaj et al. TABLE 1 \| Age, body weight, sample size and treatment dosage of each experimental group. Experimental group Age Body weight (g) [18F]-VC701 binding (n) RT-PCR (n) IHC (n) Dosage (LPS/vehicle) Adult males + LPS 2 months 26.9 ± 2.7 8 5/8 3 0.63 mg/kg Adult males vehicle 2 months 26.1 ± 1.6 8 5/8 3 50 µl saline Adult females + LPS 2 months 18.3 ± 3.6 8 4/8 3 0.63 mg/kg Adult females vehicle 2 months 19.6 ± 1.1 8 5/8 3 30 µl saline Aged males + LPS 17/18 months 35.5 ± 2.5 8 5/8 3 0.63 mg/kg Aged male vehicle 17/18 months 36.4 ± 3.3 8 5/8 3 50 µl saline Aged females + LPS 17/18 months 31.1 ± 4.2 8 5/8 3 0.63 mg/kg Aged females vehicle 17/18 months 34.2 ± 6.6 8 5/8 3 50 µl saline homogenized in the supplied homogenization buffer and stored at −80◦C. Total RNA was extracted with the Promega Maxwell® 16 LEV simplyRNA kit using the Maxwell® 16 Instrument (Promega). cDNA synthesis was performed using 250 ng of total RNA with the ThermoScript RT-PCR System (Invitrogen) and Random Primers (Promega) in a final volume of 20 µl according to the manufacturer’s instructions. The cDNA from each sample was used to perform RT-qPCR using LightCycler 480 SYBR Green I Master Mix (Roche) on the LightCycler 480 Instrument (Roche). β-actin was used as housekeeping gene for sample normalization. All analyses were performed in triplicate. The 2−∆∆CT method was used to calculate the relative changes in gene expression in LPS-treated relative to vehicle-treated mice. November 2019 \| Volume 11 \| Article 299 Immunohistochemistry (IHC) Immunohistochemistry (IHC) and histopathologic analysis were performed in distinct adult and aged female mice (n = 3 per group and treatment). Mice were sacrificed 6 h after LPS or vehicle challenge and perfused with PBS followed by 4% paraformaldehyde (PFA). Brains were removed, post-fixed in PFA 4% for 24 h, cryo-protected overnight in 20% sucrose and 10 µm sections were cut on a cryostat for histological TABLE 2 \| Genes and relative primer list used in gene expression analysis. Name Primer sequence (5′-3′) β-actin F 5′-gactcctatgtgggtgacgagg-3′ β-actin R 5′ catggctggggtgttgaaggtc-3′ Trem2 F 5′-gcacctccaggaatcaagag-3′ Trem2 R 5′-gggtccagtgaggatctgaa-3′ Trem2L F 5′-tggtggtggtgttgacatttcttcc-3′ Trem2L R 5′atccagggtttagcatagttgctgc-3′ Tspo F 5′-tcagcggctaccaacct-3′ Tspo R 5′-caggattcaggcatggtgat-3′ Il-1β F 5′-gcccatcctctgtgactcat-3′ Il-1β R 5′-aggccacaggtattttgtcg-3′ Tnf-α F 5′-cctgtagcccacgtcgtag-3′ Tnf-α R 5′-gggagtagacaaggtacaaccc-3′ Il4 F 5′-tcaacccccagctagttgtc-3′ Il4 R 5′-tgttcttcgttgctgtgagg-3′ Arg1 F 5′-ttgggtggatgctcacactg-3′ Arg1 R 5′-gtacacgatgtctttggcaga-3′ IL-6 F 5′-agttgccttcttgggactga-3′ IL-6 R 5′-tccacgatttcccagagaac-3′ Frontiers in Aging Neuroscience \| www.frontiersin.org Statistical Analysis Statistical evaluation of [18F]-VC701 tracer uptake was carried out using two-way ANOVA for Multiple Comparisons with a Tukey post hoc correction with gender, aging and treatment as covariates. Data representing radio ligand uptake in each LPS-treated and vehicle group were analyzed through parametric unpaired Student’s t-test. For gene expression studies, data were normalized to housekeeping gene followed by a fold change evaluation comparing data to internal control group. Data derived from comparison of LPS-treated groups were statistically analyzed using two-way ANOVA for Multiple Comparisons with a Tukey post hoc correction, while for those reported on Supplementary Figures, data were statistically evaluated using parametric unpaired Student’s Peripheral Exposure to LPS Inﬂuences Sex- and Age-Dependent Microglia Activation TSPO expression in different brain areas of adult and aged untreated and LPS-treated male and female mice was evaluated by ex vivo binding of [18F]-VC701 to TSPO, 6 h after LPS/vehicle injection. LPS treatment induced a statistically significant increase (p < 0.05) of tracer’s uptake in the cortex and cerebellum of aged males, and in cortex and hippocampus of aged females (Supplementary Figure S1). Higher uptake of [18F]-VC701 was observed in the cortex of aged vs. adult LPS-treated females (Figure 2). This age-dependent effect was not observed in males. The percentage of tracer’s uptake in the cortex and hippocampal areas was significantly higher in LPS-treated aged females compared to age-matched vehicle (cortex 51.6%, p ≤0.017; hippocampus 86.4% p ≤0.010; cerebellum ns, 36.1%, data not shown). Tissue to blood ratio of tracer’s uptake in LPS-treated females, expressed as % of injected dose per gram of tissue (%I.D./g), is shown in Table 3. RNA Extraction and RT-qPCR Analysis RNA Extraction and RT-qPCR Analysis RT-PCR analysis was performed on five out of the eight animals/group that was used for the [18F]-VC701 experiments. One mouse in the adult female LPS-treated group was excluded due to insufficient mRNA availability. The brain areas (cortex, striatum, hippocampus and cerebellum) were dissected, Frontiers in Aging Neuroscience \| www.frontiersin.org November 2019 \| Volume 11 \| Article 299 3 Murtaj et al. Age and Sex Inﬂuence Neuroinﬂammation FIGURE 1 \| Schematic diagram of the experimental design and readouts. \| Schematic diagram of the experimental design and readouts. t-test. Immunohistochemical analyses were performed using two-way ANOVA for Multiple Comparison with Tukey post hoc correction. analysis in bright-field microscopy, slices were stained using standard protocols for hematoxylin and eosin (using Mayer’s Hematoxylin, BioOptica #05-06002/L and Eosin, BioOptica #05-10002/L). Analyses were performed using the Prism V6.0 software (GraphPad Prism, San Diego, CA, USA). Statistical significance was accepted when ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. For IHC, slices were immuno-stained with polyclonal rabbit anti-Iba-1 antibody (Wako, #019-19741) and polyclonal rabbit anti-GFAP antibody (Novus Biologicals, #NB300-141). Any endogenous peroxidase activity was quenched by incubating the slices with 0.3% H2O2 in methanol for 10 min at RT. Both antibodies were used at a dilution of 1:1,000, incubated for 1 h at RT with EXPOSE rabbit specific HRP/DAB detection IHC kit (Abcam, #ab80437). DAB chromogen was than applied to each section for 5 min at RT and counterstained with Mayer’s hematoxylin, dehydrated and mounted with Eukitt (BioOptica, #09-00100). Positive and negative controls were run simultaneously. Slides were acquired with Aperio AT2 digital scanner at a magnification of 40× (Leica Biosystems). Region of interest (ROI) were drowned manually on half hemisphere for cortex, on the entire hippocampus, and on a selected region of the cerebellum. Percentage of positive cells were calculated using Aperio eSlide Manager (Leica Biosystems). From the data obtained (weak, medium and strong positive intensity), weak positive were not included in the analysis as considered as background. Frontiers in Aging Neuroscience \| www.frontiersin.org Sex and Age Effect on Transcript Levels of Markers of Microglia Activation Interestingly, we found an age- and sex-dependent effect on the expression of the neuro-inflammatory markers TREM2 and TREML2 after LPS administration. Our results showed that peripheral LPS injection induced a significant decrease of TREM2 transcript levels in all brain regions of adult males and females (Supplementary Figure S2D). Yet in aged males, LPS injection did not decrease TREM2 transcript levels, which were instead significantly decreased in all brain regions of aged females (Supplementary Figure S3D). When we specifically compared TREM2 transcript levels among LPS-treated mice, we found that they were significantly higher in aged males compared to aged females (p < 0.0002), while no differences were detected in adult mice of both sexes (Figure 4A). TREML2 transcript levels were significantly increased in the hippocampus of aged males compared to aged females (p < 0.01), and also in the cortex, although not reaching statistical significance (Figure 4B, Supplementary Figures S2E, S3E). Female Brain Shows a Higher Pro-inﬂammatory Response to LPS During Aging In order to confirm that the higher [18F]-VC701 uptake was associated with a pro-inflammatory response of microglia, we measured the levels of IL-1β, TNF-α, IL-6, IL-4 and Arg-1 transcripts in LPS-treated and untreated mice. In all brain regions, peripheral LPS injection induced neuroinflammation irrespective of sex and age, as shown by significantly increased November 2019 \| Volume 11 \| Article 299 Frontiers in Aging Neuroscience \| www.frontiersin.org 4 Murtaj et al. Age and Sex Inﬂuence Neuroinﬂammation TABLE 3 \| Tissue to blood [18F]-VC701 uptake ratio. Brain Region Tissue/blood ratio (%I.D./g; mean ± SD) LPS-treated adult females LPS-treated aged females Cortex 1.6 ± 0.6 2.52 ± 0.7 Hippocampus 2.31 ± 1.4 2.46 ± 0.9 Cerebellum 2.75 ± 1.3 3.75 ± 1.1 TABLE 3 \| Tissue to blood [18F]-VC701 uptake ratio. FIGURE 2 \| Translocator protein (TSPO) tracer uptake reveal microglial activation increased in aged female lipopolysaccharide (LPS)-treated animals. [18F]-VC701 brain tracer uptake in cortex (A); hippocampus (B) and cerebellum (C) on LPS-treated adult and aged male and female mice. Data were analyzed using two-way ANOVA analysis with Tukey post hoc and are expressed as percentage (%) of injected dose per gram of tissue to blood ratio, mean ± SD, eight mice per group; Mouse brain stained slices (cortex, hippocampus and cerebellum) derived from immunohistochemistry (IHC) studies. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. IL-6 in the cerebellum were detected in aged females compared to aged males (Figures 3A,C). TNF-α transcript levels were increased in the cortex and hippocampus of aged compared to adult females, although the differences were not statistically significant (Figure 3B). Microglia Activation and Astrogliosis in Aged Female Animals FIGURE 2 \| Translocator protein (TSPO) tracer uptake reveal microglial activation increased in aged female lipopolysaccharide (LPS)-treated animals. [18F]-VC701 brain tracer uptake in cortex (A); hippocampus (B) and cerebellum (C) on LPS-treated adult and aged male and female mice. Data were analyzed using two-way ANOVA analysis with Tukey post hoc and are expressed as percentage (%) of injected dose per gram of tissue to blood ratio, mean ± SD, eight mice per group; Mouse brain stained slices (cortex, hippocampus and cerebellum) derived from immunohistochemistry (IHC) studies. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. As major differences in the neuro-inflammatory response to LPS were observed in females, we further evaluated glia activation in females by IHC. In all LPS-treated females, H&E staining showed no sign of neuronal loss or morphological changes in the three brain areas examined (cortex, cerebellum and hippocampus, data not shown). In LPS-treated aged females an increased percentage of Iba-1 positive cells were detected when compared to LPS-treated adult females, and vehicle- treated adult and aged females, which is indicative of a higher inflammatory status (Figure 5A, cortex). Microglia activation was suggested by cell morphology in the cerebral and cerebellar cortex of LPS-treated aged vs. LPS-treated adult females and vehicle-treated females (Figures 5A–C, cortex and cerebellum). In the hippocampus both LPS and vehicle-treated aged females showed a higher degree of Iba-1 immunoreactivity in comparison with adult females reaching statistical significance in vehicle-treated aged vs. vehicle-treated adult mice (Figure 5C, hippocampus). transcript levels of IL-1β, IL-6 and TNF-α (Supplementary Figures S2A–C, S3A–C). Arg-1 and IL-4 transcripts were negligibly expressed in both LPS-treated and untreated mice (data not shown). In agreement with [18F]-VC701 uptake results, in female but not in male brain, age affected levels of the pro-inflammatory cytokines IL-1β, TNF-α and IL-6 in response to the LPS challenge. Aged females compared to adult females showed increased levels of IL-1β and IL-6 in all areas, which was statistically significant in the cortex for IL-1β, and in both cortex and cerebellum for IL-6 (Figures 3A,C). No age-dependent effects on IL-1β, TNF-α and IL-6 transcript levels were detected in males. Increased IL-1β transcript levels in the cortex and Astroglial cells showed an increase in GFAP immunoreactivity in aged compared to adult females (Figures 6A,B). This observation was consistent in the cortex, Astroglial cells showed an increase in GFAP immunoreactivity in aged compared to adult females (Figures 6A,B). Microglia Activation and Astrogliosis in Aged Female Animals This observation was consistent in the cortex, November 2019 \| Volume 11 \| Article 299 Frontiers in Aging Neuroscience \| www.frontiersin.org 5 Murtaj et al. Age and Sex Inﬂuence Neuroinﬂammation 3 \| Increased expression levels of pro-inﬂammatory cytokines in aged LPS treated mice. Gene expression analysis of IL-1β (A), TNF-α (B) and IL-6 (C) by n cortex, hippocampus and cerebellum of all LPS-treated animals. All the values were normalized to mouse β-actin and expressed as fold change referred control group. Bars represent the average of triplicate measurements and error bars represent ± SEM, ﬁve animals per group. Statistical analysis was using two-way ANOVA following by Tukey post hoc test of the relative mRNA expression of LPS-treated mice previously normalized to internal control < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. FIGURE 3 \| Increased expression levels of pro-inﬂammatory cytokines in aged LPS treated mice. Gene expression analysis of IL-1β (A), TNF-α (B) and IL-6 (C) by RT-qPCR in cortex, hippocampus and cerebellum of all LPS-treated animals. All the values were normalized to mouse β-actin and expressed as fold change referred to internal control group. Bars represent the average of triplicate measurements and error bars represent ± SEM, ﬁve animals per group. Statistical analysis was performed using two-way ANOVA following by Tukey post hoc test of the relative mRNA expression of LPS-treated mice previously normalized to internal control group. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. FIGURE 3 \| Increased expression levels of pro-inﬂammatory cytokines in aged LPS treated mice. Gene expression analysis of IL-1β (A), TNF-α (B) and IL-6 (C) by RT-qPCR in cortex, hippocampus and cerebellum of all LPS-treated animals. All the values were normalized to mouse β-actin and expressed as fold change referred to internal control group. Bars represent the average of triplicate measurements and error bars represent ± SEM, ﬁve animals per group. Statistical analysis was performed using two-way ANOVA following by Tukey post hoc test of the relative mRNA expression of LPS-treated mice previously normalized to internal control group. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. in LPS-treated aged females for Iba-1 and in vehicle and LPS-treated aged females for GFAP (data not shown). cerebellum and hippocampus of aged females where astrocytes also showed hyperplasia of cytoplasm and of cellular processes resembling an activated phenotype. Microglia Activation and Astrogliosis in Aged Female Animals Quantification analysis showed a significant higher percentage of GFAP positive cells in cortex and hippocampus of aged compared to adult females. A similar trend was observed also in the cerebellar region (Figure 6C). Furthermore, in the sections where olfactory bulb was present, a general increase in immunoreactivity for both Iba-1 and GFAP was observed, again more evident DISCUSSION In this study, we demonstrate for the first time that both age and sex influence an early neuro-inflammatory response after an acute mild peripheral LPS challenge in mice. In comparison to control conditions, LPS-treated aged male and female mice November 2019 \| Volume 11 \| Article 299 Frontiers in Aging Neuroscience \| www.frontiersin.org 6 Murtaj et al. Age and Sex Inﬂuence Neuroinﬂammation Murtaj et al. Age and Sex Inﬂuence Neuroinﬂammation FIGURE 4 \| Triggering receptor expressed on myeloid cells like 2 (TREM2) is differentially expressed in aged LPS treated mice. Gene expression analysis of TREM2 (A), TREML2 (B), two immunomodulatory markers by RT-qPCR in cortex, hippocampus and cerebellum of all LPS-treated animals. All the values were normalized to mouse β-actin and expressed as fold change referred to internal control group. Bars represent the average of triplicate measurements and error bars represent ± SEM, ﬁve animals per group. Statistical analysis was performed using two-way ANOVA following by Tukey post hoc test of the relative mRNA expression of LPS-treated mice previously normalized to internal control group. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. showed an increase [18F]-VC701 binding indicative of higher i f TSPO A i ff b d i (Di Grigoli et al., 2015). TSPO’s main role is to transport h l l h i h d i l b Whil FIGURE 4 \| Triggering receptor expressed on myeloid cells like 2 (TREM2) is differentially expressed in aged LPS treated mice. Gene expression analysis of TREM2 (A), TREML2 (B), two immunomodulatory markers by RT-qPCR in cortex, hippocampus and cerebellum of all LPS-treated animals. All the values were normalized to mouse β-actin and expressed as fold change referred to internal control group. Bars represent the average of triplicate measurements and error bars represent ± SEM, ﬁve animals per group. Statistical analysis was performed using two-way ANOVA following by Tukey post hoc test of the relative mRNA expression of LPS-treated mice previously normalized to internal control group. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (Di Grigoli et al., 2015). TSPO’s main role is to transport cholesterol across the outer mitochondrial membrane. While TSPO is negligibly expressed in the normal brain parenchyma, its expression increases upon microglia activation and macrophage showed an increase [18F]-VC701 binding indicative of higher expression of TSPO. A prominent effect was observed in LPS-treated aged compared to adult females. DISCUSSION [18F]-VC701 is a structural analog of PK11195, which targets 18 kDa TSPO November 2019 \| Volume 11 \| Article 299 Frontiers in Aging Neuroscience \| www.frontiersin.org Frontiers in Aging Neuroscience \| www.frontiersin.org 7 Age and Sex Inﬂuence Neuroinﬂammation Murtaj et al. were sexually differentiated in the adult brain, as male mice carried a phenotype more poised to inflammatory reactions than female microglia. Moreover, in females, microglia displayed neuroprotective features in an acute pre-clinical model of ischemic insult (Villa et al., 2018). Gene sexual dimorphism has also been reported in humans by Berchtold et al. (2008). This study shows that males display a greater number of gene expression changes (mainly downregulation) during the transition to the sixth and seventh decade of life while females show more prominent gene expression changes later in life, during the eighth and ninth decade of life. Moreover, increased immune activation was greater in the female brain, as shown by a higher percentage of up-regulated genes involved in the immune response and inflammation (34% in males and 50% in females; Berchtold et al., 2008). FIGURE 5 \| Increased IBA1 immunoreactivity and changes in glia morphology in brain areas of LPS-treated aged vs. LPS-treated adult females. (A) Immunostaining of Iba-1+ cells in cerebral cortex, hippocampus and cerebellum from one representative vehicle and one representative LPS-treated adult female mouse. (B) Immunostaining of Iba-1+ cells in cerebral cortex, hippocampus and cerebellum from one representative vehicle and one representative LPS-treated aged female mouse. (C) Quantiﬁcation of the percentage of Iba-1+ cells in cortex, hippocampus and cerebellum of all female groups used in the study. Data are expressed as mean ± SEM of selected region of interest (ROI) performed on three animals per group, scale bar = 100 µm. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. To better understand the nature of the microglia response, we evaluated the effect of the peripheral LPS challenge on transcription of a few inflammatory mediators: IL-1β, TNF-α, IL-6, Arg1 and IL-4. We showed that LPS induced a pro-inflammatory reaction in the brain of male and female mice as indicated by the up-regulation of IL-1β, TNF-α and IL-6 gene expression. However, in agreement with [18F]-VC701 studies, the effect of LPS on pro-inflammatory cytokine transcript levels was significantly increased in aged compared to adult females, as well as in aged females compared to aged males. DISCUSSION As a major age effect was observed in females, we further analyzed microglia and astrocyte status by IHC in all brain regions of adult and aged females. In agreement with TSPO expression and pro-inflammatory cytokine transcript levels, we found an increased expression of microglia and astrocyte markers in LPS-treated and vehicle-treated aged compared to adult females, with some regional differences. Increased Iba-1 expression was present particularly in the cerebral cortex of LPS-treated aged animals, whereas GFAP immunoreactivity was higher in aged females in all the cerebral areas examined, regardless of LPS administration. Finally, the higher Iba-1 and GFAP signals detected in aged females was associated with a different cellular morphology. Altogether, our results highlight the acute onset of a higher neuro-inflammatory response in aged females. A similar response was also reported in female 3xTg-AD mice, where neuroinflammation was associated with progressive cognitive decline that increased as a function of age (Belfiore et al., 2019). FIGURE 5 \| Increased IBA1 immunoreactivity and changes in glia morphology in brain areas of LPS-treated aged vs. LPS-treated adult females. (A) Immunostaining of Iba-1+ cells in cerebral cortex, hippocampus and cerebellum from one representative vehicle and one representative LPS-treated adult female mouse. (B) Immunostaining of Iba-1+ cells in cerebral cortex, hippocampus and cerebellum from one representative vehicle and one representative LPS-treated aged female mouse. (C) Quantiﬁcation of the percentage of Iba-1+ cells in cortex, hippocampus and cerebellum of all female groups used in the study. Data are expressed as mean ± SEM of selected region of interest (ROI) performed on three animals per group, scale bar = 100 µm. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. FIGURE 5 \| Increased IBA1 immunoreactivity and changes in glia morphology in brain areas of LPS-treated aged vs. LPS-treated adult GURE 5 \| Increased IBA1 immunoreactivity and changes in glia FIGURE 5 \| Increased IBA1 immunoreactivity and changes in glia morphology in brain areas of LPS-treated aged vs. LPS-treated adult females. (A) Immunostaining of Iba-1+ cells in cerebral cortex, hippocampus and cerebellum from one representative vehicle and one representative LPS-treated adult female mouse. (B) Immunostaining of Iba-1+ cells in cerebral cortex, hippocampus and cerebellum from one representative vehicle and one representative LPS-treated aged female mouse. (C) Quantiﬁcation of the percentage of Iba-1+ cells in cortex, hippocampus and cerebellum of all female groups used in the study. Frontiers in Aging Neuroscience \| www.frontiersin.org DISCUSSION Data are expressed as mean ± SEM of selected region of interest (ROI) performed on three animals per group, scale bar = 100 µm. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Microglia, as the primary source of pro-inflammatory cytokines, are pivotal mediators of neuroinflammation (Colonna and Butovsky, 2017). The amplitude and duration of the context-dependent activation of microglia are regulated by many pattern-recognition receptors, and immune receptors that deliver either activating or inhibitory signals. TREM2 and TREML2 are two microglia/monocyte regulators with potential and opposite roles in neurodegenerative disorders such as AD (Zsido et al., 2017). We show that acute peripheral administration of LPS induced an opposite modulation of TREM2 and TREML2 transcripts. In all groups analyzed, except aged male mice, we showed that LPS reduced TREM2 while increasing TREML2 transcript levels. TREM2 regulates critical functions of microglia including inhibition of pro-inflammatory responses infiltration (Betlazar et al., 2018). Thus TSPO up-regulation has been mainly associated with a pro-inflammatory status of microglia (Bonsack et al., 2016). PET-TSPO has been successfully applied to image the neuro-inflammatory reaction to LPS administration in the brain of human subjects, showing an increased binding of the TSPO radiotracer [11C]-PBR28 in all brain regions (Sandiego et al., 2015). Co-localization of TSPO with CD206-positive alternatively-activated microglia has also been reported (Liu et al., 2015). Our results, although obtained from analysis of whole tissue are in agreement with transcriptomic data of purified microglia (Villa et al., 2018). Villa et al. (2018) demonstrated that microglia cells Frontiers in Aging Neuroscience \| www.frontiersin.org November 2019 \| Volume 11 \| Article 299 Frontiers in Aging Neuroscience \| www.frontiersin.org 8 Age and Sex Inﬂuence Neuroinﬂammation Murtaj et al. FIGURE 6 \| Increased astrocytosis and changes in glia morphology in brain areas of LPS-treated and vehicle-treated aged females. (A) Immunostaining of GFAP+ cell in cerebral cortex, hippocampus and cerebellum from one representative vehicle and one representative LPS-treated adult female mouse. (B) Immunostaining of GFAP+ astrocyte cells in cerebral cortex, hippocampus and cerebellum from one representative vehicle and one representative LPS-treated aged female mouse. (C) Quantiﬁcation of percentage of GFAP+ cells in cortex, hippocampus and cerebellum of all female groups used in the study. Data are expressed as mean ± SEM of selected ROI performed on three animals per group, scale bar = 100 µm. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. 2017). DISCUSSION In particular, reduction of TREM2 was associated with a decreased microglia proliferation, an effect that was absent when TREML2 was knocked down. During aging, microglia undergo cellular senescence losing regenerative and trophic functions (Mecca et al., 2018). According to Colonna and Wang (2016) TREM2 protects the aged brain from insults. Indeed, carrying a loss of function mutations in the TREM2 gene represents an important risk factor for dementia (Gao et al., 2017). In vitro experiments demonstrated that, silencing of TREM2 exacerbated LPS-induced pro-inflammatory response in BV2 microglia (Zhong et al., 2015), In contrast, overexpression of TREM2 lowered the pro-inflammatory response, thus supporting the hypothesis that TREM2 expression modulates the acute and transient pro-inflammatory responses induced by LPS (Zhong et al., 2015). Thus, the higher TREM2 expression observed in aged male mice might represent a strategy to counterbalance the pro-inflammatory milieu of aging, partially sustained by activation of signaling pathways mediated by TREML2, which likely activates apoptosis and modulates microglial functions (de Freitas et al., 2012; Zheng et al., 2016). In aged female brains, we observed up-regulation of reactive GFAP-positive astroglia in all the regions examined independently from LPS administration. Although GFAP expression is limited to only a fraction of astrocytes with a substantial regional heterogeneity, its expression has been associated with reactive astrogliosis (Verkhratsky and Nedergaard, 2018). During aging, high levels of GFAP expression were identified as ‘‘mild to moderate astrogliosis’’ while the presence of cellular hypertrophy in addition to GFAP increase appears to be associated to ‘‘severe diffuse astrogliosis’’ (Cohen and Torres, 2019). Matias et al. (2019) reported that the astrocyte response to brain aging showed intra-regional heterogeneity (Matias et al., 2019): astrocytes in the hippocampal region presented an age-dependent hypertrophy, similar to the astrocyte morphology detected in our study, which may contribute to synapse loss and neuroinflammation. FIGURE 6 \| Increased astrocytosis and changes in glia morphology in brain areas of LPS-treated and vehicle-treated aged females. (A) Immunostaining of GFAP+ cell in cerebral cortex, hippocampus and cerebellum from one representative vehicle and one representative LPS-treated adult female mouse. (B) Immunostaining of GFAP+ astrocyte cells in cerebral cortex, hippocampus and cerebellum from one representative vehicle and one representative LPS-treated aged female mouse. (C) Quantiﬁcation of percentage of GFAP+ cells in cortex, hippocampus and cerebellum of all female groups used in the study. Frontiers in Aging Neuroscience \| www.frontiersin.org CONCLUSION This work was supported by grants from: The Italian Ministry of Education, University and Research (Ministero dell’Istruzione, dell’Universitá e della Ricerca; MIUR) Project ‘‘Identification, validation and commercial development of new diagnostic and prognostic biomarkers for complex trait diseases (IVASCOMAR, Prot. CTN01 00177 165430)’’. In conclusion, the results of this study show that the neuro- inflammatory response of a mouse’s brain to an acute peripheral LPS challenge is sex- and age-dependent, and is likely to involve multiple cellular and molecular regulators of neuronal functions. In conclusion, the results of this study show that the neuro- inflammatory response of a mouse’s brain to an acute peripheral LPS challenge is sex- and age-dependent, and is likely to involve multiple cellular and molecular regulators of neuronal functions. Our results might set the basis for further studies aimed at characterizing sex-related targets involved in the modulation of the neuro-inflammatory response in aging. This knowledge could be relevant for the treatment of conditions such as delirium and dementia. Our results might set the basis for further studies aimed at characterizing sex-related targets involved in the modulation of the neuro-inflammatory response in aging. This knowledge could be relevant for the treatment of conditions such as delirium and dementia. ACKNOWLEDGMENTS We thank Pasquale Simonelli for technical support with animal preparation and ex vivo binding experiments. DATA AVAILABILITY STATEMENT The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnagi.2019.002 99/full#supplementary-material. The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher. ETHICS STATEMENT The animal study was approved by our Institutional Guidelines for the Care and Use of Experimental Animals (IACUC) and the National Law for animals used in research (Prot. N. SK552/2012 D.lsg. 116/1992 and N. 722/2016-PR D.lsg. 26/2016). Future studies should explore chronic neuroinflammation and morphological-functional changes in glial cell upon repetitive systemic insult or an acute severe neural event. As the cortex represents in our study the most important region influenced by sex and age, we will further explore gene modulation of specific pathways trough transcriptomic analysis of cortical glia in adult and aged female and male mice. Furthermore, specific behavioral phenotypes such as attention and active inhibitory processes associated with cognitive frailty will be investigated. AUTHOR CONTRIBUTIONS VMu: data generation, collection, analysis and interpretation, and manuscript drafting. SB and GG: data generation and collection. MP: support in qRT-PCR data generation and analysis. EB, VR-M, and PM: support in IHC data analysis and interpretation. AC, VMa, and CM: production and validation of the tracer [18F]-VC701. GB: critical review of the manuscript. PP-B and RM: study conception and design, manuscript preparation. All the authors approved the final version of the manuscript. Finally, it would be appropriate to ascertain if age- and sex-specific patterns of neuroinflammation could be identifiable in humans too, and to clarify the mechanisms involved in such differences. DISCUSSION Data are expressed as mean ± SEM of selected ROI performed on three animals per group, scale bar = 100 µm. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Interestingly, increased levels of neuro-inflammatory markers, including GFAP, are present in the brain of AD patients. Recently Barroeta-Espar et al. (2019) demonstrated reduced levels of GFAP and a different pattern of cytokines expression in subjects resilient to AD pathology. This selected population of aged subjects harbor plaque and tangle loads that in some cases are equivalent to those found in AD, although in absence of the typical patterns of neuronal/synaptic loss seen in the pathology. These findings suggest that down-regulation of neuroinflammation is one of the differential traits of human brain resilience to AD pathology, a mechanism that might be less efficient in female brain. Implications of our findings may impact the development of clinical phenotypes such as delirium and dementia. Delirium is one of the most common neuropsychiatric conditions among older people, which is known to develop as an acute consequence of peripheral infection or inflammation originating outside the brain (Cerejeira et al., 2010; Franceschi and Campisi, 2014; Bu et al., 2015; Morandi et al., 2018). and stimulation of phagocytosis of apoptotic neurons (Piccio et al., 2008). TREM2-transduced myeloid precursors mediate nervous tissue debris clearance and facilitate recovery in an animal model of multiple sclerosis (Takahashi et al., 2005, 2007). Moreover, TREM2 binds poly-anionic ligands including LPS (Wang et al., 2015). Previous findings showed that LPS stimulation suppressed TREM2 levels in microglia, at the same time inducing pro-inflammatory cytokines secretion (Zheng et al., 2016). Peripheral LPS challenge (at a similar dose to that used in our study) induced up-regulation of TREML2 and down-regulation of TREM2 levels in microglia, and increase of pro-inflammatory cytokines (Zheng et al., 2016; Zhang et al., Frontiers in Aging Neuroscience \| www.frontiersin.org November 2019 \| Volume 11 \| Article 299 Frontiers in Aging Neuroscience \| www.frontiersin.org 9 Age and Sex Inﬂuence Neuroinﬂammation Murtaj et al. The higher the level of frailty, the higher the likelihood of developing delirium in reaction to an inflammatory stimulus (Bellelli et al., 2017; Persico et al., 2018). Neuroinflammation has also been recognized as a pathophysiological mechanism of dementia, especially AD, again with frailty as a modulating factor of the disease clinical expression (Tao et al., 2018; Wallace et al., 2019). REFERENCES review about the existing evidence. Front. Med. 4:188. doi: 10.3389/fmed.2017. 00188 review about the existing evidence. Front. Med. 4:188. doi: 10.3389/fmed.2017. 00188 Amateau, S. K., and McCarthy, M. M. (2002). Sexual differentiation of astrocyte morphology in the developing rat preoptic area. J. Neuroendocrinol. 14, 904–910. doi: 10.1046/j.1365-2826.2002.00858.x Belloli, S., Zanotti, L., Murtaj, V., Mazzon, C., Di Grigoli, G., Monterisi, C., et al. (2018). 18F-VC701-PET and MRI in the in vivo neuroinflammation assessment of a mouse model of multiple sclerosis. J. 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https://openalex.org/W2342762837	https://europepmc.org/articles/pmc5324630?pdf=render	English	null	Experiences of cervical screening and barriers to participation in the context of an organised programme: a systematic review and thematic synthesis	Psycho-oncology	2,016	cc-by	9,236	© 2016 The Authors. Psycho-Oncology Published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Abstract Objective: As uptake of cervical screening continues to decline, this systematic review synthesises the qualitative literature on women’s perceptions and experiences of cervical screening in the context of an organised call–recall programme, in order to understand the barriers to informed uptake. Methods: We searched nine databases for English language peer-reviewed publications reporting on qualitative data from screening-eligible women, exploring barriers to cervical screening in countries that offer a nationally organised call–recall programme. Evidence was integrated using thematic synthesis. Results: Thirty-nine papers from the UK, Australia, Sweden and Korea were included. The major- ity of participants had attended screening at least once. Two broad themes were identiﬁed: (a) should I go for screening? and (b) screening is a big deal. In considering whether to attend, women discussed the personal relevance and value of screening. Women who had previously attended described how it was a big deal, physically and emotionally, and the varied threats that screening presents. Practical barriers affected whether women translated screening intentions into action. Conclusions: The variation in women’s understanding and perceptions of cervical screening suggests that interventions tailored to decisional stage may be of value in increasing engagement with the invitation and uptake of screening in those who wish to take part. There is also a need for further research with women who have never attended screening, especially those who remain unaware or unengaged, as their perspectives are lacking in the existing literature. g g , p p g g © 2016 The Authors. Psycho-Oncology Published by John Wiley & Sons Ltd. When considering programme coverage, it is important to remember that levels of attendance rely on individual women making the decision to attend and translating that decision into action. Therefore, research exploring the experiences and attitudes of the programme-eligible popu- lation is important in informing policy and intervention design. A range of reasons for nonattendance have been identiﬁed using qualitative methods, including attitudinal factors such as embarrassment or fear of an abnormal result, as well as practical barriers such as clinic opening hours or availability of childcare. Despite the wealth of evidence available, the process of reading and interpreting original qualitative research can be arduous, and studies are not designed to be representative. Experiences of cervical screening and barriers to participation in the context of an organised programme: a systematic review and thematic synthesis Amanda J. Chorley, Laura A. V. Marlow, Alice S. Forster, Jessica B. Haddrell and Jo Waller* Cancer Research UK Health Behaviour Research Centre, Department of Epidemiology and Public Health, UCL, London, UK Correspondence to: Cancer Research UK Health Behaviour Research Centre, Department of Epidemiology and Public Health, UCL, Gower Street, London WC1E 6BT, UK. E-mail: j.waller@ucl.ac.uk Psycho-Oncology Psycho-Oncology 26: 161–172 (2017) Published online 12 April 2016 in Wi Psycho-Oncology Psycho-Oncology 26: 161–172 (2017) Published online 12 April 2016 in Wi gy ncology 26: 161–172 (2017) d online 12 April 2016 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/pon.4126 : 161 172 (2017) 2 April 2016 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/pon.4126 Received: 15 December 2015 Revised: 4 February 2016 Accepted: 7 March 2016 Correspondence to: Cancer Research UK Health Behaviour Research Centre, Department of Epidemiology and Public Health, UCL, Gower Street, London WC1E 6BT, UK. E-mail: j.waller@ucl.ac.uk © 2016 The Authors. Psycho-Oncology Published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited ology Published by John Wiley & Sons Ltd. the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, l it d Background In recent decades, many countries have had great success in reducing cervical cancer incidence and mortality through offering population-based screening programmes, using centralised registers of eligible women to generate invitations at regular intervals. For example, the national screening programme in the UK has averted an estimated 100 000 deaths from cervical cancer since its inception in 1988 [1]. Such systems remove the need for women to remember that they need screening and provide details of how to go about it. Many programmes also offer screening free at the point of delivery, reducing ﬁnancial barriers. Despite this, there are still many women invited to partic- ipate who do not attend according to recommendations, and in recent years, coverage of many programmes has been suboptimal. In the England in 2014–2015, age-appropriate coverage1 was 73.5% [2], with coverage for women aged 25–29 years just 63.3%. Heterogeneity also exists in cover- age by ethnic and socioeconomic group, with lower atten- dance in Black and Asian minority ethnic (BAME) groups [3] and women from socioeconomically deprived back- grounds [4,5]. Previous reviews have drawn together evidence on bar- riers to and experiences of screening for speciﬁc popula- tion subgroups [6–11], within a particular country [12], from speciﬁc theoretical viewpoints [13], or with a focus on a singular aspect of screening such as risk perceptions [14]. However, no attempt has been made to synthesise the existing qualitative literature in a way that incorporates all women regardless of screening history or demographic characteristics, within a shared screening context across 162 A. J. Chorley et al. Data synthesis multiple countries. Synthesising qualitative research is a relatively new method that seeks to generate a novel inter- pretation of a phenomenon through the comparison and translation of concepts across studies, while maintaining rigorous and transparent standards of analysis [15–18]. This allows for the generation of new knowledge, which ‘goes beyond’ that in any original piece of research, and produces a coherent, convincing argument about the major attributes of the phenomenon of interest. Articles were synthesised according to the principles of the- matic synthesis [21], chosen because of its explicit focus on creating an end product, which is useful and accessible to re- searchers and policy makers and in intervention design. Ini- tially, A.C., L.M. and J.W. independently carried out free coding of one third of the data, with codes generated induc- tively. The similarities and differences in identiﬁed codes were discussed and arranged into hierarchical groupings, which fully described emergent themes. A coding frame that represented concepts across all studies was developed and applied to the data by A.C. and L.M. using NVIVO 10 (QSR International, Melbourne, Australia) [22], with reﬁne- ments made after the coding of 10 articles. All text was coded, unless it met speciﬁc exclusion criteria (Supporting Information 3). Uncertainties regarding coding were re- solved through discussion. Finally, A.C., L.M. and J.W. familiarised themselves with the content of each code, and higher order themes were developed through discussion. The aim of the current study was to carry out a syn- thesis of the available qualitative research on women’s perceptions and experiences of screening, allowing the identiﬁcation of important themes across contexts, increasing generalisability and creating practically useful information. Methods This review was prospectively registered with PROSPERO (registration number: CRD42015017075) and reported according to PRISMA [19] and ENTREQ [20] guidelines. Search strategy On 12 February 2015, we conducted a comprehensive search of published qualitative literature, using MEDLINE, PsycINFO, Embase, Social Policy and Practice, CINAHL Plus, ProQuest Social Science Journals, Anthrosource, POPLINE and Web of Science. Articles were included if they reported primary qualitative data on women’s percep- tion or experiences of cervical screening and were based in a country with a well-established (10+years) call–recall programme (Supporting Information 1). Articles also had to be available in English and have a publication date after the start of the organised cervical screening programme in the country in question. Search terms (Supporting Informa- tion 2) covered cervical screening (e.g. ‘smear test’), quali- tative methodology (e.g. ‘focus groups’), barriers to screening (e.g. ‘perception’) and country of origin (e.g. ‘Australia’) and were linked using Boolean operators, with truncations and wildcards where appropriate. Additional articles were identiﬁed through the reference lists of included articles and excluded review articles, and forward citation searches. Results Overall, 450 unique articles were identiﬁed. After screen- ing of titles and abstracts, 103 articles remained, and the full texts were reviewed. From these, 32 articles met the eligibility criteria, with a further seven articles identiﬁed through reference lists and forward citation searches. Thirty-nine articles were included in the ﬁnal synthesis (Supporting Information 4). Only four articles had a CASP appraisal score of less than ﬁve (out of nine). As no theme relied on a single study, it is unlikely that inclu- sion of these studies substantially affected the results. y Included studies were published between 1988 and 2015 (see Table 1 for full characteristics). Over half were based in the UK (51.3%), with others coming from Australia (28.2%), Sweden (17.9%) and Korea (2.6%). Studies used interviews (51.3%), focus groups (30.8%) or a combination of the two (17.9%). Three studies (7.7%) also used open text responses from surveys or anonymous fax messages. Almost half of the articles (48.7%) focused on a speciﬁc subgroup of the population, mostly BAME women (n=14). Most women across studies had attended cervical screening at least once. However, a number of studies included women who had never attended. Four studies (with women from BAME groups and deaf women) included some participants who were unaware of screening [24,43,49,62]. Despite their Quality assessment The quality of included studies was assessed by A.C. and J. H. using the critical appraisal skills programme (CASP) qual- itative checklist [23]. Studies with a score of 0–4 were con- sidered to be low quality, while scores of 5–9 were considered high quality. No study was excluded on the basis of quality. © 2016 The Authors. Psycho-Oncology Published by John Wiley & Sons Ltd. Data extraction After the removal of duplicates, all articles were indexed in Microsoft Excel. A.C. and L.M. screened all titles and abstracts and, subsequently, the full text of remaining articles, to establish whether they met the inclusion criteria. At each stage, disagreement was resolved through discussion. Primary outcome data, deﬁned as participant quotes and authors’ interpretation of textual data, were extracted into a Microsoft Word document. © 2016 The Authors. Psycho-Oncology Published by John Wiley & Sons Ltd. Psycho-Oncology 26: 161–172 (2017) DOI: 10.1002/pon Psycho-Oncology 26: 161–172 (2017) DOI: 10.1002/pon Experiences of cervical screening and barriers to participation 163 Table 1. Characteristics of studies included in review Country Eligible participantsa (n) Age range (years) Population Study design Analytic method CASP score Abdullahi et al. (2009) [24] UK 50 25–64 Somali-born women Focus groups + in- depth interviews Thematic analysis 8 Armstrong (2005)b [25] UK 35 20–64c Nonspeciﬁc Depth interviews Inductive 6 Armstrong (2007)b [26] UK 35 20–64c Nonspeciﬁc In-depth interviews Inductive 5 Armstrong et al. (2012)b [27] UK 34 26–60 Nonspeciﬁc Semistructured interviews Constant comparative method 8 Blomberg et al. (2008) [28] Sweden 98 Not stated Women actively declining cervical screening participation Telephone interviews and fax messages Interpretive description 8 Blomberg et al. (2011a)d [29] Sweden 138 30 Thirty-year-old women Focus groups (face to face and Internet based) + one interview Interpretive description 8 Blomberg et al. (2011b) [30] Sweden 38 29–32 Thirty-year-old women + convenience sample of three women aged 29– 32 years Focus groups + one interview Inductive 8 Box (1998) [31] UK 17 Min. 16 BAME women In-depth interviews Not stated 4 Broughton and Thomson (2000) [32] UK 52 20–64 Women with mild to moderate learning disabilities Semistructured interviews Thematic approach 8 Bush (2000) [33] UK 35 20–64 Nonspeciﬁc Semistructured in- depth interviews + qualitative comments from a survey Not stated 7 Cadman et al. (2015) [34] UK 23 23–63 Hindu women Focus groups Framework analysis 8 Cadman et al. (2012) [35] UK 124 20–59 Women reporting a history of sexual abuse Qualitative survey responses + one focus group Content analysis 8 Chiu et al. (1999) [36] UK 27 Not stated BAME women Focus groups Discursive strategy 7 Elkind et al. Continues Data extraction (1988) [37] UK 56 Not stated Women noted as ‘did not attend’ in health authority records Interviews Not stated 5 Emami and Tishelman (2004) [38] Sweden 45 25–70c Iranian immigrant women Focus groups Not stated 8 Forss et al. (2001) [39] Sweden 66 (with three transcripts excluded) 25–60 Cervical screening attenders Unstructured interviews Modiﬁed phenomenographic approach 9 Gregory and McKie (1991) [40] UK 72 20 to mid-60s Nonspeciﬁc Focus groups Not stated 4 Jackowska et al. (2012) [41] UK 52 20–55 Polish, Romanian and Slovakian women Focus groups and interviews Framework analysis 8 Jirojwong and Manderson (2001) [42] Australia 6 Not stated Thai immigrant women In-depth interviews Content analysis 4 Kwok et al. (2011) [43] Australia 18 28–66 Chinese-Australian women In-depth interviews Content analysis 7 Logan and McIlfatrick (2011) [44] UK 48 18–65c Women living in socially deprived areas Focus groups Thematic content analysis 8 Manderson and Hoban (2006) [45] Australia 368e Not stated Indigenous women Focus groups, in-depth interviews and case histories Thematic analysis 9 McKie (1995) [46] UK 72 18–73 White British, working-class women Focus groups Not stated 6 Milburn and MacAskill (1994) [47] UK Not stated 20–60c Nonspeciﬁc Focus groups Not stated 4 Naish et al. (1994) [48] UK Not stated Not stated BAME women Focus groups Not stated 6 Continues Table 1. Characteristics of studies included in review Continues © 2016 The Authors. Psycho-Oncology Published by John Wiley & Sons Ltd. Psycho-Oncology 26: 161–172 (2017) DOI: 10.1002/pon A. J. Chorley et al. A. J. Chorley et al. 164 Table 1. Continued Country Eligible participantsa (n) Age range (years) Population Study design Analytic method CASP score Ogunsiji et al. (2013) [49] Australia 21 25–50 West African immigrant women Semistructured interviews Constant comparison 8 Oscarsson et al. (2008) [50] Sweden 14 33–64 Women with no cervical smear attendance in previous 5 years Interviews Inductive content analysis 8 Park et al. (2006) [51] Korea 23 27–37 Sexually active women aged under 40 years Focus groups Content analysis 8 Peters (2010)f [52] Australia 9 30–65 Women residing in a socioeconomically disadvantaged area Conversational interviews Feminist approach 8 Peters (2012)f [53] Australia 6 Not stated Socially disadvantaged women (i.e. from a minority cultural group, had a physical disability and/or had experienced sexual abuse) Conversational interviews Feminist approach 7 Power et al. Data extraction (2009) [54] Australia 13 22–42 Lesbian and bisexual women In-depth interviews Thematic analysis 7 Savage and Clarke (1998) [55] Australia 20 46–69 Women aged 45– 70 years Unstructured interviews Not stated 7 Stewart and Thistlethwaite (2010) [56] Australia 24 18–51 Nonspeciﬁc Semistructured interviews Deductive content analysis 5 Szarewski et al. (2009) [57] UK 28 21–65 Muslim women Focus groups Framework analysis 8 Team et al. (2013) [58] Australia 8 Min. 40 Russian immigrant women, with caregiving responsibilities Semistructured interviews Grounded theory 8 Thomas et al. (2005) [59] UK 85 20–75e BAME women Focus groups + telephone interviews Content analysis 8 Waller et al. (2012) [60] UK 46 25–64 Cervical screening never and irregular attenders Focus groups + interviews Framework analysis 8 Widmark et al. (2008) [61] Sweden 49 21–74 Nonspeciﬁc Focus groups Interpretive description 8 Wollin and Elder (2003) [62] Australia 13 Not stated Deaf women Interviews Not stated 7 BAME, Black and Asian minority ethnic; CASP, critical appraisal skills programme. aSome studies also included data from noneligible participants, that is, health professionals or men. Information on these participants has not been included in this table. bThese three articles are based upon the same sample of women but report different aspects of the data. cEligibility age range given as sample age range not reported. dIncludes secondary analysis of Blomberg et al [30] them. Cervical screening is not a one-off event but a behav- iour that is repeated at regular intervals over decades, so many of the themes discussed were not singular consider- ations or experiences but were reassessed over time. For fur- ther illustrative quotations, see Supporting Information 5. presence, the perspective of never attenders was far less explored. Because of this, the identiﬁed themes are predom- inantly shaped by the perceptions of women who had at least one experience of screening to draw upon. Experiences of cervical screening and barriers to participation 165 Figure 1. Relationship between identiﬁed themes Figure 1. Relationship between identiﬁed themes which the relevance of screening changed. Some women reported feeling more vulnerable during the menopause. For others, being post-menopausal meant that screening was no longer considered important, having been ‘more essential for them in younger days’ [A] [50]. should go for screening. Most women considered whether they should go for screening in relation to the following: (a) the relevance and (b) the value of screening. The relevance of screening – who is it for? Most studies discussed beliefs regarding who needs to be screened. For some women, the prospect of cervical can- cer was considered unlikely, and screening was given little further thought: ‘I just don’t believe that it will happen to me and it’s not of concern to me’ [P] [49].2 Among women who had considered screening, its relevance was largely inﬂuenced by four subthemes (causal beliefs, life stage, current health state and family history), and beliefs about relevance appeared to ﬂuctuate over time. Current health state: Some studies discussed how partic- ipants’ general feelings of being healthy inﬂuenced their perceived need for screening. Other women explicitly discussed the presence or absence of symptoms as an indi- cator that medical help including screening was needed. Interestingly, this was raised regardless of age, ethnicity or attendance status. For some, lack of symptoms was clearly stated as a reason for nonattendance: ‘If there was anything wrong I think I’d have a discharge or some- thing so I am not so worried about that either’ [P] [61]. Other women acknowledged that cancer could be asymptomatic. Causal beliefs: Discussion about who needs screening was heavily inﬂuenced by beliefs about the causes of cer- vical cancer. Discussions about the numbers of sexual partners and perceptions of promiscuity predominated, but while many women were aware of the link between cervical cancer and sexual behaviour, they were often unclear about the underlying mechanisms. Women often related the risk of cervical cancer to their current sexual behaviour, often emphasising the stability and length of their current relationships, positioning themselves as be- ing at low risk and therefore not needing screening. For BAME women, factors including religion, ethnicity and marital status were seen as synonymous with low-risk sex- ual behaviour indicating that screening was unnecessary. Family history: Family history was often identiﬁed as a risk factor for cervical cancer, and women interpreted its absence as an indication that screening was less important for them. Should I go for screening? Two overarching themes were identiﬁed: (a) should I go for screening? and (b) screening is a big deal. Figure 1 repre- sents these themes and the main subthemes inﬂuencing The ﬁrst major theme related to women’s thoughts about who needs screening and, consequently, whether they Psycho-Oncology 26: 161–172 (2017) DOI: 10.1002/pon © 2016 The Authors. Psycho-Oncology Published by John Wiley & Sons Ltd. © 2016 The Authors. Psycho-Oncology Published by John Wiley & Sons Ltd. The value of screening – what is the point? In assessing the value of screening, women fell into one of three groups: those who felt that screening had value, those who did not and those who were aware of screening but unsure of its importance. These assessments were informed by beliefs regarding who needs to be screened, as well as beliefs about the consequences of cervical cancer. Life stage: Many women felt that their age and general feeling of mortality were indicators for screening: ‘Sick- ness was described as something that could occur at older ages, but not at age 30 years’ [A] [30]. For others, repro- ductive stages and social roles such as motherhood determined screening relevance; for some women, consid- ering having children made them think about their gynaecological health. Menopause was also a time in Screening has value: Women who believed that screen- ing had value expressed the view that it allowed can- cer to be detected early, which was beneﬁcial. These women also valued the reassurance that a negative screening result could provide, giving them ‘peace of mind’ [A/P] [33,37,43]. There was evidence that some © 2016 The Authors. Psycho-Oncology Published by John Wiley & Sons Ltd. Psycho-Oncology 26: 161–172 (2017) DOI: 10.1002/pon 166 A. J. Chorley et al. of one study based in Korea [51], this theme was only found in studies with BAME women. For some, screening was considered harmful because it could lead to further investigation or unnecessary treatment. of one study based in Korea [51], this theme was only found in studies with BAME women. For some, screening was considered harmful because it could lead to further investigation or unnecessary treatment. women did not understand the limitations of screening and felt that a negative result provided a ‘certiﬁcate of health’ [A] [59], that there was ‘nothing untoward hap- pening’ [P] [26] and that they were ‘free of cancer’ [A] [56]. These statements seemed to relate to misper- ceptions about the purpose of screening, with women seeing it as a general cancer test, test for infections or a reproductive checkup. Screening causes anxiety: A more widespread concern was that screening could cause ill health by increasing anxiety and worry, causing an emotional imbalance or making individuals ruminate on the possibility of cancer. For others, the wait for results or threat of a positive result made them anxious. The procedure – physical and emotional experiences The actual procedure was often considered physically and emotionally signiﬁcant. For some women, cervical screen- ing was simply another health check that needs to be car- ried out, and while not pleasant, it was not seen as a problem: ‘it’s not worse than going to the dentist’ [P] [39]. Other women described the test as ‘awful’ [P] [36], ‘pretty revolting’ [P] [55], ‘intrusive’ [A] [24] and ‘daunt- ing’ [A] [47]. Women also seemed to be inﬂuenced by stories of unpleasant experiences shared by others, espe- cially those who had never attended screening as they did not have access to positive experiences that might counteract negative accounts. Unsure of the importance of screening: The ﬁnal group appeared to have no opinion of the value of screening. These women had heard of screening but had never con- sidered it to be important. The studies that identiﬁed this group were predominantly with women from BAME or lower socioeconomic status backgrounds. Screening is a big deal The second major theme related to women’s perceptions that screening is a big deal, both physically and emotionally. This theme relates to perceptions of cervical screening as posing a threat and negative experiences of the procedure. Physical experiences: Cervical screening was described as a highly embodied experience. Some women described the procedure as uncomfortable or even painful and reported side effects of the test, including lasting pain and bleeding. The value of screening – what is the point? Screening was also valued because it allowed women’s knowledge of the health of a hidden part of their body: ‘The cervix is an organ inside my body that I cannot see. I can’t tell whether it is normal or abnormal, unlike my face, or my hand that I see everyday’ [P] [42]. Screening causes a social threat: By attending screening and creating the possibility that they may receive a posi- tive result, women risked facing ‘public distaste and fear of labelling as promiscuous’ [A] [46]. This was also related to cultural beliefs about sex before marriage where some women described how they would ‘bin the screen- ing invitation letters to prevent suspicion’ [P] [59]. It was acknowledged that some unmarried women were sexually active, often without their parents’ knowledge, and that fear of parental disapproval could be a barrier to attendance. Screening does not have value: A second group of women believed that screening was not important. Some felt that they would know if they were ill or that if there was something wrong, it would resolve by itself. Some described a lack of trust in the test results, potentially based on experience of false positives. For others, there was a general cynicism about the motives of cervical screening programmes, with women suggesting that they were being ‘used to fulﬁl quotas’ [A] [31] and that screen- ing programmes were ‘trying to get women all in one mind’ [P] [33]. Psycho-Oncology 26: 161–172 (2017) DOI: 10.1002/pon Experiences of cervical screening and barriers to participation 167 [P] [30] or that ‘pieces are cut from the womb with the speculum’ [A] [48]. [P] [30] or that ‘pieces are cut from the womb with the speculum’ [A] [48]. this ideal had not been met. Poor communication seemed to exacerbate the loss of control that some women associ- ated with screening. Where clear information about the procedure was provided, women reported much more sat- isfactory experiences. Emotional experiences: Women reported strong emo- tional responses to the procedure, including embarrass- ment, shame and vulnerability. Some perceived the test as degrading and violating and described a loss of power and control over the situation. Experiencing negative emo- tions during the procedure also worsened the physical experience for some women, through increasing tension. Some women reported situations in which they felt that their experience of the test had been dismissed or ignored by the smear taker. Others described inconsiderate or dis- respectful behaviour by the smear taker, including ‘igno- rance, rough treatment, insensitivity… joking, crudeness and shouting at [the] patient’ [P] [35]. Further, BAME women and women with disabilities sometimes found smear takers to be prejudiced or insensitive to their spe- ciﬁc needs, and both groups suggested specialised training to improve clinical encounters. Negative emotional experiences were also associated with events outside the actual appointment. Some women described how receiving an invitation resulted in anxiety, and they felt that it was necessary to ‘psych themselves up’ [A] [47] or ‘pluck up the courage’ [P] [26] for the encounter. Efforts by health professionals to reduce or deny the emotional signiﬁcance of the procedure by emphasising its routine nature were largely disliked. In doing so, women felt that the smear taker was disregarding the sig- niﬁcance it held for them: ‘Doctors may perform the same test many times, but it is not such a routine test to an indi- vidual woman’ [P] [51]. In particular, women wanted the smear taker to acknowledge the emotional signiﬁcance that cervical screening held for them. In some cases, women found the experience de-individualising, espe- cially when the smear taker emphasised that they’d ‘seen it all before’: ‘Doctors have seen a lot of vaginas but they didn’t see mine and that’s it for me. […] So I just saw it as an invasion of my person and my privacy’ [P] [27]. Experiences of cervical screening and barriers to participation In some cases, women used language that suggests that they found the experience to be dehumanising, referring to feeling like ‘a piece of meat’ [P] [31,40] and being ‘treated like…cattle’ [P] [28]. Many of the negative emotions women described involved embarrassment and shame, stemming from a sit- uation in which social norms surrounding nudity are broken: ‘Even though she is a female doctor but I was still ashamed the ﬁrst time I did it, of opening up my private part to somebody, but after that I got used to it’ [P] [49]. Some older women and women who had experienced abuse had particular concerns about the appearance of their body, which made them reluctant to attend. Some Somali women, although not ashamed of it, were worried about the smear taker’s response to the fact that they had been circumcised and considered that it may cause further embarrassment. There was particular concern from some BAME women about exposing their bodies, because of the belief that their naked body should only be seen by their husband. Concerns about nudity and the sexual connotations of the procedure resulted in a strong preference across stud- ies for female smear takers. Although this was often still seen as embarrassing, seeing a male health professional resulted in the most severe negative emotional reactions: ‘I hate the idea of going to a man doctor. It’s a lot of embarrassment for me. I’m not going back to him. Even though it lasts only a couple of minutes I think it’s, it’s very undigniﬁed’ [P] [44]. Smear taker preferences: In addition to a female smear taker (see previous text), women expressed other prefer- ences, which they hoped would improve the screening experience. Some were concerned about having a trusting relationship with the smear taker, which usually meant seeking continuity of care. Others preferred the relative anonymity of having cervical screening carried out by an unfamiliar health professional. Less commonly, women expressed preferences regarding professional background. Some women also believed that a female smear taker would be more sympathetic to women’s health issues and that having experienced it themselves, they would be more likely to be gentle and considerate. The BAME women sometimes expressed a preference for health professionals who were of the same ethnic back- ground. Others preferred to visit health professionals of a different background, as a strategy to avoid potential breaches of conﬁdentiality. Experiences of cervical screening and barriers to participation Health professionals: Women’s experiences of screening were often shaped by the quality of their interaction with the health professional carrying out the procedure. Some women reported positive encounters with these health profes- sionals, but more commonly, women recalled negative en- counters, with poor communication often cited as the reason. Cervical screening as a threat Potential for screening to reveal cancer: For some women, cervical screening was seen as a signiﬁcant and emotional experience because of the potential for harm to occur to themselves; the most obvious being a diagnosis of cervical cancer. Fear and anxiety stemming from the potential for cervical screening to identify cancer were related to ideas of pain, suffering and death, as well as concerns about the impact upon fertility among younger women. Women also commented on two aspects of the proce- dure, which they found particularly unpleasant: the taking of the sample and, in particular, the speculum: ‘I don’t like the metal thingamajig they use actually. I think that’s what puts me off more than anything’ [P] [60]. Discussion of the speculum also related its coldness and the act of penetration. Some women had a good understanding of how the sample was taken but felt highly aware of what had been done to their bodies. Other women had misconceptions about the procedure, believing that the cervix was ‘frozen’ Screening causes physical harm: Some women worried about clinic hygiene, leading to concern about acquiring an infection or even cervical cancer. With the exception © 2016 The Authors. Psycho-Oncology Published by John Wiley & Sons Ltd. Psycho-Oncology 26: 161–172 (2017) DOI: 10.1002/pon Psycho-Oncology 26: 161–172 (2017) DOI: 10.1002/pon © 2016 The Authors. Psycho-Oncology Published by John Wiley & Sons Ltd. Conclusions Experiences with healthcare provision in general: Experiences of healthcare outside of screening, in particu- lar gynaecological examinations, inﬂuenced intentions to have cervical screening. A single negative experience of a gynaecological examination could result in women avoiding screening. Other experiences of healthcare, such as breaches of conﬁdentiality, affected trust in the system and willingness to attend screening. Through synthesising the qualitative research on women’s perceptions and experiences of cervical screening, we identi- ﬁed two novel overarching themes; the ﬁrst, ‘should I go for screening?’, is indicative of women who are undecided about attending screening, after the ﬁrst invitation and on subse- quent occasions, as they consider both the relevance and the value of screening. The second theme, ‘screening is a big deal’, is reﬂective of the experiences of women who have already attended screening at least once, relating in particular to the ways in which screening presented a threat, and the largely negative physical and emotional experiences of the procedure. These negative experiences were both inherent to the procedure and inﬂuenced by the conduct of health pro- fessionals. As screening is not a one-time event but an action that should be repeated at regular intervals over decades, neg- ative experiences clearly inform women’s decisions about fu- ture attendance. Previous experiences of cervical screening: Previous screening experiences seemed to inﬂuence decisions regarding re-attendance in two ways. Firstly, previous normal results were sometimes interpreted as being given an ‘all clear’ with no further need to attend. Conversely, one woman reported how a previous positive result reduced her desire to attend screening because of the increased anxiety that she now felt. Secondly, even when screening was still considered rel- evant, previous experiences inﬂuenced willingness to re-attend. For some women, a single negative experience prevented them from re-attending screening, even if they had multiple positive previous experiences to draw upon. There were also instances of women who wished to attend but had difﬁculty translating those intentions into action be- cause of competing priorities and practical barriers and the re- sources available to overcome these. As well as women who intended to act and those who were undecided, there were also women who had made an active decision to not attend screening, although this does not necessarily mean that they had not previously attended or would deﬁnitely not attend in the future. Practical barriers Women reported a number of practical barriers to accessing screening, which were often associated with their life circumstances and the resources available to them. Some of these women wanted to attend screening but felt prevented by practical barriers. For others, practi- cal barriers were indicated as reasons for nonattendance in addition to one or more of the barriers outlined previously. We also identiﬁed women within the synthesis who were unaware of or unengaged with screening, although these themes were far less developed than those previ- ously outlined. It is clear that these perspectives are much less explored within the literature than others, as the majority of women across studies had attended screening at least once. One reason for this may be the way in which women are recruited for studies. It is unlikely that unaware or unengaged women would be motivated to take part in a study about screening. As this may be a target group for interventions aimed at increasing engagement with the screening invitation, further research may need to seek a different method of recruitment and approach to the subject of screening in order to ensure that the perspectives of these groups are included in intervention design and evaluation. Women described how screening was yet another demand on their time and often competed with daily tasks such as work and childcare, which were given higher pri- ority. For others, screening was seen as inaccessible, because of features of the clinic such as inconvenient loca- tion or appointment times. There were also speciﬁc barriers reported by BAME women. Some women felt that they had received racist treat- ment by health professionals, which made them less willing to attend. Others reported difﬁculties in accessing informa- tion. For some women, this was due to low competency in the language of their country of residence and a lack of trans- lated materials. For others, low literacy levels or a cultural preference for verbal information meant that they could not access information even when translated. The impact of these factors was largely related to the screening context of women’s country of origin. Women from Somalia [24] and west Africa [49] reported being unaware of cervical These ﬁndings show that there are a number of reasons why women may not attend cervical screening as recom- mended and highlight the fact that screening nonattenders should not be viewed as a homogenous group. Previous experiences Previous experiences included general experiences in healthcare settings as well as more speciﬁc experiences with screening. Conclusions It is unclear whether these women are making an informed choice not to attend or are basing their decisions on lack of understanding or misconceptions. Additional contributing factors Women expressed a desire for explanation about the procedure as it is carried out and the opportunity to ask questions, but many reported negative experiences where In addition to the two main themes identiﬁed, some minor themes played additional roles. These included the follow- ing: (a) previous experiences and (b) practical barriers. Psycho-Oncology 26: 161–172 (2017) DOI: 10.1002/pon © 2016 The Authors. Psycho-Oncology Published by John Wiley & Sons Ltd. Psycho-Oncology 26: 161–172 (2017) DOI: 10.1002/pon 168 A. J. Chorley et al. screening before migrating, while women from eastern Eu- rope [41,58] had prior knowledge of screening because of participating in their home country. Previous experiences © 2016 The Authors. Psycho-Oncology Published by John Wiley & Sons Ltd. Psycho-Oncology 26: 161–172 (2017) DOI: 10.1002/pon Implications This review points to some clear ways for practitioners to minimise the negative aspects of the screening expe- rience, for example, providing the option of a female smear taker, acknowledging that the procedure is not ex- perienced as routine for women, taking steps to ensure that dignity is maintained and being sensitive to the spe- ciﬁc needs of women from BAME groups, women with disabilities and those who have experienced abuse. The wide variety of barriers to screening identiﬁed makes it clear that nonparticipants are a heterogeneous group. Conceptualising screening nonparticipants along a con- tinuum of screening adoption may be a useful way of targeting appropriate interventions, from awareness- raising initiatives for women who are unaware of screening in order to promote informed choice about screening to addressing practical barriers for women who have difﬁculty translating positive intentions into attendance. Our ﬁndings identify the need for more re- search to understand how and why some women in countries with established screening programmes remain unaware of screening. There is some evidence that BAME women may be more likely to be unaware or unengaged [70], but the issue should be explored further with a view to reducing social inequalities in screening participation. Our identiﬁcation of the themes of ‘personal relevance’ and ‘value’ in women’s decision-making is a new way of conceptualising this stage of the process and can now be used to develop appropriate information for women deciding whether to take part. Designing information to address the issues that women across studies grapple with may be a way to make it more relevant and helpful. In addition, our ﬁnding that ‘screening is a big deal’ was such a major theme in women’s accounts speaks to a strong need to acknowledge this within programmes and to develop ways to minimise negative physical and psychological consequences. Experiences of cervical screening and barriers to participation 169 of studies from Sweden and Korea supports the idea that our ﬁndings might apply to other countries with similar screening programmes, but it is possible that women’s perceptions of cervical screening may differ in other contexts. There is also a need for further research to en- sure that the perspectives of women who have never attended are included. associated reasons for nonattendance, particularly when de- veloping interventions to promote uptake. Stage-based models of behaviour change, such as the transtheoretical model (TTM) [63] and the precaution adoption process model (PAPM) [64], offer ways of conceptualising these differences in engagement with cervical screening. Find- ings from this synthesis suggest that the PAPM, with its more detailed categorisation of those with no intention to act into ‘unaware’, ‘unengaged’ and ‘decided not to act’ groups, may be of greater value than the TTM with its sin- gular ‘precontemplation’ stage, when considering ways to support women in making an informed choice about cervi- cal screening. Previous studies of mammography [65,66] and colorectal cancer screening [67–69] readiness have shown that the stages of the PAPM are associated with particular sociodemographic and psychological correlates, which could be used to inform the development of targeted interventions. While our ﬁndings offer support for the value of using the PAPM to categorise women in terms of their engagement with cervical screening, further re- search is needed to establish the proportion of women within each group in the general population and to exam- ine stage-speciﬁc differences in sociodemographic and psy- chological characteristics. Practical barriers There may be value in considering different subgroups and the Psycho-Oncology 26: 161–172 (2017) DOI: 10.1002/pon Psycho-Oncology 26: 161–172 (2017) DOI: 10.1002/pon © 2016 The Authors. Psycho-Oncology Published by John Wiley & Sons Ltd. © 2016 The Authors. Psycho-Oncology Published by John Wiley & Sons Ltd. 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Conﬂict of interest suggest that interventions tailored to decisional stage may be of value in increasing informed uptake of cervi- cal screening. The authors have no conﬂicts of interest to declare. Limitations To our knowledge, this is the ﬁrst time that the methods of thematic synthesis have been applied to studies on women’s perceptions of cervical screening. Doing so identiﬁed two broad themes in women’s narratives of cervical screening, which were not wholly apparent in any given study. Many women considered both the per- sonal relevance and the value of screening when decid- ing whether they should attend. Those who had previously been screened reported largely negative expe- riences of screening, which informed their decisions about future attendance. We also identiﬁed practical bar- riers to attendance, which may prevent women from realising their screening intentions. In addition to these themes, drawing together multiple studies highlighted the scarcity of accounts from women who have never attended screening and identiﬁed the need for further re- search. Combined, these ﬁndings highlight the varying reasons that women may have for nonattendance and Inherent to qualitative synthesis are the limitations that arise from working with published articles. It is impossible to know whether the reported data and authors’ interpretations were representative of the data as a whole or the extent to which reported quotes were spontaneous from participants. Further, through the process of synthesis, the speciﬁc con- text of studies – a valuable component of qualitative re- search – may be compromised. 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https://openalex.org/W2562598326	https://munin.uit.no/bitstream/10037/10187/2/article.pdf	English	null	A novel non-canonical Wnt signature for prostate cancer aggressiveness	Oncotarget	2,016	cc-by	10,616	A novel non-canonical Wnt signature for prostate cancer aggressiveness Elise Sandsmark1, Ailin Falkmo Hansen1, Kirsten M. Selnæs1, Helena Bertilsson2,3, Anna M. Bofin4, Alan J. Wright5, Trond Viset6, Elin Richardsen7,8, Finn Drabløs3, Tone F. Bathen1, May-Britt Tessem1,, Morten B. Rye2,3,9, Elise Sandsmark1, Ailin Falkmo Hansen1, Kirsten M. Selnæs1, Helena Bertilsson2,3, Anna M. Bofin4, Alan J. Wright5, Trond Viset6, Elin Richardsen7,8, Finn Drabløs3, Tone F. Bathen1, May-Britt Tessem1,, Morten B. Rye2,3,9, Elise Sandsmark1, Ailin Falkmo Hansen1, Kirsten M. Selnæs1, Helena Bertilsson2,3, Anna M. Bofin4, Alan J. Wright5, Trond Viset6, Elin Richardsen7,8, Finn Drabløs3, Tone F. Bathen1, May-Britt Tessem1,, Morten B. Rye2,3,9, 1Department of Circulation and Medical Imaging, Faculty of Medicine, NTNU - Norwegian University of Science and Technology, Trondheim, Norway Department of Urology, St. Olavs Hospital, Trondheim University Hospital, Norway 2Department of Urology, St. Olavs Hospital, Trondheim University Hospital, Norway 3Department of Cancer Research and Molecular Medicine, Faculty of Medicine, NTNU - Norwegian University of Science and Technology, Trondheim, Norway 4Department of Laboratory Medicine, Children’s and Women’s Health, Faculty of Medicine, NTNU - Norwegian University of Science and Technology, Trondheim, Norway 6Department of Pathology and Medical Genetics, St. Olavs Hospital, Trondheim University Hospital, Norway Department of Medical Biology, UiT - The Arctic University of Norway, Tromsø, Norway 8Department of Clinical Pathology, University Hospital of North Norway, Tromsø, Norway 9St. Olavs Hospital, Trondheim University Hospital, Norway These authors have contributed equally to this work Correspondence to: Elise Sandsmark, email: elise.sandsmark@gmail.com Morten B. Rye, email: morten.rye@ntnu.no Correspondence to: Elise Sandsmark, email: elise.sandsmark@gmail.com M t B R il t @ t Correspondence to: Elise Sandsmark, email: elise.sandsmark@gmail.com Morten B. Rye, email: morten.rye@ntnu.no Keywords: EMT, gene expression signature, biochemical recurrence, spectroscopy, MR Keywords: EMT, gene expression signature, biochemical recurrence, spectroscopy, MRSI Keywords: EMT, gene expression signature, biochemical recurrence, spectroscopy, MRSI Received: August 26, 2016 Accepted: November 23, 2016 Published: December 24, 2016 Received: August 26, 2016 Accepted: November 23, 2016 Published: December 24, 2016 Oncotarget, Advance Publications 2016 Oncotarget, Advance Publications 2016 www.impactjournals.com/oncotarget/ INTRODUCTION aggressiveness [16]. Several studies support the activation of CWP in advanced and metastatic prostate cancer [7, 17], but little evidence exists for localized and locally advanced prostate cancer. Increased activation of the Wnt signaling pathway (WP) is associated with development, progression, and metastasis of many cancers [1]. In prostate cancer, the WP has been associated with aggressive, late stage disease, and metastasis [2–5]; however, its potential for early prediction of aggressiveness is still unclear. Previous studies are mainly performed in prostate cancer cell lines [6–9], and proper validation in human tissue is lacking. The WP is proposed as a therapeutic target in prostate cancer treatment [10], and reduced proliferation has been detected as a result of targeted Wnt-inhibitor drugs in cell lines [11, 12]. However, to develop Wnt-targeted drugs for human prostate cancer, an increased understanding of the molecular mechanisms in vivo is needed. The NCWP is commonly divided into two pathways, the planar cell polarity (PCP), and the Wnt/Calcium pathway (Figure 1B-1C). Few studies have addressed the significance of NCWP in prostate cancer. Most attention has been focused on the role of the non-canonical ligand Wnt5a, a key activator of the NCWP. Wnt5a is generally found to be upregulated in prostate cancer, but results are inconsistent regarding its association with good [18–20] or poor prognosis [21]. Recently, a new NCWP involving Wnt5a and the receptor Frizzled2 (Fzd2) was discovered (Figure 1D) and shown to promote tumor progression and EMT in several cancer cell lines and a mouse xenograft model [22]. In the same study, a Wnt5/Fzd2 based gene set was also shown to accurately predict metastasis and survival in a small cohort (n=46) of patients with hepatocellular carcinoma. However, this study did not address the in vivo relevance of the NCWP in larger patient cohorts or in prostate cancer tissue. Wnt ligands bind to Frizzled (Fzd) receptors to activate the WP, which then induces signal transduction cascades. The WP is generally divided into a β-catenin- dependent canonical WP (CWP), and a β-catenin- independent non-canonical WP (NCWP). The importance of the CWP in carcinogenesis was first discovered in colorectal cancer, where mutations of the APC gene, a part of the β-catenin destruction complex (Figure 1A), resulted in stabilization and nuclear translocation of β-catenin [13]. This β-catenin translocation is a hallmark of CWP activation, and can drive tumor invasion and metastasis through a process of epithelial-to-mesenchymal transition (EMT) [14]. ABSTRACT Activation of the Canonical Wnt pathway (CWP) has been linked to advanced and metastatic prostate cancer, whereas the Wnt5a-induced non-canonical Wnt pathway (NCWP) has been associated with both good and poor prognosis. A newly discovered NCWP, Wnt5/Fzd2, has been shown to induce epithelial-to-mesenchymal transition (EMT) in cancers, but has not been investigated in prostate cancer. The aim of this study was to investigate if the CWP and NCWP, in combination with EMT, are associated with metabolic alterations, aggressive disease and biochemical recurrence in prostate cancer. An initial analysis was performed using integrated transcriptomics, ex vivo and in vivo metabolomics, and histopathology of prostatectomy samples (n=129), combined with at least five-year follow-up. This analysis detected increased activation of NCWP through Wnt5a/ Fzd2 as the most common mode of Wnt activation in prostate cancer. This activation was associated with increased expression of EMT markers and higher Gleason score. The transcriptional association between NCWP and EMT was confirmed in five other publicly available patient cohorts (1519 samples in total). A novel gene expression signature of concordant activation of NCWP and EMT (NCWP-EMT) was developed, and this signature was significantly associated with metastasis and shown to be a significant predictor of biochemical recurrence. The NCWP-EMT signature was also associated with decreased concentrations of the metabolites citrate and spermine, which have previously been linked to aggressive prostate cancer. Our results demonstrate the importance of NCWP and EMT in prostate cancer aggressiveness, suggest a novel gene signature for improved risk stratification, and give new molecular insight. www.impactjournals.com/oncotarget Oncotarget 1 INTRODUCTION To date, metabolic alterations associated with the WP have not been investigated in prostate cancer. low and high histopathological Gleason score have previously been detected using in vivo MRSI of patients [27], and citrate and spermine are suggested as the main contributors to discriminating on the basis of tumor aggressiveness from tissue HR-MAS MRS analysis [28]. To date, metabolic alterations associated with the WP have not been investigated in prostate cancer. are reduced expression of the receptor FZD1, increased expressions of the antagonist SFRP4 and casein kinase CSNK1E, which support the absence of CWP activation. Although some variations were observed (Figure 2A), the lack of upregulation of the main CWP genes suggests no increased expression activity of the CWP in prostate cancer in our main cohort. g p The aim of this study was to investigate if the CWP and NCWP, in combination with EMT markers, are activated and associated with aggressive disease and metabolic alterations in human prostate cancer. To approach these questions, we first used a patient cohort where integrated omics analyses were performed on the same samples from fresh-frozen prostatectomy-tissue slices, including transcriptomics, tissue ex vivo and in vivo patient metabolomics, and detailed histopathological evaluation [29]. Histopathology allowed us to control for tissue heterogeneity, particularly the fraction of stroma, which is a major complicating factor when analyzing tissue samples [30]. The findings were confirmed in publicly available prostate cancer cohorts (n=1519 samples in total), and in a separate immunohistochemistry cohort. The analysis suggests that the NCWP, and not the CWP, is the most active WP for in vivo prostate cancer, and that this activity correlates with markers for EMT. Our approach allowed for the development of a novel NCWP-EMT gene signature significantly associated with recurrent and metastatic cancer and metabolic biomarkers. This signature may help differentiate aggressive from indolent prostate cancer. Translocation of β-catenin from the membrane to the nucleus is the hallmark of CWP activation, and to validate the findings above, we performed β-catenin immunohistochemistry (IHC) on the immunohistochemistry cohort (Figure 3A-3B). All the samples (n=40) had weak or non-detectable nuclear staining (SI≤2). Most of the samples (n=30) had strong membranous β-catenin staining (SI=9), indicating no activation of the CWP. Ten samples had weak or moderate membranous staining (SI≤6), indicating reduced membranous expression without increased nuclear expression of β-catenin. RESULTS AND DISCUSSION Patient and sample characteristics of the main and the immunohistochemistry cohorts are presented in Table 1. The five validation cohorts (in total 1519 samples) are presented in the methods section with more information listed in Supplementary Table S1. INTRODUCTION These findings demonstrate that the CWP is not activated in prostate cancer in our immunohistochemistry cohort, which is in concordance with the gene expression results from the main cohort. We therefore conclude that there is little evidence of CWP activation in prostate cancer compared to normal prostate tissue investigated in two independent cohorts. We further investigated alterations in the CWP between low Gleason (≤3+4) and high Gleason (≥4+3) samples (Figure 2A). There were no significant gene expression alterations detected for β-catenin (CTNNB1), the Wnt ligands, the receptor-complex and the destruction complex (Supplementary Table S2). Of the CWP inhibitors, both SFRP2 and SFRP4 were upregulated in high Gleason compared to low Gleason cancer samples, which is contradictory to CWP activation. However, the inhibitor of β-catenin translocation, ICAT (CTNNBIP1), was downregulated, and the CWP transcription factors LEF1 and TCF were upregulated in high Gleason cancer, which could indicate activation of downstream components of the pathway independently of the β-catenin destruction complex. To conclude, the overall analysis suggests no significant increase in CWP activation through the canonical destruction complex, neither in cancer compared to normal nor in high Gleason cancer. INTRODUCTION During EMT, epithelial cancer cells develop into less adhesive and more motile mesenchymal- like cells, which increases the cancer’s potential for invasion and metastasis [15]. There is mounting evidence associating EMT in prostate cancer with increased Metabolic reprogramming is a hallmark of cancer [23], and the WP has been suggested as an emerging mediator of cancer cell metabolism [24, 25]. Wnt5a- mediated NCWP has been directly related to alterations of the energy metabolism in melanoma and breast cancer cells [26]. Selected metabolic alterations detected in tissue samples by high resolution magic angle spinning magnetic resonance spectroscopy (HR-MAS MRS) can be translated for use in a clinical setting by magnetic resonance spectroscopy imaging (MRSI). Differences in (choline + creatine + spermine)/citrate ratio between Figure 1: Schematics of Wnt signaling pathways in cancer cells. A. Canonical Wnt pathway. In the absence of Wnt signaling, the β-catenin destruction complex labels β-catenin for proteasomal degradation. In the presence of Wnt signaling, the destruction complex is inhibited, resulting in stabilization and nuclear translocation of β-catenin, activating transcription of target genes. B. Non-canonical planar cell polarity (PCP) pathway activates signaling cascades resulting in cytoskeletal changes, as well as alterations in cell polarity, movement and survival. C. Non-canonical Wnt/Calcium pathway signaling activates intracellular calcium, which in turn reduce cell adhesion through further signaling. D. Non-canonical Wnt5/Fzd2 pathway. Wnt5 signals via the FZD2 receptor and FYN activates STAT3 transcription leading to epithelial-mesenchymal transition (EMT) in cancer cells. Figure 1: Schematics of Wnt signaling pathways in cancer cells. A. Canonical Wnt pathway. In the absence of Wnt signaling, the β-catenin destruction complex labels β-catenin for proteasomal degradation. In the presence of Wnt signaling, the destruction complex is inhibited, resulting in stabilization and nuclear translocation of β-catenin, activating transcription of target genes. B. Non-canonical planar cell polarity (PCP) pathway activates signaling cascades resulting in cytoskeletal changes, as well as alterations in cell polarity, movement and survival. C. Non-canonical Wnt/Calcium pathway signaling activates intracellular calcium, which in turn reduce cell adhesion through further signaling. D. Non-canonical Wnt5/Fzd2 pathway. Wnt5 signals via the FZD2 receptor and FYN activates STAT3 transcription leading to epithelial-mesenchymal transition (EMT) in cancer cells. www.impactjournals.com/oncotarget Oncotarget 2 low and high histopathological Gleason score have previously been detected using in vivo MRSI of patients [27], and citrate and spermine are suggested as the main contributors to discriminating on the basis of tumor aggressiveness from tissue HR-MAS MRS analysis [28]. The canonical Wnt pathway is not activated in prostate cancer 50 benign samples were excluded from further analysis in the immunohistochemistry cohort sPSA – serum PSA, pT stage – pathological tumor stage. the pathway components apart from downregulation of the ligand WNT5B, and upregulation of the calcium pathway component PLCB2, suggesting no increased activation of the NCWP in prostate cancer in general. However, when high Gleason samples were compared with low Gleason samples, significantly increased expressions were detected for three of the four key genes of the Wnt5/Fzd2 pathway; the ligand WNT5A (p<0.001), the receptor FZD2 (p=0.003) and the midstream kinase component FYN (p<0.001) (Figure 2B). No significant expression change was detected for the last key component, the transcription factor STAT3. For the Wnt/Calcium pathway, only PLCB2 was upregulated in high Gleason cancer (Figure 2B), and none of the central components of the PCP pathway were altered (Supplementary Table S2). In summary, these data suggest upregulation of the Wnt5/Fzd2 pathway in high Gleason prostate cancer. were using prostate cancer cell lines [6–9]. The disparity could therefore also highlight a difference between in vitro cell lines and human prostate tissue, emphasizing the importance of validation studies in human tissue, especially for identification of potential targets for personalized drug therapy. py In our main cohort, the central CWP genes showed an expression pattern that was indicative of substantial stromal influence when comparing normal against cancer tissue (Figure 2A). This trend was particularly strong for genes that, directly or partly, regulate the activity of the β-catenin destruction complex, and indicates a difference of CWP activity when cancer is compared to stroma, but not when compared to benign epithelium. Thus, at least some of the discrepancies from previous studies of CWP in prostate cancer may be explained by uneven sampling of stroma content between cancer and normal samples which has previously been observed in tissue samples from prostate cancer patient cohorts [30, 31]. For validation, IHC of WNT5A was performed on the immunohistochemistry cohort (Figure 3C-3D). Of the 40 cancer samples, 32 had strong (SI=9) and 8 had moderate or weak staining (SI≤6). There was no association between the staining intensity and Gleason grade for this cohort. The canonical Wnt pathway is not activated in prostate cancer To investigate if the CWP is activated in prostate cancer, we compared gene expression of the central CWP genes between cancer and normal samples of the main cohort using sample subsets balanced and unbalanced for stroma content according to histopathology (Figure 2A, Supplementary Table S2, Methods). The level of β-catenin (CTNNB1), the key component of the CWP pathway, showed no significant altered expression in cancer compared to normal, and two of the main components of the β-catenin destruction complex, GSK3B and AXIN1, were significantly upregulated in cancer. This may suggest increased activity of β-catenin destruction in prostate cancer, contrary to what is expected when the CWP is turned on. Additionally, the Wnt ligand genes associated with the CWP were not significantly changed in cancer compared to normal samples. Other important findings There is currently no consensus in the literature regarding CWP activation in prostate cancer, and our findings are contradictory to several previous studies suggesting increased CWP in prostate cancer [7, 9, 17]. The CWP has previously been associated with advanced disease such as androgen resistant prostate cancer in cell lines [7], and prostate cancer bone metastasis in human tissue and cell lines [8, 17]. The fact that our cohorts consist of radical prostatectomy tissue, from localized or locally advanced disease, may explain the absence of CWP activation. The CWP may therefore still be of importance in advanced, metastatic prostate cancer, but might not prove useful for early risk stratification. Furthermore, several previous studies reporting increased CWP signaling www.impactjournals.com/oncotarget Oncotarget 3 Table 1: Patients and sample characteristics of the two cohorts Main cohort Immunohistochemistry cohort Patients n=41 n=40 Age (median, range) Years 64 (48-69) 62 (48-73) sPSA (median, range) Before Surgery (ng/mL) 9.1 (4.0-45.8) 8.9 (5.2-18.0) Clinical pT stage (patients) pT1c - 7 pT2 28 20 pT3 13 10 Unknown - 3 Tissue samples n=129 n=40 Sample weight (mean, range) (mg) 12.7 (3.0-21.9) 12.6 (7.6-21.0) Gleason score of tissue samples Benign 34 - * 6 24 5 7 41 25 8 15 5 9 15 4 10 - 1 Gleason grade groups Low Gleason (≤3+4) 48 21 High Gleason (≥4+3) 47 19 * 50 benign samples were excluded from further analysis in the immunohistochemistry cohort sPSA – serum PSA, pT stage – pathological tumor stage. * 50 benign samples were excluded from further analysis in the immunohistochemistry cohort sPSA – serum PSA, pT stage – pathological tumor stage. Wnt5a-induced non-canonical Wnt signaling is increased in high Gleason prostate cancer The NCWP, including the Wnt/Calcium, PCP and the new Wnt5/Fzd2 pathways, were investigated (Figure 2B, Supplementary Table S2). When comparing cancer with normal samples, we found no alterations in any of Wnt5a has been suggested as a biomarker in prostate cancer, but its prognostic outcome has been inconsistent [18–21]. The increased WNT5A gene expression in high Gleason cancer samples compared to low Gleason samples www.impactjournals.com/oncotarget Oncotarget 4 5 www.impactjournals.com/oncotarget Figure 2: Alterat in prostate canc (balanced for st low Gleason pros compared with lo samples. The x-a multiplied by −1 for genes. P-values for balanced for stroma Supplementary Tabl pattern of no further but show a confound destruction complex show an upregulatio C. The central epith genes indicate ongoi The central EMT ne Oncotarget 5 www impactjournals com/oncotarget Figure 2: Alterations in central Wnt and EMT genes in prostate cancer compared with normal samples (balanced for stroma), high Gleason compared with low Gleason prostate cancer, and high stroma content compared with low stroma content (unbalanced) tissue samples. The x-axis displays log10(p-value) fold change, multiplied by −1 for upregulated genes, and 1 for downregulated genes. P-values for prostate cancer vs. normal prostate tissue are balanced for stroma content; unbalanced p-values are available in Supplementary Table S2. A. The central canonical genes show a pattern of no further activation in cancer or high Gleason cancer, but show a confounding stroma effect, especially of the genes of the destruction complex. B. The central non-canonical genes generally show an upregulation of Wnt5/Fzd2 genes in high Gleason cancer. C. The central epithelial-mesenchymal transition (EMT) positive genes indicate ongoing EMT, especially in high Gleason cancer. D. The central EMT negative genes. www impactjournals com/oncotarget Figure 2: Alterations in central Wnt and EMT genes in prostate cancer compared with normal samples (balanced for stroma), high Gleason compared with low Gleason prostate cancer, and high stroma content compared with low stroma content (unbalanced) tissue samples. The x-axis displays log10(p-value) fold change, multiplied by −1 for upregulated genes, and 1 for downregulated genes. P-values for prostate cancer vs. normal prostate tissue are balanced for stroma content; unbalanced p-values are available in Supplementary Table S2. A. The central canonical genes show a pattern of no further activation in cancer or high Gleason cancer, but show a confounding stroma effect, especially of the genes of the destruction complex. B. Wnt5a-induced non-canonical Wnt signaling is increased in high Gleason prostate cancer In melanoma, pancreatic and gastric cancer, Wnt5a expression is associated with worse prognosis, but in colon and thyroid cancer Wnt5a expression is associated with better prognosis as reviewed by McDonald and Silver, and Zhu et al. [34, 35]. The tumor-promoting role of Wnt5a can be caused by activation of NCWP [35], whereas the tumor- suppressing role may be caused by inhibition of the CWP [36]. Because of this conflicting role in different cancer types, we suspect that Wnt5a alone may not be a useful biomarker for prostate cancer. is in agreement with results from Yamamoto et al. who reported increased Wnt5a IHC staining of prostatectomy tissue samples with high Gleason grade [21]. This oncogenic effect of Wnt5a in prostate cancer progression is also supported by studies of cell lines, where Wnt5a has been shown to improve migration capacity [32], induce androgen resistance in prostate cancer metastases [33], and induce bone metastasis [8]. Contrary to this, other IHC studies of prostatectomy tissue samples have detected a tumor- suppressing role of Wnt5a in prostate cancer; increased Wnt5a IHC expression has been associated with increased Figure 3: Immunohistochemical staining of the immunohistochemistry cohort. A. Strong membranous β-catenin staining and B. weak β-catenin staining. C. Strong Wnt5a staining and D. weak Wnt5a staining. E. Positive membranous N-cadherin staining and F. negative N-cadherin staining. G. Strong membranous E-cadherin staining and H. weak E-cadherin staining. Magnification x400. Bar 50μm. Wnt5a-induced non-canonical Wnt signaling is increased in high Gleason prostate cancer The central non-canonical genes generally show an upregulation of Wnt5/Fzd2 genes in high Gleason cancer. C. The central epithelial-mesenchymal transition (EMT) positive genes indicate ongoing EMT, especially in high Gleason cancer. D. The central EMT negative genes. Figure 2: Alterations in central Wnt and EMT genes in prostate cancer compared with normal samples (balanced for stroma), high Gleason compared with low Gleason prostate cancer, and high stroma content compared with low stroma content (unbalanced) tissue samples. The x-axis displays log10(p-value) fold change, multiplied by −1 for upregulated genes, and 1 for downregulated genes. P-values for prostate cancer vs. normal prostate tissue are balanced for stroma content; unbalanced p-values are available in Supplementary Table S2. A. The central canonical genes show a pattern of no further activation in cancer or high Gleason cancer, but show a confounding stroma effect, especially of the genes of the destruction complex. B. The central non-canonical genes generally show an upregulation of Wnt5/Fzd2 genes in high Gleason cancer. C. The central epithelial-mesenchymal transition (EMT) positive genes indicate ongoing EMT, especially in high Gleason cancer. D. The central EMT negative genes. www.impactjournals.com/oncotarget Oncotarget 5 10 years survival [18], and a lower risk of biochemical recurrence [19, 20]. This was, however, only true for low Gleason grade samples in one of the studies [20]. This apparent opposing role of Wnt5a in prostate cancer may be explained by the paradoxical effect of Wnt5a in other cancers. In melanoma, pancreatic and gastric cancer, Wnt5a expression is associated with worse prognosis, but in colon and thyroid cancer Wnt5a expression is associated with better prognosis as reviewed by McDonald and Silver, and Zhu et al. [34, 35]. The tumor-promoting role of Wnt5a can be caused by activation of NCWP [35], whereas the tumor- suppressing role may be caused by inhibition of the CWP [36]. Because of this conflicting role in different cancer types, we suspect that Wnt5a alone may not be a useful biomarker for prostate cancer. 10 years survival [18], and a lower risk of biochemical recurrence [19, 20]. This was, however, only true for low Gleason grade samples in one of the studies [20]. This apparent opposing role of Wnt5a in prostate cancer may be explained by the paradoxical effect of Wnt5a in other cancers. www.impactjournals.com/oncotarget The NCWP-EMT gene signature is associated with metabolic alterations Using all cancer samples in the main cohort, we calculated an average Pearson’s correlation r of 0.34 between all 15 gene using pairwise correlations. This is comparable or higher than the average correlation between genes in previously validated prostate cancer signatures [38, 39] (Figure 4B), including signatures for the established TMPRSS2-ERG gene fusion (average Pearson’s r=0.30). The pattern of the NCWP-EMT gene set from the main cohort was validated in PCA analysis of the Wnt-genes in the five publicly available cohorts (n=1519 samples in total, Supplementary Table S1). The same 48 central Wnt- genes, in addition to WNT1, WNT3 and WNT3A which were lacking data in the main cohort, were used. All cohorts confirm the NCWP-EMT component as the most important source of variation in the gene expression, although there were some variations in the highlighted genes (Figure 4C- 4G and Supplementary Figure S1). The CWP was either insignificant or spanning a separate axis of variation with little correlation to EMT. Interestingly, WNT5A expression pattern varied considerably with respect to the NCWP-EMT axis. Overall, these data show the NCWP-EMT gene cluster to be robust over large prostate cancer patient cohorts, and the 15 NCWP-EMT genes to be accessible for a concordant NCWP-EMT gene expression signature. We further investigated the metabolic alterations of 23 metabolites between samples with low, intermediate, and high activation of the developed NCWP-EMT gene expression signature (Supplementary Table S4) in the main cohort. The most prominent alterations were observed for the metabolites citrate and the polyamine spermine (Table 2), which showed significantly decreased concentration in the high NCWP-EMT compared to low NCWP-EMT samples. This alteration was also observed for high NCWP-EMT samples when compared with intermediate NCWP-EMT samples, but not when comparing intermediate with low NCWP-EMT samples. This suggests citrate and spermine alterations to be more profound in the samples with high NCWP-EMT score compared to low and intermediate score NCWP-EMT. In addition, there were alterations in the concentration of phosphoethanolamine and taurine between the low and the intermediate score group (p=0.002, p=0.028 respectively). Decreased concentrations of citrate and spermine have been associated with aggressive prostate cancer [28, 40], and our results therefore suggest the NCWP-EMT signature to be associated with an aggressive metabolic profile. Reduced citrate can be a result of increased energy production through the Krebs cycle in prostate cancer [41]. A novel 15 gene non-canonical Wnt pathway - EMT (NCWP-EMT) signature useful addition to the selection criteria for active surveillance in prostate cancer patients. The novel NCWP-EMT signature also showed significant association with previously published mesenchyme and cytokine gene signatures (Supplementary Figure S2), and highly significant gene ontology (GO) terms related to cell adhesion, extracellular matrix, inflammation and immune response which are features commonly associated with EMT (Supplementary Table S3). The same analysis based on the expression level of WNT5A alone, did not produce any significant GO terms, further supporting the hypothesis that Wnt5a alone is an ambiguous biomarker in prostate cancer. To further investigate the relationship between the expression of Wnt and EMT genes, PCA analysis was performed on the expression profiles of 48 central Wnt and EMT genes (Methods). The first two principal components clearly highlighted a separate cluster of 15 genes related to the Wnt5a/Fzd2 pathway and EMT (Figure 4A). This gene set included 11 genes, which were also upregulated in high Gleason samples. In addition, two inhibitors of the CWP (NKD2 and SFRP1), and two EMT positive markers (CDH3 and MMP9) were part of the PCA cluster and included in the gene set. Because of the clear relationship to Wnt5/Fzd2 NCWP and EMT, we will refer to this set of genes collectively as the NCWP-EMT genes. EMT markers are upregulated in high Gleason prostate cancer The Wnt5/Fzd2 NCWP has previously been linked with EMT studies on various cancer cell-lines, but not in prostate cancer [22]. We therefore evaluated the gene expression of the most central EMT positive and negative markers in prostate cancer in the main cohort (Figure 2C and 2D). When comparing high Gleason with low Gleason samples, significant upregulations were detected for the expression of EMT positive markers in high Gleason; N-cadherin (CDH2), OB-cadherin (CDH11), vimentin (VIM) and Delta-2-catenin (CTNND2) (Figure 2C). In addition, a non-significant downregulation of E-cadherin (CDH1), an EMT negative marker, was observed in high Gleason samples (fold-change=-0.25, p=0.07; Figure 2D), suggesting ongoing EMT in high Gleason samples. In the immunohistochemistry cohort, IHC of N-cadherin showed membranous staining (SI≥2) in only two, both high Gleason, of the forty cancer samples (Figure 3E-3F). Reduced, moderate membranous staining of E-cadherin (SI=6), was detected in five samples while the remaining samples had strong membranous staining (SI=9) (Figure 3G-3H). However, the reduced E-cadherin staining did not correspond to N-cadherin staining, as hypothesized for the N- to E-cadherin switch proposed to be important for EMT in prostate cancer [37]. Inspection of the principal component analysis (PCA) score plots for the main and validation cohorts also confirmed consistent N-cadherin upregulation correlating with high Gleason and EMT genes, while the anticorrelation to E-cadherin was inconsistent between the cohorts, in accordance with observations in the immunohistochemistry cohort (Figure 4A, 4C-4G). In conclusion, the increased levels of several EMT positive genes, suggests ongoing EMT in a subset of mainly high Gleason prostate cancer samples. This was partly supported by the IHC, although the number of samples in the immunohistochemistry cohort was too few to make a conclusion. Figure 3: Immunohistochemical staining of the immunohistochemistry cohort. A. Strong membranous β-catenin staining and B. weak β-catenin staining. C. Strong Wnt5a staining and D. weak Wnt5a staining. E. Positive membranous N-cadherin staining and F. negative N-cadherin staining. G. Strong membranous E-cadherin staining and H. weak E-cadherin staining. Magnification x400. Bar 50μm. www.impactjournals.com/oncotarget Oncotarget 6 The NCWP-EMT gene signature is associated with metabolic alterations Two component PCA plot reveals a group of 15 of 48 genes, mainly connected to Wnt5a/Fzd2 non-canonical Wnt pathway, epithelial-mesenchymal transition (EMT), and inhibitors of the canonical Wnt pathway, collectively termed NCWP-EMT (CDH2, CDH3, CDH11, FYN, FZD2, LEF1, MMP9, NKD2, PLCB2, SFRP1, SFRP2, SFRP4, VIM, TCF4 WNT5A). B. The Pearson correlation of co-expression of the genes in the NCWP-EMT signature is as good or better compared with other recognized genes expression signatures in prostate cancer. Random marks 200 randomly selected genes for validation. C-G. The NCWP-EMT signature confirmed in the validation cohorts, although there were some variations in the highlighted genes. High-resolution versions of the PCA plots including all gene names, and Pearson correlation of the validation cohorts are available in Supplementary Figure S1. Figure 4: The NCWP-EMT gene expression signature. A. Two component PCA plot reveals a group of 15 of 48 genes, mainly connected to Wnt5a/Fzd2 non-canonical Wnt pathway, epithelial-mesenchymal transition (EMT), and inhibitors of the canonical Wnt pathway, collectively termed NCWP-EMT (CDH2, CDH3, CDH11, FYN, FZD2, LEF1, MMP9, NKD2, PLCB2, SFRP1, SFRP2, SFRP4, VIM, TCF4 WNT5A). B. The Pearson correlation of co-expression of the genes in the NCWP-EMT signature is as good or better compared with other recognized genes expression signatures in prostate cancer. Random marks 200 randomly selected genes for validation. C-G. The NCWP-EMT signature confirmed in the validation cohorts, although there were some variations in the highlighted genes. High-resolution versions of the PCA plots including all gene names, and Pearson correlation of the validation cohorts are available in Supplementary Figure S1. The NCWP-EMT gene signature is associated with metabolic alterations Previously, Wnt5a signaling has been identified as a regulator of the energy metabolism in melanoma cancer cells [26], and alterations of this metabolism have also been associated with EMT in cancer [42]. Another study detected that reduced polyamine content promoted EMT in non-tumor MDCK cells [43]. We therefore hypothesize that NCWP-EMT activation is associated with alterations in citrate and spermine metabolism in prostate cancer, although the direct mechanisms require further investigation. The continuous single sample gene set enrichment analysis (GSEA) score of the novel NCWP-EMT signature was significantly correlated with the Gleason score of the samples (Pearson’s r of 0.49, p<0.001). When the samples were categorized according to the NCWP-EMT score as low, intermediate, and high, the distribution of low/high Gleason samples in the groups were as following: NCWP- EMT low (n=25/n=7), NCWP-EMT intermediate (n=17/ n=14), and NCWP-EMT high (n=6/n=26). As expected most samples with high NCWP-EMT score also were high Gleason samples; however, some samples were low Gleason, and vice versa for samples with low NCWP-EMT score. This indicates that the NCWP-EMT signature might add an additional dimension for stratification, compared to Gleason grade alone. The NCWP-EMT signature may therefore, with further refinements and validation, be a To investigate the potential clinical translation of the metabolic findings, we inspected the gene signature score with matched pre-surgical in vivo MRSI from the same patients. Reduced citrate/creatine and spermine/creatine ratios were detected for high NCWP-EMT score samples when compared with low NCWP-EMT score (Table 2). Although we had a limited number of matched samples www.impactjournals.com/oncotarget Oncotarget 7 : The NCWP-EMT gene expression signature. A. Two component PCA plot reveals a group of 15 of 48 gen to Wnt5a/Fzd2 non-canonical Wnt pathway, epithelial-mesenchymal transition (EMT), and inhibitors of the cano collectively termed NCWP-EMT (CDH2, CDH3, CDH11, FYN, FZD2, LEF1, MMP9, NKD2, PLCB2, SFRP1, SFRP 4 WNT5A). B. The Pearson correlation of co-expression of the genes in the NCWP-EMT signature is as good or better recognized genes expression signatures in prostate cancer. Random marks 200 randomly selected genes for validation MT signature confirmed in the validation cohorts, although there were some variations in the highlighted genes. High Figure 4: The NCWP-EMT gene expression signature. A. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 8 Table 2: Alterations in citrate and spermine metabolism Table 2: Alterations in citrate and spermine metabolism Table 2: Alterations in citrate and spermine metabolism Metabolite concentration (mmol/kg wet weight) ex vivo and metabolites amount/ratios in vivo p-valuesa Signature score Low Intermediate(Int) High Low vs. High Int. vs High Low vs. Int. Median (IQR) Median (IQR) Median (IQR) Ex vivo (n=95) (n=32) (n=31) (n=32) Citrate 7.31 (5.57-11.56) 6.38 (4.56-11.58) 3.55 (2.08-7.25) 3.38·10-4* 0.018* 0.282 Spermine 1.55 (1.02-2.36) 1.23 (0.67-2.27) 0.75 (0.39-1.43) 3.38·10-4* 0.028* 0.113 In vivo (n=22) (n=10) (n=7) (n=5) Citrate/Creatine 7.36 (5.81-8.79) 4.45 (3.34-7.79) 2.77 (1.48-3.00) 0.0056* 0.027* 0.030* Spermine/ Creatine 0.83 (0.44-1.04) 0.50 (0.04-1.11) 0.00 (0.00-0.02) 0.0057* 0.027* 0.101 IQR – Interquartile range * Indicates significance at p<0.05 a P-values from LMM adjusting for multiple samples per patient, and corrected for multiple testing by Benjamini and Hochberg procedure. IQR – Interquartile range * Indicates significance at p<0.05 a P-values from LMM adjusting for multiple samples per patient, and corrected for multiple testing by Benjamini and Hochberg procedure. * Indicates significance at p<0.05 a P-values from LMM adjusting for multiple samples per patient, and corrected for multiple testing by Benjamini and Hochberg procedure plot showed a significant separation between the groups (log-rank p=0.035) (Figure 5A). Validation of recurrence was possible in the GSE21034 cohort (131 samples, 27 with recurrence), and showed a similar pattern with 10-year biochemical recurrence free rates of 81%, 73% and 57% in patients with low, intermediate and high NCWP-EMT score, respectively. However, there was no significant separation in the Kaplan-Meier curves for this cohort (log-rank p=0.522) (Figure 5B). For this validation dataset there was only one sample per patient, not necessarily extracted from the most aggressive cancer foci, which may reduce the precision of the NCWP-EMT grouping for biochemical recurrence analysis. In addition, many of the patients in the validation dataset were lost to follow-up early, and therefore censored in the analysis (Figure 5B), causing reduces reliability of the curves. In the GSE46691 cohort, samples with high NCWP-EMT scores were significantly associated with metastases (545 samples, 212 with metastasis, p-value<0.001, chi-square test, Supplementary Figure S3). With the significant separation in our data, and the similar trend in the validation datasets, we therefore suggest that increased NCWP-EMT signature score is associated with an increased risk of biochemical recurrence and metastases. www.impactjournals.com/oncotarget This strengthens the NCWP-EMT signature, and the activation of the Wnt5/Fzd2 pathway, as markers of aggressive prostate cancer. in the main cohort (n=22), the results support our findings from the tissue analysis, and demonstrates that the MR biomarkers can reflect the NCWP-EMT signature also in non-invasive MRSI examinations. Citrate and spermine are stored within the luminal space of the glands in prostate tissue, and the observed metabolic alterations can be due to cell metabolism or morphological changes. In the main cohort, the citrate and spermine concentrations were correlated with luminal space (Spearman’s rho=0.30/p=0.003, rho=0.31/p=0.003, respectively). This was a weaker correlation than between citrate and spermine concentrations and the NCWP-EMT signature score (Spearman’s rho=0.42/p<0.001, rho=0.38/ p<0.001, respectively). LMM, adjusting for luminal space as well as other tissue heterogeneity and Gleason score, still showed the same metabolic alterations to be significant (Supplementary Table S5). These results suggest the alterations observed in citrate and spermine concentrations are a combination of changes in both luminal space and reprogramming of metabolism in samples with high NCWP-EMT score. There was no relationship between Wnt5a expression and metabolite concentrations in either the main or immunohistochemistry cohort (Supplementary Table S6). This supports that Wnt5a should be used as a biomarker in combination with other pathway components, such as our NCWP-EMT signature. www.impactjournals.com/oncotarget NCWP-EMT signature may help predict biochemical recurrence Patients in the main cohort with a post-operative Gleason score of 7 showed a five-year biochemical recurrence free survival of 100%, 89% and 67% with low, intermediate and high NCWP-EMT score, respectively (Figure 5C). Although not statistically significant, possibly due to the low number of patients (n=23), this separation with no crossing indicates that the NCWP-EMT gene In the main cohort the five-year biochemical recurrence free rates were 100%, 75% and 46% for the patients in the low, intermediate and high NCWP- EMT score groups, respectively, and the Kaplan-Meier www.impactjournals.com/oncotarget Oncotarget 9 Gleason score (≤ 7), the hazard ratio for NCWP- EMT was 1.61, indicating that increased NCWP- EMT signature score gives a significant higher risk of biochemical recurrence for this group. To compare the NCWP-EMT and post-operative Gleason score as predictors of biochemical recurrence, two additional Cox proportional hazards models, each excluding either NCWP-EMT or post-operative Gleason score, were tested (Supplementary Table S8). The Akaike information criterion (AIC) represent the goodness of fit as well as the complexity of the model, and can be compared between models, where the lower AIC provides a better model fit. The model including post-operative Gleason Gleason score (≤ 7), the hazard ratio for NCWP- EMT was 1.61, indicating that increased NCWP- EMT signature score gives a significant higher risk of biochemical recurrence for this group. To compare the NCWP-EMT and post-operative Gleason score as predictors of biochemical recurrence, two additional Cox proportional hazards models, each excluding either NCWP-EMT or post-operative Gleason score, were tested (Supplementary Table S8). The Akaike information criterion (AIC) represent the goodness of fit as well as the complexity of the model, and can be compared between models, where the lower AIC provides a better model fit. The model including post-operative Gleason signature might be useful for improved risk stratification in the challenging group of patients with Gleason score 7. Univariate cox proportional hazards analyses identified NCWP-EMT, Gleason score and pathological T-stage as significant predictors of biochemical recurrence (Table 3). Multivariate analysis showed both NCWP-EMT and post-operative Gleason score to be significant predictors of biochemical recurrence (Table 3). The multivariate model included a significant interaction term between NCWP-EMT and post- operative Gleason score, implying that the hazards ratio of these variables were dependent on the value of the other variable. For patient with low post-operative ure 5: Kaplan-Meier and ROC curves of biochemical recurrence. A. NCWP-EMT signature may help predict biochemical recurrence Low GS – Hazard ratio/p-value for patients with post-operative Gleason score ≤7 High GS – Hazard ratio/p-value for patients with post-operative Gleason score ≥8 Δ Indicates the category used as a reference in each analysis.i Δ Indicates the category used as a reference in each analysis. * Indicates significant p-value. gi p Low GS – Hazard ratio/p-value for patients with post-operative Gleason score ≤7 High GS – Hazard ratio/p-value for patients with post-operative Gleason score ≥8 Low GS – Hazard ratio/p-value for patients with post-operative Gleason score ≤7 High GS – Hazard ratio/p-value for patients with post-operative Gleason score ≥8 had a slightly lower AIC (AIC=64.24) compared to the model including NCWP-EMT (AIC=65.61), suggesting post-operative Gleason to be a slightly better predictor of biochemical recurrence than NCWP-EMT. However, the model containing all variables, had the lowest AIC (AIC=60.15) demonstrating improved prediction of biochemical recurrence when NCWP-EMT and post- operative Gleason score were modelled together.i associated with reduced concentrations of the metabolites citrate and spermine both ex vivo, and in a clinical non- invasive setting using in vivo patient MRSI. The novel NCWP-EMT signature was also shown to be a predictor of biochemical recurrence and was associated with metastasis, indicating that upregulation of the NCWP and EMT is linked to more aggressive prostate cancer. The novel NCWP-EMT signature may therefore be useful for risk stratification and molecular subtyping of prostate cancer patients. The NCWP and its relation to EMT, cancer aggressiveness and tumor metabolism warrants further attention in prostate cancer studies. Similar findings were also visualized by using logistic regression and receiver operating characteristic (ROC) curves with the depended variable being biochemical recurrence after 5-year follow-up. The area under the curve (AUC) of the ROC-curve were the same for NCWP-EMT and post-operative Gleason score (AUC=80.9), and in combination they provided increased sensitivity and specificity (AUC=90.4) (Figure 5D). In conclusion, our results suggest that the NCWP- EMT signature could be a useful addition in prediction of biochemical recurrence in prostate cancer. Patients and tissue samples In the main cohort, human prostate tissue was collected from 41 localized and locally advanced prostate cancer patients. The tissue harvesting was performed on fresh-frozen prostatectomy specimens using a standardized method thoroughly described by Bertilsson et al. [29]. A total of 95 cancer tissue samples, and 34 adjacent normal tissue samples were collected (median 3, range 1-6 samples per patient). At least five years of follow-up data were successfully retrieved for 33 patients in the main cohort, including the date of biochemical recurrence (PSA of at least 0.2 ng/mL) and/or last negative PSA measurement. To validate the results of the main cohort, an additional www.impactjournals.com/oncotarget NCWP-EMT signature may help predict biochemical recurrence The main cohort shows clear separation in biochemica rrence free survival between the low, intermediate and high NCWP-EMT signature groups. B. A validation cohort (GSE21034) shows ame pattern, although not a significant separation. C. A similar pattern was also shown for the patient of the main cohort with a post ative Gleason score of 7. D. The ROC curves of biochemical recurrence after 5 years show the same AUC of post-operative Gleason e and NCWP-EMT, but an increased AUC when combined. Δ Continuous NCWP-EMT signature score, * continuous post-operative son score. Abbreviations: BCR - biochemical recurrence, RP – radical prostatectomy, ROC – Receiver operating characteristic, and C – area under the curve. Figure 5: Kaplan-Meier and ROC curves of biochemical recurrence. A. The main cohort shows clear separation in biochemical recurrence free survival between the low, intermediate and high NCWP-EMT signature groups. B. A validation cohort (GSE21034) shows the same pattern, although not a significant separation. C. A similar pattern was also shown for the patient of the main cohort with a post- operative Gleason score of 7. D. The ROC curves of biochemical recurrence after 5 years show the same AUC of post-operative Gleason score and NCWP-EMT, but an increased AUC when combined. Δ Continuous NCWP-EMT signature score, * continuous post-operative Gleason score. Abbreviations: BCR - biochemical recurrence, RP – radical prostatectomy, ROC – Receiver operating characteristic, and AUC – area under the curve. www.impactjournals.com/oncotarget Oncotarget 10 ate and multivariate Cox proportional hazards analyses of biochemical recurrence Table 3: Univariate and multivariate Cox proportional hazards analyses of biochemical recurrence Univariate Multivariate – All variables (AIC = 60.15) Variables Hazard ratio (95% CI) P-values Hazard ratio (95% CI) P-values Post-operative Gleason score (≤7Δ and ≥8) 7.66 (2.20-26.62) 0.001* 19.46 (2.67-142.9) 0.003* Pathological T-stage (≤T2cΔ and ≥T3a) 6.88 (2.06-23.01) 0.002* 8.27 (0.89-77.15) 0.064 Pre-operative PSA (<10Δ and ≥10) 2.17 (0.69-7.13) 0.204 2.89 (0.72-11.67) 0.14 NCWP-EMT Continuous score/100 (-4.4–5.4) 1.37 (1.08-1.73) 0.009* Low GS 1.61 (1.06-2.44) Low GS 0.028* High GS 0.59 (0.35-0.99) High GS 0.044* NCWP-EMT and Post-operative Gleason score (≤7Δ and ≥8) (interaction term) - - 0.37 (0.18-0.74) 0.005* Δ Indicates the category used as a reference in each analysis. * Indicates significant p-value. Histopathology In the main cohort, gene expression analysis was performed after HR-MAS MRS on the exact same tissue sample, using an Illumina TotalPrep RNA Amplification Kit (Ambion Inc.) and an Illumina Human HT-12v4 Expression Bead Chip (Illumina), as described by Bertilsson et al. [51]. The microarray data has previously been published in Array Expression with access number: E-MTAB-1041. Genes relevant to both the WP and EMT were carefully chosen by investigating literature and publicly available pathway maps (KEGG as per March 2015) [2, 3, 5, 22]], resulting in 196 genes (Supplementary Table S2). To control for the effect of confounding stroma tissue when identifying differentially expressed genes, we used a recently published strategy of balancing the stroma content between sample groups [30]. This strategy makes it possible to separate molecular signals relevant to cancer from signals originating due to different stroma fractions between the sample groups. Briefly described, the strategy selects samples to ensure an equal average fraction of stroma tissue (according to histopathology) in each sample group termed a balanced differential expression analysis. In contrast, an unbalanced analysis is also performed to highlight differentially expressed gene due to different average fractions of stroma tissue. In the main cohort, tissue slices for histopathological evaluation were cryosectioned from each tissue sample prior to HR-MAS MRS [29]. All cryosections were stained with Haematoxylin and Eosin, and the histopathological evaluations were performed according to the clinical criteria for prostate cancer, by an experienced pathologist specialized in uropathology (TV). The percentage of Gleason grades, cancer, normal glandular epithelia, and stromal tissue were reported for each sample. Reproducibility of the histopathological scoring was assessed independently by a second pathologist specialized in uropathology (ER), and the overall kappa (κ) coefficient for interobserver agreement of Gleason score was 0.66 indicating substantial agreement. The first reading was used in this study due to slight degradation of the cryosections between the readings (5 years, slides kept dry and dark). Luminal space was quantified in each sample by a color- based segmentation method (Positive Pixel Count algorithm in ImageScope v.8, Aperio Technologies) [49]. The samples in the immunohistochemistry cohort were formalin fixed and paraffin embedded for sectioning after HR-MAS MRS analysis, and histopathological evaluation was done according to the same protocol as the main cohort. CONCLUSIONS Absolute quantification of the spectra was performed using LCModel [50] with a basis set of 23 metabolites, and reported in mmol/kg wet weight. Full procedure and parameters of the HR-MAS MRS acquisition and LCModel quantification have earlier been described by Giskeødegård et al. [28]. In vivo patient MRSI examination of the prostate, performed using a 3T system (Magnetom Trio, Siemens, Germany) prior to prostatectomy, was available on a subset of the patients in the main cohort (n=9). Choline, citrate, creatine and spermine were quantified using LCModel, and creatine was used as an internal standard for normalization (metabolites to creatine ratios). HR-MAS cancer samples from the same patients were spatially matched to an in vivo voxel (n=22). Further details on the MRSI acquisition, quantification, and spatial matching are previously described by Selnæs et al. [27]. Immunohistochemistry (IHC) In the immunohistochemistry cohort, IHC was performed with mouse monoclonal antibodies against Wnt5a (Sigma-Aldrich, clone 3A4, dilution 1:50), N-cadherin (Dako, clone 6G11, dilution 1:30), and E-cadherin/NCH-38 (Dako, clone NCH-38, dilution 1:100) and polyclonal rabbit antibodies against β-catenin/CTNNB1 (PRESTIGE antibodies Sigma, dilution 1:300). The sections were counter-stained with Histopathology In both cohorts, we investigated differences between low and high Gleason grade by sorting the tissue samples into two groups, where samples in the low Gleason group had a Gleason score ≤ 3+4 and samples in the high Gleason group had a Gleason score ≥ 4+3 (Table 1). CONCLUSIONS The present study showed no alterations in the CWP in prostate cancer, but revealed an increased expression of NCWP and EMT markers in a subgroup of mainly high Gleason grade prostate cancer samples. A novel gene expression signature (NCWP-EMT) for this expression profile was presented and confirmed in several publicly available patient cohorts. High NCWP-EMT score was www.impactjournals.com/oncotarget Oncotarget 11 Bruker Avance DRX600 Spectrometer (Bruker Biospin, Germany) equipped with a dual 1H/13C MAS probe. Absolute quantification of the spectra was performed using LCModel [50] with a basis set of 23 metabolites, and reported in mmol/kg wet weight. Full procedure and parameters of the HR-MAS MRS acquisition and LCModel quantification have earlier been described by Giskeødegård et al. [28]. In vivo patient MRSI examination of the prostate, performed using a 3T system (Magnetom Trio, Siemens, Germany) prior to prostatectomy, was available on a subset of the patients in the main cohort (n=9). Choline, citrate, creatine and spermine were quantified using LCModel, and creatine was used as an internal standard for normalization (metabolites to creatine ratios). HR-MAS cancer samples from the same patients were spatially matched to an in vivo voxel (n=22). Further details on the MRSI acquisition, quantification, and spatial matching are previously described by Selnæs et al. [27]. cohort of 90 needle biopsies from 90 localized and locally advanced cancer patients were harvested and snap frozen within seconds after prostatectomy. Of these, only the samples with histopathological confirmed cancer were used as the immunohistochemistry cohort for this study (n=40). The patients in both cohorts received no prostate cancer treatment prior to surgery and had no detected metastasis at diagnosis. The Regional Committee of Medical and Health Research Ethics (REC), Central Norway approve both cohorts, and all patients gave written, informed consent. Validation was performed in four prostate cancer microarray datasets available through the Gene Expression Omnibus with GEO accessions GSE8218 (65 samples) [44], GSE16560 (281 samples) [45], GSE21034 (131 samples) [46], GSE46691 (545 samples) [47], and one data set from The Cancer Genome Atlas (TCGA, 497 samples) [48], in total 1519 samples (Supplementary Table S1). These datasets are collectively termed the validation cohorts. Biochemical recurrence was validated in the GSE21034 cohort, and metastasis in the GSE46691 cohort. Bruker Avance DRX600 Spectrometer (Bruker Biospin, Germany) equipped with a dual 1H/13C MAS probe. GRANT SUPPORT The study was supported by grants from the Medical Student’s Research Programme, Norwegian University of Science and Technology (NTNU), the Norwegian Cancer Society, Central Norway Regional Health Authority (RHA), and the Liaison Committee between the RHA and NTNU. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Statistical analysis The WP and EMT genes were compared for differential expression between normal and cancer samples, and between low and high Gleason samples by t-test. All the 196 genes were considered, but to ease data analysis and presentation a subgroup 48 key and/or significantly altered genes are presented as the central genes, however, a full table of the p-values is given in Supplementary Table S2. PCA was used to further investigate and visualize the unsupervised relationship between the expressions of these central WP and EMT genes. Based on the PCA score plot, a distinct set of genes was selected to make a gene expression signature termed NCWP-EMT. The co- expression between the signature genes was investigated by Pearson’s correlation, and compared to other recognized gene expression signatures in prostate cancer. The distinct gene-signature pattern from PCA and Pearson’s correlation between signature genes were confirmed in the validation cohorts. Single sample GSEA was performed to give each of the cancer samples in the main and validation cohorts a score representing the expression of the genes in the NCWP- EMT signature [52]. The samples in each cohort were sorted into three equal sized groups of low, intermediate, and high NCWP-EMT signature scores, where the high score group had the highest pathway activity. Features associated with NCWP-EMT were investigated by Gene Ontology (GO) using the Database for Annotation and Visualization and Integrated Discovery (DAVID). Biochemical recurrence free survival for the NCWP-EMT score groups were plotted by Kaplan-Meier curves and tested by log-rank test in the main and GSE21034 cohort, where for the individual patient’s highest NCWP-EMT score was used in the main cohort. The association between NCWP-EMT and metastasis in the GSE46691 cohort was tested using a contingency table and chi-squared test. Univariate and multivariate cox proportional hazards statistics were used to investigate the role of the NCWP-EMT signature in prediction of biochemical recurrence. Prior to analysis, post-operative Gleason score, pathological T-stage and pre-operative PSA were dichotomized (Table 3), and together with the continuous NCWP-EMT signature score selected for multivariate analysis. Biochemical recurrence at five-year follow-up was selected to plot ROC curves of NCWP-EMT score, post-operative Gleason score and both combined. Linear mixed model (LMM) was used to account for multiple samples per patient, when investigating the relationship between NCWP-EMT score groups and HR-MAS MRS and MRSI experiments and quantification For both the main and the immunohistochemistry cohort, proton HR-MAS MRS was acquired using a www.impactjournals.com/oncotarget Oncotarget 12 metabolite concentrations. The analyses were repeated with additional adjustment for Gleason grade, and tissue heterogeneity including the proportion of cancer, benign epithelium, stroma and luminal space in the individual tissue sample. The immunohistochemistry cohort consisted of one sample per patient, and t-test was used to investigate the association between IHC and metabolite concentrations. Prior to analysis, all metabolite values were log transformed to obtain normalized residuals, and p-values were corrected for multiple testing using Benjamini-Hochberg false discovery rate. P-values <0.05 were considered significant. The statistical analyses were performed in R (version 3.2.0, R Foundation for Statistical Computing). Haematoxylin. Assessment was performed manually, and all the IHC sections were evaluated based on the average staining intensity (0-3) multiplied by the percentage of positive cancer cells (0-3), obtaining a total staining index (SI) (0-9). A SI of 0 was regarded as negative, 1-2 as weak positive staining; 3-6 as moderate, and 9 as strong positive staining (Supplementary Table S7). An experienced pathologist (AMB) validated the scoring. ACKNOWLEDGMENTS The tissue samples in the Main cohort were collected and stored by Biobank1, St. Olavs Hospital, HR-MAS MRS was performed at the MR Core Facility, Norwegian University of Science and Technology (NTNU), histopathological preparation and staining was performed at the Cellular & Molecular Imaging Core Facility (CMIC), NTNU, and the microarray service was provided by the Genomics Core facility NTNU and Norwegian Microarray Consortium (NMC), a national platform supported by the functional genomics program (FUGE) of the research Council of Norway. The authors thank Turid Follestad for assistance with LMM and survival statistical analyses, and Deborah K. Hill for her useful comments and discussions. The Authors do not have any conflicts of interest. The Authors do not have any conflicts of interest. REFERENCES Nature reviews Cancer. 2013; 13:11-26. 23. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011; 144:646-674. 24. Sherwood V. 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https://openalex.org/W4297369949	https://egusphere.copernicus.org/preprints/2022/egusphere-2022-110/egusphere-2022-110.pdf	English	null	Towards vertical wind and turbulent flux estimation with multicopter uncrewed aircraft systems	Atmospheric measurement techniques	2,022	cc-by	14,659	Towards vertical wind and turbulent ﬂux estimation with multicopter UAS Norman Wildmann1 and Tamino Wetz1 The spectra of variances and covariances show good agreement with the sonic anemometer up to 1 Hz temporal resolution. A case study of continuous measurements in a morning transition of a convective boundary layer with ﬁve UAS illustrates the potential of such measurements for ABL 10 research. 10 Towards vertical wind and turbulent ﬂux estimation with multicopter UAS Norman Wildmann1 and Tamino Wetz1 multicopter UAS Norman Wildmann1 and Tamino Wetz1 1Deutsches Zentrum für Luft- und Raumfahrt e.V., Institut für Physik der Atmosphäre, Oberpfaffenhofen, Germany Correspondence: Norman Wildmann (norman.wildmann@dlr.de) Norman Wildmann1 and Tamino Wetz1 1Deutsches Zentrum für Luft- und Raumfahrt e.V., Institut für Physik der Atmosphäre, Oberpfaffenhofen, Germany Correspondence: Norman Wildmann (norman.wildmann@dlr.de) Abstract. Vertical wind velocity and its ﬂuctuations are essential parameters in the Atmospheric Boundary Layer (ABL) to determine turbulent ﬂuxes and scaling parameters for ABL processes. The typical instrument to measure ﬂuxes of momentum and heat in the surface layer are sonic anemometers. Without the infrastructure of meteorological masts and above their typical heights, in-situ point measurements of the three-dimensional wind vector are hardly available. We present a method to obtain the three-dimensional wind vector from avionic data of small multicopter unmanned aerial systems (UAS). To achieve a good 5 accuracy in both, average and ﬂuctuating parts of the wind components, calibrated motor thrust and measured accelerations by 1 Abstract. Vertical wind velocity and its ﬂuctuations are essential parameters in the Atmospheric Boundary Layer (ABL) to determine turbulent ﬂuxes and scaling parameters for ABL processes. The typical instrument to measure ﬂuxes of momentum and heat in the surface layer are sonic anemometers. Without the infrastructure of meteorological masts and above their typical heights, in-situ point measurements of the three-dimensional wind vector are hardly available. We present a method to obtain the three-dimensional wind vector from avionic data of small multicopter unmanned aerial systems (UAS). To achieve a good 5 accuracy in both, average and ﬂuctuating parts of the wind components, calibrated motor thrust and measured accelerations by the UAS are used. In a validation campaign, in comparison to sonic anemometers on a 99-m mast, accuracies below 0.2 m s−1 are achieved for the mean wind components and below 0.2 m2s−2 for their variances. The spectra of variances and covariances show good agreement with the sonic anemometer up to 1 Hz temporal resolution. A case study of continuous measurements the three-dimensional wind vector from avionic data of small multicopter unmanned aerial systems (UAS). To achieve a good 5 accuracy in both, average and ﬂuctuating parts of the wind components, calibrated motor thrust and measured accelerations by the UAS are used. In a validation campaign, in comparison to sonic anemometers on a 99-m mast, accuracies below 0.2 m s−1 are achieved for the mean wind components and below 0.2 m2s−2 for their variances. https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. Towards vertical wind and turbulent ﬂux estimation with multicopter UAS Norman Wildmann1 and Tamino Wetz1 1Deutsches Zentrum für Luft- und Raumfahrt e.V., Institut für Physik der Atmosphäre, Oberpfaffenhofen, Germany Correspondence: Norman Wildmann (norman.wildmann@dlr.de) 1 Introduction The nature of turbulence in the atmospheric boundary layer (ABL) is multifaceted and depends on internal and external pa- rameters such as thermal stratiﬁcation and shear, surface heat ﬂuxes and surface roughness. Turbulent ﬂuxes of momentum 15 and heat are the main driver for mixing in the ABL and thus determine its diurnal cycle. Determining turbulent ﬂuxes requires 15 measuring the three-dimensional wind vector and temperature with a temporal resolution that covers all turbulent scales that contribute signiﬁcantly to the process. The eddy covariance (EC) method is a way to determine the momentum ﬂuxes from the covariances of the wind components u′w′, v′w′, u′v′ and the heat ﬂux from the covariance between potential temperature and vertical wind θ′w′ (Aubinet et al., 2012). From the ﬂuxes, scaling parameters for the ABL such as shear stress u∗, convective velocity scale w∗as well as Obukhov length L and Richardson number Ri can be derived (Stull, 1988) which are essential for 20 a profound understanding of boundary-layer processes. It is thus evident that the measurement of vertical wind velocity and its ﬂuctuations is crucial. In practice, the most prominent instrument to measure turbulent ﬂuxes in the ABL is the three-dimensional sonic anemometer (Kaimal and Businger, 1963) which has undergone extensive development and commercialization since the 1990s and is nowa- days the essential part of every surface energy balance station (Mauder and Zeeman, 2018). Operating sonic anemometers is 25 velocity scale w∗as well as Obukhov length L and Richardson number Ri can be derived (Stull, 1988) which are essential for 20 a profound understanding of boundary-layer processes. It is thus evident that the measurement of vertical wind velocity and its ﬂuctuations is crucial. ﬂuctuations is crucial. In practice, the most prominent instrument to measure turbulent ﬂuxes in the ABL is the three-dimensional sonic anemometer (Kaimal and Businger, 1963) which has undergone extensive development and commercialization since the 1990s and is nowa- d th ti l t f f b l t ti (M d d Z 2018) O ti i t i 25 In practice, the most prominent instrument to measure turbulent ﬂuxes in the ABL is the three-dimensional sonic anemometer (Kaimal and Businger, 1963) which has undergone extensive development and commercialization since the 1990s and is nowa- days the essential part of every surface energy balance station (Mauder and Zeeman, 2018). 1 Introduction Operating sonic anemometers is 25 In practice, the most prominent instrument to measure turbulent ﬂuxes in the ABL is the three-dimensional sonic anemometer (Kaimal and Businger, 1963) which has undergone extensive development and commercialization since the 1990s and is nowa- days the essential part of every surface energy balance station (Mauder and Zeeman, 2018). Operating sonic anemometers is 25 days the essential part of every surface energy balance station (Mauder and Zeeman, 2018). Operating sonic anemometers is 25 1 1 In order to operate a ﬂeet of small and lightweight multicopter UAS, we propose a method to obtain the three-dimensional wind vector with avionic data alone, including calibrated motor thrust measurement. This is the ﬁrst method that allows vertical wind and turbulent ﬂux measurement with multicopter UAS without an external sensor. In Section 2, we describe the UAS and the methodology to ﬁlter sensor data, calculate the forces that act on the UAS and the rotors, measurements of vertical wind is feasible. However, such a design increases the complexity of the ﬂight system, 45 its weight and cost. In order to operate a ﬂeet of small and lightweight multicopter UAS, we propose a method to obtain the three-dimensional wind vector with avionic data alone, including calibrated motor thrust measurement. This is the ﬁrst method that allows vertical wind and turbulent ﬂux measurement with multicopter UAS without an external sensor. In Section 2, we describe the UAS and the methodology to ﬁlter sensor data, calculate the forces that act on the UAS and In Section 2, we describe the UAS and the methodology to ﬁlter sensor data, calculate the forces that act on the UAS and calibrate the vertical velocity measurements. Section 3 describes the setting of the calibration and validation campaign at the 50 boundary layer ﬁeld site (Grenzschichtmessfeld, GM) Falkenberg of the German Meteorological Service (DWD). Section 4 contains the results of three-dimensional wind measurements, second-order statistics and the derivation of momentum ﬂuxes in comparison to measurements of sonic anemometers on a 99 m mast. A case study with measurements during a morning transition of a convective boundary layer illustrates the potential of the method. We conclude and give an outlook on future research in Sect. 5. 55 gy calibrate the vertical velocity measurements. Section 3 describes the setting of the calibration and validation campaign at the 50 boundary layer ﬁeld site (Grenzschichtmessfeld, GM) Falkenberg of the German Meteorological Service (DWD). Section 4 contains the results of three-dimensional wind measurements, second-order statistics and the derivation of momentum ﬂuxes in comparison to measurements of sonic anemometers on a 99 m mast. A case study with measurements during a morning transition of a convective boundary layer illustrates the potential of the method. We conclude and give an outlook on future calibrate the vertical velocity measurements. https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. mostly limited to the surface layer Baldocchi et al. (2001) or requires meteorological masts to reach heights up to 300 m above ground which involves signiﬁcant costs and infrastructure (Wolfe and Lataitis, 2018). To obtain representative quantities for a certain area with single point measurements, assumptions of homogeneity and ergodicity need to be made. It has recently been shown that coherent structures such as secondary circulations can violate the ergodicity assumption and lead to errors mostly limited to the surface layer Baldocchi et al. (2001) or requires meteorological masts to reach heights up to 300 m above ground which involves signiﬁcant costs and infrastructure (Wolfe and Lataitis, 2018). To obtain representative quantities for a certain area with single point measurements, assumptions of homogeneity and ergodicity need to be made. It has recently been shown that coherent structures such as secondary circulations can violate the ergodicity assumption and lead to errors in the EC method (Mauder et al., 2020; Morrison et al., 2022). Only few experiments exist where multiple sonics have been 30 installed over a wide area and heights above the surface layer, due to the large logistical effort. Some examples are the Great Belt Coherence experiment over water (Mann et al., 1991) and the Perdigão 2017 experiment in complex terrain (Fernando et al., 2019). One possibility to obtain area-averaged ﬂuxes are airborne measurements with research aircraft incorporating ﬂow probes and in the EC method (Mauder et al., 2020; Morrison et al., 2022). Only few experiments exist where multiple sonics have been 30 installed over a wide area and heights above the surface layer, due to the large logistical effort. Some examples are the Great Belt Coherence experiment over water (Mann et al., 1991) and the Perdigão 2017 experiment in complex terrain (Fernando et al., 2019). O ibilit t bt i d ﬂ i b t ith h i ft i ti ﬂ b d 30 One possibility to obtain area-averaged ﬂuxes are airborne measurements with research aircraft incorporating ﬂow probes and fast temperature sensors. Such measurements can cover many square kilometers in a relatively short amount of time, but also 35 require assumptions of stationarity throughout the ﬂight which can lead to sampling errors (Mahrt, 1998). To overcome lim- itations of manned aircraft to ﬂy at low altitudes and at lower costs, ﬁxed-wing unmanned aircraft systems (UAS) have been deployed in the past to measure turbulent ﬂuxes (van den Kroonenberg et al., 2008; Wildmann et al., 2015). To combine the beneﬁts of the ﬂexible deployment of UAS and allow point measurements instead of area-averaged measure- fast temperature sensors. Such measurements can cover many square kilometers in a relatively short amount of time, but also 35 require assumptions of stationarity throughout the ﬂight which can lead to sampling errors (Mahrt, 1998). To overcome lim- itations of manned aircraft to ﬂy at low altitudes and at lower costs, ﬁxed-wing unmanned aircraft systems (UAS) have been deployed in the past to measure turbulent ﬂuxes (van den Kroonenberg et al., 2008; Wildmann et al., 2015). To combine the beneﬁts of the ﬂexible deployment of UAS and allow point measurements instead of area-averaged measure- To combine the beneﬁts of the ﬂexible deployment of UAS and allow point measurements instead of area-averaged measure- ments, multicopter UAS are a possible solution. Wetz et al. (2021) showed that such systems can be deployed in small ﬂeets 40 and obtain distributed horizontal wind measurements with a good accuracy. They become increasingly popular for vertical proﬁling of the ABL when temperature and humidity sensors are carried (Koch et al., 2018; Segales et al., 2020). The mea- surement of vertical velocity with such systems is however challenging due to the obvious distortion of the ﬂow by the thrust of the rotors. Thielicke et al. (2021) showed that with a sonic anemometer installed on a multicopter in sufﬁcient distance to ments, multicopter UAS are a possible solution. Wetz et al. (2021) showed that such systems can be deployed in small ﬂeets 40 and obtain distributed horizontal wind measurements with a good accuracy. They become increasingly popular for vertical proﬁling of the ABL when temperature and humidity sensors are carried (Koch et al., 2018; Segales et al., 2020). The mea- surement of vertical velocity with such systems is however challenging due to the obvious distortion of the ﬂow by the thrust of the rotors. Thielicke et al. (2021) showed that with a sonic anemometer installed on a multicopter in sufﬁcient distance to the rotors, measurements of vertical wind is feasible. However, such a design increases the complexity of the ﬂight system, 45 its weight and cost. 2.1 System description We operate a ﬂeet of 35 UAS of type Holybro QAV250 within the SWUF-3D project (Simultaneous Wind measurements with Unmanned Flight Systems in 3D). The main characteristics are described in Wetz et al. (2021). With a weight below 1 kg and a frame size of 0.25 m, the single UAS are small enough to be operated in the open category of the European Regulation 60 2019/947. Operation in a ﬂeet with fewer pilots than UAS requires special permission from the ﬂight authorities. A Pixhawk®4 Mini autopilot is used as the ﬂight controller and allows access to all avionic data which is stored at high sampling rates on an internal SD card. The sampling rate depends on the type of data and is presented in more detail in Sect. 2.5. In the current conﬁguration, ﬂight times up to 17 minutes can be achieved. Figure 1 shows two UAS in ﬂight. 60 Section 3 describes the setting of the calibration and validation campaign at the 50 boundary layer ﬁeld site (Grenzschichtmessfeld, GM) Falkenberg of the German Meteorological Service (DWD). Section 4 contains the results of three-dimensional wind measurements, second-order statistics and the derivation of momentum ﬂuxes in comparison to measurements of sonic anemometers on a 99 m mast. A case study with measurements during a morning transition of a convective boundary layer illustrates the potential of the method. We conclude and give an outlook on future 2 2 Figure 1. Two Holybro QAV250 UAS in ﬂight. In the background the 99-m mast with a sonic anemometer on the boom at 50 m can be seen. https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure 1. Two Holybro QAV250 UAS in ﬂight. In the background the 99-m mast with a sonic anemometer on V250 UAS in ﬂight. In the background the 99-m mast with a sonic anemometer on the boom at 50 m can be see Figure 1. Two Holybro QAV250 UAS in ﬂight. In the background the 99-m mast with a sonic anemometer on the boom at 50 m can be seen. 2.2 Motor thrust calibration 65 A schematic of the setup is shown in 80 Fig. 2. We can thus determine the motor constant kV and the thrust coefﬁcient Ct experimentally. In order to determine this relationship experimentally, as well as the thrust coefﬁcient of the rotor, which relates thrust to revolution speed, a hardware-in-the-loop test stand for a single motor/rotor setup is used. A schematic of the setup is shown in 80 Fig. 2. We can thus determine the motor constant kV and the thrust coefﬁcient Ct experimentally. revolution speed, a hardware-in-the-loop test stand for a single motor/rotor setup is used. A schematic of the setup is shown in 80 Fig. 2. We can thus determine the motor constant kV and the thrust coefﬁcient Ct experimentally. 2.3 Quadrotor dynamics and rotor aerodynamics To describe the motion of a quadrotor in three dimensions in its body-ﬁxed frame of reference (x to the front, y to the right, z downwards), the rigid body model as described in Wetz et al. (2021) can be used: To describe the motion of a quadrotor in three dimensions in its body-ﬁxed frame of reference (x to the front, y to the right, z downwards), the rigid body model as described in Wetz et al. (2021) can be used: m(¨x + q ˙z −r ˙y) = −mg[sin(θ)] + Fx (4) 85 m(¨y + p ˙z −r ˙x) = −mg[cos(θ)sin(ϕ)] + Fy (5) m(¨z + p ˙y −q ˙x) = mg[cos(θ)cos(ϕ)] + Fz −T , (6) m(¨x + q ˙z −r ˙y) = −mg[sin(θ)] + Fx 85 m(¨y + p ˙z −r ˙x) = −mg[cos(θ)sin(ϕ)] + Fy m(¨z + p ˙y −q ˙x) = mg[cos(θ)cos(ϕ)] + Fz −T , (5) (6) (5) (6) with m the mass of the UAS, p, q and r the gyroscopic terms, g the acceleration due to gravity and θ and ϕ the pitch and roll angle. The external forces F in all three components are caused by wind. Eq. 6 contains the total actuator thrust T which counteracts all the external forces due to wind and gravity. The concept of operation for the UAS ﬂeet is to hover at ﬁxed 90 positions, so that motions due to maneuvering can be neglected. Gyroscopic terms become very small and can be neglected in the following. In Wetz et al. (2021), only the forces in x-direction in Eq. 4 were considered, since the UAS yaws into the main wind direction and the wind speed magnitude can thus be approximated by this one dimensional simpliﬁcation In Wetz with m the mass of the UAS, p, q and r the gyroscopic terms, g the acceleration due to gravity and θ and ϕ the pitch and roll angle. The external forces F in all three components are caused by wind. Eq. 6 contains the total actuator thrust T which counteracts all the external forces due to wind and gravity. The concept of operation for the UAS ﬂeet is to hover at ﬁxed 90 with m the mass of the UAS, p, q and r the gyroscopic terms, g the acceleration due to gravity and θ and ϕ the pitch and roll angle. 2.2 Motor thrust calibration 65 In order to determine the forces that act on the UAS, it is important to know the actuator forces of the four rotors. There are no direct sensors for rotational speed, torque or even thrust in the system. However, the motor controller PPM (pulse-position modulation) signal as well as the battery voltage and current are measured by the autopilot. The PPM signal is directly related to the voltage which is applied to the motor and is in theory directly proportional to the revolution speed. The voltage at each rotor is 70 Ui = Ub si −smin smax −smin , (1) 3 3 Figure 2. Schematic of the motor test stand. The rotor is producing thrust in the free stream and the counter-acting force of the equal length lever-arm is measured with a scale on a table. https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure 2. Schematic of the motor test stand. The rotor is producing thrust in the free stream and the counter-acting force of the equal length lever-arm is measured with a scale on a table. with Ub the battery voltage and si the servo PPM command (smin for zero and smax for full throttle). It is important to include the battery voltage here, as it decreases throughout the ﬂight. Assuming a linear relationship for the electric servo motors, the revolution speed of each motor can be determined as with Ub the battery voltage and si the servo PPM command (smin for zero and smax for full throttle). It is important to include the battery voltage here, as it decreases throughout the ﬂight. Assuming a linear relationship for the electric servo motors, the revolution speed of each motor can be determined as Ωi = kV Ui . 75 (2) The motor thrust Ti can be calculated from the rotational speed Ωi, the rotor radius R, the rotor swept area A = πR2, air density ρ and the thrust coefﬁcient Ct (Leishman, 2016, Eq 2.31): Ti = CtρAΩ2 i R2 (3) In order to determine this relationship experimentally, as well as the thrust coefﬁcient of the rotor, which relates thrust to revolution speed, a hardware-in-the-loop test stand for a single motor/rotor setup is used. That means that Glauert’s approximation can not be used for hover ﬂight and wind speeds of the same order of magnitude. Nevertheless, lift effects are observed by reduced power consumption and less required thrust in dependence of wind speed. We do thus include a lift term FL in Eq. 6 to account for this effect, but need to determine the relationship experimentally. 105 to account for this effect, but need to determine the relationship experimentally. 105 With the assumption of hover state and the additional term for aerodynamic lift, we can then solve Eq. 4-6 for the external wind forces, so that With the assumption of hover state and the additional term for aerodynamic lift, we can then solve Eq. 4-6 for the external wind forces, so that With the assumption of hover state and the additional term for aerodynamic lift, we can then solve Eq. 4-6 for the external wind forces, so that Fx = mgsin(θ) + m¨x (8) Fy = mgcos(θ)sin(ϕ) + m¨y (9) Fz = −mgcos(θ)cos(ϕ) + m¨z + T + FL(Fx) . (10) 110 2.4 Determination of the three-dimensional wind vector Fx = mgsin(θ) + m¨x Fy = mgcos(θ)sin(ϕ) + m¨y Fz = −mgcos(θ)cos(ϕ) + m¨z + T + FL(Fx) . 110 (10) 2.3 Quadrotor dynamics and rotor aerodynamics The external forces F in all three components are caused by wind. Eq. 6 contains the total actuator thrust T which with m the mass of the UAS, p, q and r the gyroscopic terms, g the acceleration due to gravity and θ and ϕ the pitch and roll angle. The external forces F in all three components are caused by wind. Eq. 6 contains the total actuator thrust T which counteracts all the external forces due to wind and gravity. The concept of operation for the UAS ﬂeet is to hover at ﬁxed 90 positions, so that motions due to maneuvering can be neglected. Gyroscopic terms become very small and can be neglected in the following. In Wetz et al. (2021), only the forces in x-direction in Eq. 4 were considered, since the UAS yaws into the main wind direction and the wind speed magnitude can thus be approximated by this one dimensional simpliﬁcation. In Wetz counteracts all the external forces due to wind and gravity. The concept of operation for the UAS ﬂeet is to hover at ﬁxed 90 positions, so that motions due to maneuvering can be neglected. Gyroscopic terms become very small and can be neglected in the following. In Wetz et al. (2021), only the forces in x-direction in Eq. 4 were considered, since the UAS yaws into the main wind direction and the wind speed magnitude can thus be approximated by this one dimensional simpliﬁcation. In Wetz 4 4 https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. and Wildmann (2022), the two-dimensional wind vector was determined, including Eq. 5 in the algorithm and improving the dynamic response by the inclusion of the acceleration terms. In order to measure the full three-dimensional wind vector, Eq. 6 95 needs to be included. The single motor thrusts Ti can be obtained from calibration as described in Sect. 2.2 and can be summed to obtain the total acting force T = PTi. With increasing wind speed, the quadrotor lift will not only be generated by the rotor thrust, but aerodynamic effects start to occur. In theory, these effects are well described if forward ﬂight speed is much larger than the velocity which is induced by the rotor vi (V∞>> vi). Glauert’s high-speed approximation (Leishman, 2016) gives an equation for the total resulting thrust for a single-rotor helicopter: 100 and Wildmann (2022), the two-dimensional wind vector was determined, including Eq. 5 in the algorithm and improving the dynamic response by the inclusion of the acceleration terms. In order to measure the full three-dimensional wind vector, Eq. 6 95 needs to be included. The single motor thrusts Ti can be obtained from calibration as described in Sect. 2.2 and can be summed to obtain the total acting force T = PTi. With increasing wind speed, the quadrotor lift will not only be generated by the rotor thrust, but aerodynamic effects start to occur. In theory, these effects are well described if forward ﬂight speed is much larger than the velocity which is induced by the rotor vi (V∞>> vi). Glauert’s high-speed approximation (Leishman, 2016) gives an i f h l l i h f i l h li equation for the total resulting thrust for a single-rotor helicopter: 100 (7) (7) T = 2ρAviV∞ For the quadrotors used in this study, the induced velocity is on the order of 10 m s−1. That means that Glauert’s approximation can not be used for hover ﬂight and wind speeds of the same order of magnitude. Nevertheless, lift effects are observed by reduced power consumption and less required thrust in dependence of wind speed. We do thus include a lift term FL in Eq. 6 For the quadrotors used in this study, the induced velocity is on the order of 10 m s−1. 2.4 Determination of the three-dimensional wind vector To translate the forces that act on the quadrotor to wind speeds, a straight forward approach which was applied in Wetz et al. (2021) is the Rayleigh drag equation. It is however evident, that the complex aerodynamic system of a multicopter is not perfectly represented by the physical relationships for solid bodys. Wetz and Wildmann (2022) have therefore allowed a calibration curve which is a power law with a free exponent. With this approach, higher accuracies are achievable. We apply 115 this method here for all three dimensions, so that the wind components in the body frame of the quadrotor are To translate the forces that act on the quadrotor to wind speeds, a straight forward approach which was applied in Wetz et al. (2021) is the Rayleigh drag equation. It is however evident, that the complex aerodynamic system of a multicopter is not perfectly represented by the physical relationships for solid bodys. Wetz and Wildmann (2022) have therefore allowed a calibration curve which is a power law with a free exponent. With this approach, higher accuracies are achievable. We apply 115 this method here for all three dimensions, so that the wind components in the body frame of the quadrotor are calibration curve which is a power law with a free exponent. With this approach, higher accuracies are achievable. We apply 115 this method here for all three dimensions, so that the wind components in the body frame of the quadrotor are     ub vb wb     =     cxF bx x cyF by y czF bz z     , (11)     ub vb wb     =     cxF bx x cyF by y czF bz z     , (11) (11) where the parameters b and c need to be calibrated for the respective direction. For the vertical direction, it is conceivable that upward and downward motion of the quadrotor relative to the air is subject to signiﬁcantly different drag. For this reason, a distinction of cases is done for the z-component so that 120 120 wb =      cz↑F bz↑ z Fz ≥0 cz↓F bz↓ z Fz < 0 wb =      cz↑F bz↑ z Fz ≥0 cz↓F bz↓ z Fz < 0 . 2.4 Determination of the three-dimensional wind vector (12) wb =      cz↑F bz↑ z Fz ≥0 cz↓F bz↓ z Fz < 0 . (12) (12) 5 5 https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. Table 1. Sensor data overview and intial raw sampling rates. Table 1. Sensor data overview and intial raw sampling rates. p g type of data variables sampling rate accelerometer data ax,ay,az 250 Hz vehicle attitude ϕ,θ,ψ 20 Hz vehicle local velocities vx,vy,vz 10 Hz actuator outputs si 10 Hz battery data Ub 2 Hz temperature and humidity T,q 1 Hz barometric pressure P 75 Hz The ﬁnal step to obtain the meteorological wind vector U = (u,v,w) is a rotation of the wind speeds from the body frame into the geodetic coordinates with the Euler angles of the quadrotor and a subtraction of the linear velocities (vx, vy, vz) which are measured by the autopilot on the basis of GNSS (global navigation satellite system) information: 125     u v w     = R(ϕ,θ,ψ)     ub vb wb    −     vx vy vz         u v w     = R(ϕ,θ,ψ)     ub vb wb    −     vx vy vz     , (13)     u v w     = R(ϕ,θ,ψ)     ub vb wb    −     vx vy vz     , (13) 2 5 Data preparation and ﬁltering (13) 2.6 Calculation of turbulence parameters From the three-dimensional wind data, we can calculate momentum ﬂuxes, friction velocity and turbulence kinetic energy (TKE). Momentum ﬂuxes are calculated as the covariances between the wind components, i.e. u′w′,v′w′ and u′v′. The friction velocity u∗is calculated from the momentum ﬂuxes according to Stull (1988): 140 140 u∗= u′w′2 + v′w′21/4 (14) u∗= u′w′2 + v′w′21/4 (14) TKE is deﬁned as half of the sum of the variances of the three wind components: TKE = 1 2 u′2 + v′2 + w′2 . (15) TKE = 1 2 u′2 + v′2 + w′2 (15) 2.5 Data preparation and ﬁltering The raw sensor data of the UAS are stored on the SD card with different sampling rates, depending on the data type, but with a synchronized timestamp. Table 1 presents the raw data types and variables that are used for the wind calculation and their initial sampling rate. Reasons for the different sampling rates are the capabilities of the sensors and the requirements for the 130 ﬂight controller. Except for the battery data, all data are sampled with a rate of 10 Hz or higher. The raw data can be subject to signiﬁcant noise in the high frequency range due to sensor noise or vibrations. Preconditioning steps are taken before further processing of the data to obtain clean and synchronized data. Accelerometer data is ﬁltered with a Butterworth ﬁlter to prevent anti-aliasing effects from frequencies above 10 Hz. All data is then interpolated and synchronized to a common time step with initial sampling rate. Reasons for the different sampling rates are the capabilities of the sensors and the requirements for the 130 ﬂight controller. Except for the battery data, all data are sampled with a rate of 10 Hz or higher. The raw data can be subject to signiﬁcant noise in the high frequency range due to sensor noise or vibrations. Preconditioning steps are taken before further processing of the data to obtain clean and synchronized data. Accelerometer data is ﬁltered with a Butterworth ﬁlter to prevent anti-aliasing effects from frequencies above 10 Hz. All data is then interpolated and synchronized to a common time step with ∆t = 0.1 s. After this step, noise can still be recognized in accelerometer and actuator data, so that for the wind calculation, 135 frequencies above 2 Hz are cut off with an FFT ﬁlter in the frequency domain. 6 https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. 3 Field experiment A calibration and validation campaign for the SWUF-3D ﬂeet was carried out in the framework of the FESSTVaL (Field 145 Experiment on submesoscale spatio-temporal variability in Lindenberg) campaign in June/July 2021 (Hohenegger et al., in preparation) at the GM Falkenberg of the DWD. Figure 3 gives a map of the location with an indication of the measurement positions, including the location of the 99 m meteorological mast at the site. Two sonic anemometers (Metek UAS-1) are installed at 50 m and 90 m height which serve as the reference for calibration and validation. Their measurements are distorted by the tower for wind directions between 345◦and 50◦via north and are thus disregarded for such conditions. 150 Calibration ﬂights were done with ﬁve UAS at 50 m and 90 m respectively, in close proximity (∆x = 20 m) to the mast (see black stars in Fig. 3 and Wetz and Wildmann (2022)). During a morning transition of a convective boundary layer on 28 June 2021, continuous measurements were done with two sets of ﬁve UAS stacked in ﬁve heights (10 m, 50 m, 90 m, 150 m, 200 m) at the location of the blue star in Fig. 3. The two sets were measuring alternately in close separation of 5 m and with a short overlap of one minute to guarantee continuous observations. Spatially distributed measurements (red stars) were performed to 155 calculate coherence of the ﬂow, but are not analysed in this study. 4.1 Motor test stand calibration Distributed under a Creative Commons BY-SA License. Figure 3. Map of the GM Falkenberg with the location of measurement points for the different ﬂight patterns (stars) and the meteorological mast (black triangle). Background map ©OpenTopoMap contributors 2022. Distributed under a Creative Commons BY-SA License. Figure 4. Calibration results of the motor test stand. a) The calibration curve of revolution speed versus applied voltage and b) the thrust coefﬁcient curve. The ﬁtted calibration curves are shown as red lines. Figure 4. Calibration results of the motor test stand. a) The calibration curve of revolution speed versus applied voltage and b) the thrust coefﬁcient curve. The ﬁtted calibration curves are shown as red lines. calibration curve as: calibration curve as: Ti = 4.6 · 10−5Ω2 i −1.68 · 10−3Ωi 165 (16) 4.1 Motor test stand calibration With Eq. 1, the voltage at the motor can be estimated from the actuator outputs. Results from the hardware-in-the-loop exper- iment (Fig. 4a) reveal a nearly linear relationship between voltage and revolution speed. The coefﬁcient that relates voltage to 0 With Eq. 1, the voltage at the motor can be estimated from the actuator outputs. Results from the hardware-in-the-loop exper- iment (Fig. 4a) reveal a nearly linear relationship between voltage and revolution speed. The coefﬁcient that relates voltage to 160 revolution speed is kV ≈30.7 s−1V−1. With Eq. 1, the voltage at the motor can be estimated from the actuator outputs. Results from the hardware-in-the-loop exper- iment (Fig. 4a) reveal a nearly linear relationship between voltage and revolution speed. The coefﬁcient that relates voltage to 160 revolution speed is kV ≈30.7 s−1V−1. From Fig. 4b) we ﬁnd that the thrust curve Ti as a function of revolution speed Ωi is best approximated with a polynom of second order. Such a behaviour is quite common and has been described before (Bangura et al., 2016)). We determine th iment (Fig. 4a) reveal a nearly linear relationship between voltage and revolution speed. The coefﬁcient that relates voltage to 160 revolution speed is kV ≈30.7 s−1V−1. From Fig. 4b) we ﬁnd that the thrust curve Ti as a function of revolution speed Ωi is best approximated with a polynom of second order. Such a behaviour is quite common and has been described before (Bangura et al., 2016)). We determine the From Fig. 4b) we ﬁnd that the thrust curve Ti as a function of revolution speed Ωi is best approximated with a polynom of second order. Such a behaviour is quite common and has been described before (Bangura et al., 2016)). We determine the 7 7 https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure 3. Map of the GM Falkenberg with the location of measurement points for the different ﬂight patterns (stars) and the meteorological mast (black triangle). Background map ©OpenTopoMap contributors 2022. Distributed under a Creative Commons BY-SA License. https://doi.org/10.5194/egusphere 2022 110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure 3. Map of the GM Falkenberg with the location of measurement points for the different ﬂight patterns (stars) and the meteorological mast (black triangle). Background map ©OpenTopoMap contributors 2022. https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. components are presented in Wetz and Wildmann (2022). In this study, we focus on the calibration of the vertical velocity component. This calibration is done in two steps. First, the lift force which reduces the necessary thrust in hover ﬂight is 170 determined as a function of the wind speed in x-direction. Second, thrust minus lift is calibrated against the vertical wind velocity as measured by the sonic anemometers at the 99-m mast. components are presented in Wetz and Wildmann (2022). In this study, we focus on the calibration of the vertical velocity component. This calibration is done in two steps. First, the lift force which reduces the necessary thrust in hover ﬂight is 170 determined as a function of the wind speed in x-direction. Second, thrust minus lift is calibrated against the vertical wind velocity as measured by the sonic anemometers at the 99-m mast. 4.2 In-ﬂight wind vector calibration For calibration of the dynamic equations as described in Sect. 2.3, we use UAS#12, because it was operating in most of the calibration ﬂights (i.e. 14 ﬂights, with a wind speed range of 1–8 m/s). The results of the calibration of the horizontal wind 8 4.2.1 Lift force To estimate the lift force FL, we analyse the measured thrust force during all calibration ﬂig To estimate the lift force FL, we analyse the measured thrust force during all calibration ﬂights in dependency of the horizontal wind. Horizontal wind is not known a priori, so we use the drag force in x-direction in the body frame Fx as a proxy which 175 depends on the wind speed as described by Eqs. 8 and 11. It is not possible in a ﬁeld experiment to perfectly decouple the lift effects from the wind in x-direction from the drag and lift in z-direction. For calibration purposes, we assume that the x and z- component of the wind are uncorrelated and that vertical wind is normally distributed around zero. To reduce the scatter, a 10 s moving average is applied to the data. Figure 5 shows the point cloud of lift versus drag force in x-direction for all calibration ﬂights of UAS#12. The red line gives a polynomial ﬁt. It can be seen that signiﬁcant lift is generated up to Fx ≈1 N lift, before 180 it abruptly decreases, as it is typical for stall conditions. This force in x-direction corresponds to a horizontal wind speed of approximately 8 m s−1. Beyond that wind speed, only few data are available which yields a high uncertainty of the calibration at higher wind speed conditions. The determined polynomial for the lift correction is: ﬂights of UAS#12. The red line gives a polynomial ﬁt. It can be seen that signiﬁcant lift is generated up to Fx ≈1 N lift, before 180 it abruptly decreases, as it is typical for stall conditions. This force in x-direction corresponds to a horizontal wind speed of approximately 8 m s−1. Beyond that wind speed, only few data are available which yields a high uncertainty of the calibration at higher wind speed conditions. The determined polynomial for the lift correction is: 0.00 0.25 0.50 0.75 1.00 1.25 1.50 Fx / N 0.5 0.0 0.5 1.0 1.5 FL / N 0.00 0.25 0.50 0.75 1.00 1.25 1.50 Fx / N 0.5 0.0 0.5 1.0 1.5 FL / N Figure 5. Calibration curve of thrust generated by lift FL versus drag force in x-direction Fx in the body frame. Greyscale represents the probability of occurence. Figure 5. https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. 4.2.2 Thrust offset We observe that the absolute value of thrust T0 that is needed to maintain altitude in the hovering state varies between single ﬂights and especially between individual UAS. The reasons can be differences in UAS mass, motor performance or aerody- namic effects. At the present state of development and hardware robustness, we ﬁnd that T0 needs to be determined in each 190 single ﬂight as the average T over the whole ﬂight for best performance. This is justiﬁed in ﬂat terrain where zero average vertical velocity can be assumed in most cases. For cases with non-zero vertical velocity, determining T0 from the average over a single ﬂight can yield errors in the absolute value of w, but will have little effect on the variance, which is most important for the calculation of TKE and ﬂuxes. 4.2.1 Lift force Calibration curve of thrust generated by lift FL versus drag force in x-direction Fx in the body frame. Greyscale represents the probability of occurence. Figure 5. Calibration curve of thrust generated by lift FL versus drag force in x-direction Fx in the body frame. Greyscale represents the probability of occurence. FL = T0 + 0.9Fx −0.27F 5 x , (17) where T0 is a thrust offset value that can depend on changes in UAS mass, changes in motor thrust coefﬁcients or unmodelled 185 aerodynamic effects. 9 4.2.3 Vertical velocity 195 In the second step of the calibration, vertical wind is calibrated against thrust (including aerodynamic lift, see above) with data from the FESSTVaL ﬁeld campaign. Figure 6 shows the scatterplot of all calibration ﬂights of UAS#12. Since measurements of UAS and sonic anemometer are comparatively far apart (≈20 m) and absolute values of vertical wind are rather small, it is not surprising that signiﬁcant scatter is found in the comparison. It is nevertheless possible to ﬁt an optimal curve according to the theory of Eq. 18. The ﬁtted equation is: 200 wb =      3.3\|Fz\|0.85 Fz ≤0 −1.6\|Fz\|0.6 Fz > 0 . (18) wb =      3.3\|Fz\|0.85 Fz ≤0 −1.6\|Fz\|0.6 Fz > 0 . (18) 1.5 1.0 0.5 0.0 0.5 1.0 1.5 Fz / N 3 2 1 0 1 2 3 wb / ms 1 Figure 6. Scatter plot of vertical wind of the sonic anemometer versus the acting force on the UAS in the body frame. Greyscale represents the probability of occurence in the dataset. The red line shows the estimated calibration curve. Figure 6. Scatter plot of vertical wind of the sonic anemometer versus the acting force on the UAS in the body frame. Greyscale represents the probability of occurence in the dataset. The red line shows the estimated calibration curve. 10 Figure 7. Example of time series of u, v and w for a ﬂight on 02 July 2021 by UAS#13. UAS data (red) is compared to the sonic anemometer at the same height (black). https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure 7. Example of time series of u, v and w for a ﬂight on 02 July 2021 by UAS#13. UAS data (red) is compared to the sonic anemometer at the same height (black). https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure 7. Example of time series of u, v and w for a ﬂight on 02 July 2021 by UAS#13. UAS data (red) is compared to the sonic anemometer at the same height (black). 4.3 Wind vector validation The parameters that are determined for UAS#12 are now applied to the whole ﬂeet. We now want to compare the wind components u, v and w in the geodetic coordinate system according to Eq. 13. Nevertheless, it is more meaningful to rotate 205 the vector horizontally into the main wind direction for both, sonic anemometer and UAS in order to evaluate the different calibration coefﬁcients. The time series in Fig. 7 demonstrates the quality of the wind retrieval in all three dimensions and at a high resolution for a ﬂight in a turbulent ABL with horizontal wind speeds ranging from u = 2.5 −7.5 m s−1. Figure 8 gives scatter plots of the comparison of ﬂight-averaged wind components, their variances and the three covariances u′w′, v′w′ and u′v′. Since the wind vector is rotated into the main wind direction during the ﬂight time, the lateral wind component v is 210 zero by deﬁnition (Fig. 8b). Flight-averaged vertical velocities are smaller than the uncertainty of the measurement so that a linear regression does not yield a good correlation (P-value for test of null-hypothesis P > 0.1). The root-mean squared (RMS) deviation is ϵw = 0.25 m s−1. Better parameters to evaluate the quality of the wind retrieval algorithm are the variances of the wind components in lateral and vertical direction. For those, the linear regressions show a very good correlation between UAS and sonic anemometer (R2 > 0.9, Fig. 8e,f). The covariance measurements are a good indicator if an independent measurement 215 of horizontal and vertical wind can be guaranteed with the proposed method. The high correlation proves the validity of the method and the RMS deviation of approximately ε = 0.1 m2s−2 for all directions can be considered the prevailing uncertainty 11 https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. of the UAS measurements (Fig. 10g,h). The lower R2-value for v′w′ is likely due to the imperfect rotation into wind direction for both, UAS and sonic anemometer which introduces larger relative errors for the overall small values. of the UAS measurements (Fig. 10g,h). The lower R2-value for v′w′ is likely due to the imperfect rotation into wind direction for both, UAS and sonic anemometer which introduces larger relative errors for the overall small values. 4.3 Wind vector validation 10 0 10 sonic u / ms 1 10 5 0 5 10 UAS u / ms 1 R2 = 1.0 P = 0.0 u = 0.16 a 2 0 2 sonic v / ms 1 2 1 0 1 2 UAS v / ms 1 R2 = -0.11 P = 0.28 v = 0.0 b 2 0 2 sonic w / ms 1 2 1 0 1 2 UAS w / ms 1 R2 = 0.46 P = 0.0 w = 0.13 c 0 1 2 3 sonic 2 u / m2s 2 0 1 2 3 UAS 2 u / m2s 2 R2 = 0.93 P = 0.0 , u = 0.16 d 0 1 2 3 sonic 2 v / m2s 2 0 1 2 3 UAS 2 v / m2s 2 R2 = 0.96 P = 0.0 , v = 0.14 e 0 1 2 3 sonic 2 w / m2s 2 0 1 2 3 UAS 2 w / m2s 2 R2 = 0.98 P = 0.0 , w = 0.06 f 1 0 1 sonic u′w′ / m2s 2 1.0 0.5 0.0 0.5 1.0 UAS u′w′ / m2s 2 R2 = 0.77 P = 0.0 uw = 0.12 g 1 0 1 sonic v′w′ / m2s 2 1.0 0.5 0.0 0.5 1.0 UAS v′w′ / m2s 2 R2 = 0.57 P = 0.0 vw = 0.08 h 1 0 1 sonic u′v′ / m2s 2 1.0 0.5 0.0 0.5 1.0 UAS u′v′ / m2s 2 R2 = 0.93 P = 0.0 uv = 0.06 i Figure 8. Scatter plots of ﬂight-averaged UAS measurements versus sonic anemometer for u, v and w (a-c), as well as the correspon variances (d-f) and covariances (g-i). https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. 4.4 Validation of resolved turbulence scales 220 For conditions with low turbulence, the noise contributions are limiting the resolution of variance and covariance measurements with the UAS. From the noise level, we estimate this limit in resolution for the variances and covariances to ≈0.1 m2s−2 which is almost 230 equal to the RMSE that was determined in Sect. 4.3. From the noise level, we estimate this limit in resolution for the variances and covariances to ≈0.1 m2s−2 which is almost 230 equal to the RMSE that was determined in Sect. 4.3. From the noise level, we estimate this limit in resolution for the variances and covariances to ≈0.1 m2s−2 which is almost 230 equal to the RMSE that was determined in Sect. 4.3. 4.5 Momentum ﬂux during a morning transition Figure 11 shows the results of the achieved by launching two separate sets of ﬁve UAS in turns, with a short overlap of 60 s. The UAS were located close to 240 the meteorological mast as indicated by the blue star in Fig. 3. In total, 23 ﬂights with ﬁve UAS each, were performed in this pattern. Additionally, a sixth UAS was operating in close proximity (≈5 m apart), proﬁling the same height range with continuous ascents and descents. The ascents of these ﬂights allow to obtain proﬁles of wind speed, wind direction, temperature and humidity with a high vertical resolution, but are not used for the turbulence retrieval. Figure 11 shows the results of the achieved by launching two separate sets of ﬁve UAS in turns, with a short overlap of 60 s. The UAS were located close to 240 the meteorological mast as indicated by the blue star in Fig. 3. In total, 23 ﬂights with ﬁve UAS each, were performed in this pattern. Additionally, a sixth UAS was operating in close proximity (≈5 m apart), proﬁling the same height range with continuous ascents and descents. The ascents of these ﬂights allow to obtain proﬁles of wind speed, wind direction, temperature and humidity with a high vertical resolution, but are not used for the turbulence retrieval. Figure 11 shows the results of the measurements during the morning transition. On the left, the time series of the UAS at 50 m are shown in comparison to 245 the sonic anemometer at the mast at the same level. The data are post-processed with a 10-minute moving average for better visualization. Between 0730 UTC and 0745 UTC, data are missing because one UAS did not take off as planned. The dots in the plots show the 30-minute average with error bars representing the uncertainties that were estimated in Sect. 4.3. We ﬁnd a good agreement between UAS and sonic anemometer for all variables. Around 0530 UTC, the mixed CBL is growing measurements during the morning transition. On the left, the time series of the UAS at 50 m are shown in comparison to 245 the sonic anemometer at the mast at the same level. The data are post-processed with a 10-minute moving average for better visualization. Between 0730 UTC and 0745 UTC, data are missing because one UAS did not take off as planned. 4.4 Validation of resolved turbulence scales 220 A closer evaluation of the resolved turbulence scales by the UAS can be achieved with the analyses of power spectra of the three wind components in comparison to the sonic anemometer. We choose two representative ﬂights of UAS#12, one in low wind and low turbulence conditions in the morning of the 26 June (blue), the other in a turbulent convective boundary layer on 2 July (red). The spectra of the three wind components in Fig. 9 show that an inertial subrange of locally isotropic turbulence with a slope of -5/3 (dashed grey line) can be observed in both cases. For the low turbulence case, the spectra as measured by 225 the UAS deviates earlier from the curve as measured by the sonic anemometer than in the high turbulence case. It can be read from the plots that frequencies above 1 Hz and spectral power below 10−2 m2 s−2 Hz−1 can barely be resolved by the UAS, but are dominated by noise. This effect is also observed for the cospectra of the wind components (Fig. 10). For conditions with low turbulence, the noise contributions are limiting the resolution of variance and covariance measurements with the UAS. with a slope of -5/3 (dashed grey line) can be observed in both cases. For the low turbulence case, the spectra as measured by 225 the UAS deviates earlier from the curve as measured by the sonic anemometer than in the high turbulence case. It can be read from the plots that frequencies above 1 Hz and spectral power below 10−2 m2 s−2 Hz−1 can barely be resolved by the UAS, but are dominated by noise. This effect is also observed for the cospectra of the wind components (Fig. 10). For conditions with low turbulence, the noise contributions are limiting the resolution of variance and covariance measurements with the UAS. with a slope of -5/3 (dashed grey line) can be observed in both cases. For the low turbulence case, the spectra as measured by 225 the UAS deviates earlier from the curve as measured by the sonic anemometer than in the high turbulence case. It can be read from the plots that frequencies above 1 Hz and spectral power below 10−2 m2 s−2 Hz−1 can barely be resolved by the UAS, but are dominated by noise. This effect is also observed for the cospectra of the wind components (Fig. 10). 4.3 Wind vector validation 2 0 2 sonic w / ms 1 2 1 0 1 2 UAS w / ms 1 R2 = 0.46 P = 0.0 w = 0.13 c 10 0 10 sonic u / ms 1 10 5 0 5 10 UAS u / ms 1 R2 = 1.0 P = 0.0 u = 0.16 a 2 0 2 sonic v / ms 1 2 1 0 1 2 UAS v / ms 1 R2 = -0.11 P = 0.28 v = 0.0 b 0 1 2 3 sonic 2 v / m2s 2 0 1 2 3 UAS 2 v / m2s 2 R2 = 0.96 P = 0.0 , v = 0.14 e 0 1 2 3 sonic 2 w / m2s 2 0 1 2 3 UAS 2 w / m2s 2 R2 = 0.98 P = 0.0 , w = 0.06 f f v 1 0 1 sonic v′w′ / m2s 2 1.0 0.5 0.0 0.5 1.0 UAS v′w′ / m2s 2 R2 = 0.57 P = 0.0 vw = 0.08 h 1 0 1 sonic u′w′ / m2s 2 1.0 0.5 0.0 0.5 1.0 UAS u′w′ / m2s 2 R2 = 0.77 P = 0.0 uw = 0.12 g h g Figure 8. Scatter plots of ﬂight-averaged UAS measurements versus sonic anemometer for u, v and w (a-c), as well as the corresponding variances (d-f) and covariances (g-i). 12 4.5 Momentum ﬂux during a morning transition A good case to study the validity and capability of the turbulence and ﬂux measurements from the UAS is a transition phase of a convective boundary layer (CBL), which features some signiﬁcant variability and increase of turbulence at constant heights. A good case to study the validity and capability of the turbulence and ﬂux measurements from the UAS is a transition phase of a convective boundary layer (CBL), which features some signiﬁcant variability and increase of turbulence at constant heights. In the morning of 28 June 2021, conditions were present which allowed to observe a morning transition of a CBL which was 235 mainly driven by surface heating. In the cloudless night, a stable nighttime inversion had developed and still no clouds were present during the period of observation from 0400 UTC to 0800 UTC. A steady increase of shortwave downward radiation from ≈100 W m−2 to ≈700 W m−2 was observed. Continuous measurements during this period with ﬁve stacked UAS at the heights 10 m, 50 m, 90 m, 150 m and 200 m were In the morning of 28 June 2021, conditions were present which allowed to observe a morning transition of a CBL which was 235 mainly driven by surface heating. In the cloudless night, a stable nighttime inversion had developed and still no clouds were present during the period of observation from 0400 UTC to 0800 UTC. A steady increase of shortwave downward radiation from ≈100 W m−2 to ≈700 W m−2 was observed. Continuous measurements during this period with ﬁve stacked UAS at the heights 10 m, 50 m, 90 m, 150 m and 200 m were Continuous measurements during this period with ﬁve stacked UAS at the heights 10 m, 50 m, 90 m, 150 m and 200 m were achieved by launching two separate sets of ﬁve UAS in turns, with a short overlap of 60 s. The UAS were located close to 240 the meteorological mast as indicated by the blue star in Fig. 3. In total, 23 ﬂights with ﬁve UAS each, were performed in this pattern. Additionally, a sixth UAS was operating in close proximity (≈5 m apart), proﬁling the same height range with continuous ascents and descents. The ascents of these ﬂights allow to obtain proﬁles of wind speed, wind direction, temperature and humidity with a high vertical resolution, but are not used for the turbulence retrieval. https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. The friction velocity u∗is quite variable, also increasing after 0630 UTC. In the right panel of Fig. 11, Hovmoeller diagrams show the transition period in dependency of time and height. From the proﬁles of potential temperature, an estimate of the boundary-layer height as the height of the mixed layer below the lowest 255 inversion is derived and shown as a black line. A low-level jet (LLJ) was present at 100 m above ground level (a.g.l.) which disappears as the mixed layer grows and is the reason for the observed higher wind speeds in the early morning at 50 m and above. It can be clearly seen how low turbulence and high wind speeds above the inversion are separated from low wind speeds and higher turbulence in the mixed layer. Despite the large number of individual UAS that were used to take these measurements (i.e. 12 UAS), no signiﬁcant biases or discontinuities can be found in the data, which is a good validation of the 260 accuracy of the calibration. 5 Conclusions In this work, we present a method to obtain the three-dimensional wind vector with small multicopter UAS. To obtain the vertical wind velocity, motor thrust is included in the equations of motion and the resulting forces that act on the UAS are calibrated against sonic anemometers to obtain the wind velocity with an RMS deviation below 0.2 m s−1 in all three dimen- 265 sions. We showed that the variance of the wind components can be obtained particularly well with a remaining RMS deviation below 0.2 m2 s−2 in all dimensions. The range and limits of the resolvable scales of turbulence are presented with spectra and cospectra of the wind components. It is evident that there is a noise limit in the temporal resolution of approximately 1 Hz and a limit for low turbulence conditions with spectral power density below 10−2m2 s−2 Hz−1. The fact that we could exercise the calibrated against sonic anemometers to obtain the wind velocity with an RMS deviation below 0.2 m s−1 in all three dimen- 265 sions. We showed that the variance of the wind components can be obtained particularly well with a remaining RMS deviation below 0.2 m2 s−2 in all dimensions. The range and limits of the resolvable scales of turbulence are presented with spectra and cospectra of the wind components. It is evident that there is a noise limit in the temporal resolution of approximately 1 Hz and a limit for low turbulence conditions with spectral power density below 10−2m2 s−2 Hz−1. The fact that we could exercise the calibration with a single UAS and apply it to a ﬂeet of more than 20 UAS without a big loss of accuracy shows the robustness 270 of the method and the applicability in practice. From these ﬁndings, it can be concluded that UAS of the presented size and weight are capable of resolving eddies of only few meters. Bigger and heavier UAS will necessarily disturb the ﬂow more, so that a small and lightweight design is essential for a high resolution of wind measurements. In the presented dataset, the range of horizontal wind velocities is limited to ≈8 m s−1. We see that above such wind speeds, In the presented dataset, the range of horizontal wind velocities is limited to ≈8 m s−1. 5 Conclusions We see that above such wind speeds, aerodynamic effects change and more studies will be necessary in future to extend the calibration to higher wind speeds. We 275 remove a thrust bias for every individual ﬂight in this study, because the assumption of zero average vertical wind speed can be made in the ﬂat terrain in which the experiment was carried out, and this approach will lead to best estimates of vertical velocity variance. For conditions with a signiﬁcant average vertical velocity component, different strategies will need to be de- veloped in future, which may incorporate more robust hardware conﬁgurations, an integrated weight estimator in the Kalman aerodynamic effects change and more studies will be necessary in future to extend the calibration to higher wind speeds. We 275 remove a thrust bias for every individual ﬂight in this study, because the assumption of zero average vertical wind speed can be made in the ﬂat terrain in which the experiment was carried out, and this approach will lead to best estimates of vertical velocity variance. For conditions with a signiﬁcant average vertical velocity component, different strategies will need to be de- veloped in future, which may incorporate more robust hardware conﬁgurations, an integrated weight estimator in the Kalman aerodynamic effects change and more studies will be necessary in future to extend the calibration to higher wind speeds. We 275 remove a thrust bias for every individual ﬂight in this study, because the assumption of zero average vertical wind speed can be made in the ﬂat terrain in which the experiment was carried out, and this approach will lead to best estimates of vertical velocity variance. For conditions with a signiﬁcant average vertical velocity component, different strategies will need to be de- veloped in future, which may incorporate more robust hardware conﬁgurations, an integrated weight estimator in the Kalman Filter or individual calibration of motor thrusts for each UAS. For both constraints, the obtained dataset from the FESSTVaL 280 campaign can not be used to improve the calibration. In general, calibration with ﬁeld data is prone to large uncertainties due to the complex ABL ﬂow. The calibration dataset is thus subject to signiﬁcant scatter and the polynomials that are used for the relationship between forces and wind speed are not strictly based on physical principles, as simplifying assumptions are made. 4.5 Momentum ﬂux during a morning transition The dots in the plots show the 30-minute average with error bars representing the uncertainties that were estimated in Sect. 4.3. We ﬁnd a good agreement between UAS and sonic anemometer for all variables. Around 0530 UTC, the mixed CBL is growing through the measurement height, wind becomes very small and thus the estimation of wind direction deviates by almost twenty 250 degrees between UAS and sonic anemometer. Also, temperature measurements differ most during this period. TKE only picks up signiﬁcantly after 0630 UTC with a large peak just before 0800 UTC which can be seen in both UAS and sonic anemometer. 13 https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. for this purpose. We also prepare temperature sensors with a faster response time to be able to measure sensible heat ﬂux in a broad range of atmospheric conditions in future. for this purpose. We also prepare temperature sensors with a faster response time to be able to measure sensible heat ﬂux in a broad range of atmospheric conditions in future. We showed the potential of multicopter UAS measurements for fundamental ABL research with a case study during a morning transtition of a CBL. In this case study, the calibration was applied to 12 individual UAS and nevertheless, a seamless time series over 4.5 hours could be realized in six measurement heights. The evolution of wind speed and temperature, as well as 290 TKE and shear stress is well captured in comparison to the reference from sonic anemometers throughout the transition period. This example demonstrates the advantage of operating a ﬂeet of multiple UAS to obtain spatio-temporal in-situ data at ﬂexible measurement points throughout the ABL. The possibilities to deploy such a system for ABL studies in complex ﬂow such as wind farms or mountainous terrain are numerous and an observational gap can potentially be closed with this approach. g p We showed the potential of multicopter UAS measurements for fundamental ABL research with a case study during a morning transtition of a CBL. In this case study, the calibration was applied to 12 individual UAS and nevertheless, a seamless time series over 4.5 hours could be realized in six measurement heights. The evolution of wind speed and temperature, as well as 290 TKE and shear stress is well captured in comparison to the reference from sonic anemometers throughout the transition period. This example demonstrates the advantage of operating a ﬂeet of multiple UAS to obtain spatio-temporal in-situ data at ﬂexible measurement points throughout the ABL. The possibilities to deploy such a system for ABL studies in complex ﬂow such as wind farms or mountainous terrain are numerous and an observational gap can potentially be closed with this approach. 290 Data availability. Data is available from the authors upon request and will shortly be made available through the FESSTVaL data archive. 295 Data availability. Data is available from the authors upon request and will shortly be made available through the FESSTVaL data archive. 295 Author contributions. 5 Conclusions In future, we plan to conduct wind tunnel experiments to study the aerodynamic effects in a broader range of horizontal d i l i d l i i d i d il H d diﬁ i h ll h ﬂi h i d l d Filter or individual calibration of motor thrusts for each UAS. For both constraints, the obtained dataset from the FESSTVaL 280 campaign can not be used to improve the calibration. In general, calibration with ﬁeld data is prone to large uncertainties due to the complex ABL ﬂow. The calibration dataset is thus subject to signiﬁcant scatter and the polynomials that are used for the relationship between forces and wind speed are not strictly based on physical principles, as simplifying assumptions are made. In future, we plan to conduct wind tunnel experiments to study the aerodynamic effects in a broader range of horizontal and vertical wind velocities and in more detail. Hardware modiﬁcations that allow hover ﬂights indoor are currently prepared 285 14 https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. References Aubinet, M., Vesala, T., and Papale, D.: Eddy Covariance - A Practical Guide to Measurement and Data Analysis, Springer, Dordrecht, 2012. Baldocchi, D., Falge, E., Gu, L., Olson, R., Hollinger, D., Running, S., Anthoni, P., Bernhofer, C., Davis, K., Evans, R., Fuentes, J., Goldstein, A., Katul, G., Law, B., Lee, X., Malhi, Y., Meyers, T., Munger, W., Oechel, W., U, K. T. P., Pilegaard, K., Schmid, H. 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TW developed the calibration for horizontal wind speed. Author contributions. Both authors jointly developed and carried out the ﬁeld experiment. NW developed the ideas to retrieve vertical velocity and carried out the motor calibration. TW developed the calibration for horizontal wind speed. Competing interests. The authors declare that there are no competing interests. Competing interests. The authors declare that there are no competing interests. Acknowledgements. We acknowledge the support of DWD and the GM Falkenberg in the conduction of the experiment. We thank May Bohmann and Josef Zink for their assistance in the execution of the experiments. Many thanks to Frank Holzäpfel for an internal review of 300 the manuscript. Acknowledgements. We acknowledge the support of DWD and the GM Falkenberg in the conduction of the experiment. We thank May Bohmann and Josef Zink for their assistance in the execution of the experiments. 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J.: Boulder Atmospheric Observatory: 1977–2016: The End of an Era and Lessons Learned, Bulletin of the 350 American Meteorological Society, 99, 1345 – 1358, https://doi.org/10.1175/BAMS-D-17-0054.1, 2018. Wolfe, D. E. and Lataitis, R. J.: Boulder Atmospheric Observatory: 1977–2016: The End of an Era and Lessons Learned, Bulletin of the 350 American Meteorological Society, 99, 1345 – 1358, https://doi.org/10.1175/BAMS-D-17-0054.1, 2018. 17 17 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 2Hz 1 u case#1, UAS case#2, UAS case#1, sonic case#2, sonic 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 2Hz 1 v 10 3 10 1 101 103 S / m2s 2Hz 1 w https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. CC BY 4.0 License. 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 1 u′w′ case#1, UAS case#2, UAS case#1, sonic case#2, sonic 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 1 v′w′ 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 1 u′v′ pectra between sonic anemometer and UAS. 19 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 1 u′w′ case#1, UAS case#2, UAS case#1, sonic case#2, sonic 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 1 v′w′ 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 1 u′v′ . Comparison of power cospectra between sonic anemometer and UAS. 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 1 u′w′ case#1, UAS case#2, UAS case#1, sonic case#2, sonic 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 1 v′w′ 101 103 1 u′v′ 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 1 u′w′ case#1, UAS case#2, UAS case#1, sonic case#2, sonic 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 S / m2s 1 case#1, UAS case#2, UAS case#1, sonic case#2, sonic 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 1 v′w′ 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 1 u′v′ Figure 10. Comparison of power cospectra between sonic anemometer and UAS. 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 2Hz 1 u case#1, UAS case#2, UAS case#1, sonic case#2, sonic 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 2Hz 1 v 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 2Hz 1 w tra of all three wind components between sonic anemometer and 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 2Hz 1 u case#1, UAS case#2, UAS case#1, sonic case#2, sonic 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 2Hz 1 v 103 w 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 2Hz 1 u case#1, UAS case#2, UAS case#1, sonic case#2, sonic 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 2Hz 1 v 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 2Hz 1 w Comparison of power spectra of all three wind components between sonic anemometer and UAS. 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 2Hz 1 u case#1, UAS case#2, UAS case#1, sonic case#2, sonic 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 2Hz 1 u case#1, UAS case#2, UAS case#1, sonic case#2, sonic frequency / Hz 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 2Hz 1 v frequency / Hz 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 2Hz 1 w Figure 9. Comparison of power spectra of all three wind components between sonic anemometer and UAS. 18 https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. frequency / Hz 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 1 v′w′ frequency / Hz 10 3 10 2 10 1 100 101 frequency / Hz 10 5 10 3 10 1 101 103 S / m2s 1 u′v′ Figure 10. Comparison of power cospectra between sonic anemometer and UAS. 19 0 2 4 6 U / ms 1 100 150 200 / deg 20 25 T / °C UAS, 10-min sonic, 10-min sonic, 30-min average UAS, 30-min average 0.0 0.2 0.4 0.6 u * / ms 1 04:00 04:30 05:00 05:30 06:00 06:30 07:00 07:30 08:00 UTC time 0.0 0.5 1.0 TKE / m2s 2 0 50 100 150 200 altitude a.g.l. / m 0 50 100 150 200 altitude a.g.l. / m 0 50 100 150 200 altitude a.g.l. / m 0 50 100 150 200 altitude a.g.l. / m 04:00 04:30 05:00 05:30 06:00 06:30 07:00 07:30 08:00 UTC time 0 50 100 150 200 altitude a.g.l. / m 0 2 4 6 8 U / m s 1 0 100 200 300 / deg 290.0 292.5 295.0 297.5 300.0 / K 0.0 0.1 0.2 0.3 0.4 0.5 u * / ms 1 0.0 0.1 0.2 0.3 0.4 0.5 TKE / m2s 2 Figure 11. Time series of horizontal wind speed U, wind direction Ψ, temperature T, friction velocity u∗and TKE at 50 m above ground https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-110 Preprint. Discussion started: 13 May 2022 c⃝Author(s) 2022. CC BY 4.0 License. 0 2 4 6 U / ms 1 100 150 200 / deg 20 25 T / °C UAS, 10-min sonic, 10-min sonic, 30-min average UAS, 30-min average 0.0 0.2 0.4 0.6 u * / ms 1 04:00 04:30 05:00 05:30 06:00 06:30 07:00 07:30 08:00 UTC time 0.0 0.5 1.0 TKE / m2s 2 0 50 100 150 200 altitude a.g.l. / m 0 50 100 150 200 altitude a.g.l. / m 0 50 100 150 200 altitude a.g.l. / m 0 50 100 150 200 altitude a.g.l. / m 04:00 04:30 05:00 05:30 06:00 06:30 07:00 07:30 08:00 UTC time 0 50 100 150 200 altitude a.g.l. / m 0 2 4 6 8 U / m s 1 0 100 200 300 / deg 290.0 292.5 295.0 297.5 300.0 / K 0.0 0.1 0.2 0.3 0.4 0.5 u * / ms 1 0.0 0.1 0.2 0.3 0.4 0.5 TKE / m2s 2 Figure 11. Time series of horizontal wind speed U, wind direction Ψ, temperature T, friction velocity u∗and TKE at 50 m above ground level during the morning transition of 28 June 2021 in comparison between UAS and sonic (left). the continuous lines show 10-minute moving averages, whereas the dots give the 30-minute mean for each quantity. On the right, Hovmoeller diagrams for the observed height range from 10-200 m are shown. The black line indicates the height of the mixed layer below the lowest temperature inversion as observed from vertical proﬁles of potential temperature θ. For wind speed U, wind direction Ψ and θ, continuous ascent vertical proﬁles from a single drone are shown in the background of the measurements from the hovering UAS at 10 m, 50 m, 90 m, 150 m and 200 m. 0 50 100 150 200 altitude a.g.l. / m 0 50 100 150 200 altitude a.g.l. / m 0 50 100 150 200 altitude a.g.l. / m 0 50 100 150 200 altitude a.g.l. / m 04:00 04:30 05:00 05:30 06:00 06:30 07:00 07:30 08:00 UTC time 0 50 100 150 200 altitude a.g.l. / m 0 2 4 6 8 U / m s 1 0 100 200 300 / deg 290.0 292.5 295.0 297.5 300.0 / K 0.0 0.1 0.2 0.3 0.4 0.5 u * / ms 1 0.0 0.1 0.2 0.3 0.4 0.5 TKE / m2s 2 Figure 11. Time series of horizontal wind speed U, wind direction Ψ, temperature T, friction velocity u∗and TKE at 50 m above ground level during the morning transition of 28 June 2021 in comparison between UAS and sonic (left). the continuous lines show 10-minute moving averages, whereas the dots give the 30-minute mean for each quantity. On the right, Hovmoeller diagrams for the observed height range from 10-200 m are shown. The black line indicates the height of the mixed layer below the lowest temperature inversion as observed from vertical proﬁles of potential temperature θ. For wind speed U, wind direction Ψ and θ, continuous ascent vertical proﬁles from a single drone are shown in the background of the measurements from the hovering UAS at 10 m, 50 m, 90 m, 150 m and 200 m. 20
W3174597874.txt	https://journals.wlb-stuttgart.de/ojs/index.php/wlbf/article/download/169/183	de		Regimentsgeschichten online	WLB-Forum	2,017	cc-by	628	Regimentsgeschichten online Digitalisierung einer wiederentdeckten Quelle zum Ersten Weltkrieg 18 Im Jahr 2016 hat die Bibliothek für Zeitgeschichte ein umfangreiches Projekt zur Digitalisierung von Truppengeschichten des Ersten Weltkriegs gestartet. Langfristiges Ziel ist es, dass alle in der WLB vorhandenen Regimentsschriften online zur Verfügung stehen. In diesem Zusammenhang wurden 2016 erste Bände der mehr als 1.000 Titel umfassenden Serie „Erinnerungsblätter deutscher Regimenter“ digitalisiert. Bereits 2014 wurden die gemeinfreien Titel der Reihe „Die württembergischen Regimenter im Weltkrieg“ online gestellt (42 von 55 Bänden). Alle digitalisierten Titel sind über den Katalog der WLB oder überregional über den Karlsruher Virtuellen Katalog (KVK) abrufbar. Während die Regimentsgeschichten in der Forschung lange nur wenig Beachtung gefunden haben, erfuhren sie in letzter Zeit vermehrt Aufmerksamkeit. Zum einen wird das Genre zum Gegenstand kultur- und mentalitätsgeschichtlicher Untersuchungen – ein Dissertationsprojekt an der Universität Düsseldorf beschäftigt sich zum Beispiel anhand der Regimentsgeschichten mit den Denkmustern und Mentalitäten der besiegten Kriegsteilnehmer in der Zwischenkriegszeit.1 Andere sehen in den Darstellungen der Regimentsgeschichtsschreiber eine Ersatzüberlieferung zu den im Zweiten Weltkrieg verlorenen Aktenbeständen des Potsdamer Heeresarchivs. Zu ihnen zählt beispielsweise Gunter Spraul, der in seinem viel diskutierten Werk „Der Franktireurkrieg 1914“2 anhand der Regimentsgeschichten zu klären versucht, ob deutsche Truppen 1914 beim Einmarsch ins neutrale Belgien von Freischärlern angegriffen wurden und ob bestimmte deutsche Einheiten in Kriegsverbrechen verwickelt waren. Die ersten gedruckten Regimentsgeschichten entstanden im Anschluss an die napoleonischen Kriege in den 1820er Jahren. Diese Truppengeschichten schilderten den Werdegang eines Regiments, seinen Dienst und seine Einsätze im Krieg. Sie richteten sich an die aktiven und ehemaligen Angehörigen der jeweiligen Einheit und dienten der Traditionspflege und damit der Schaffung eines Zusammengehörigkeitsgefühls. Die Zahl der publizierten Regimentsgeschichten nahm bis 1914 immer weiter zu, um dann nach dem Ersten Weltkrieg ihren Höhepunkt zu erreichen. Nach der Niederlage 1918 und inmitten eines radikal veränderten Umfeldes sollten sie für den Kriegsteilnehmer einen wichtigen Orientierungs- Abb. 1: Einklebeblatt für die persönlichen Daten eines Regimentsangehörigen. Die Geschichte des Oldenburgischen Infanterie-Regiments Nr. 91. Oldenburg 1930. WLBforum Ausgabe 2017/1 1) Dissertationsprojekt von Sara-Verena Adamsky M.A.: Der Erste Weltkrieg in den „Erinnerungsblättern deutscher Regimenter“. Ein Beitrag zur Mentalitätsgeschichte der Weimarer Republik“, http://www. geschichte.hhu.de/lehrstuehle/neuere-geschichte/unsere-forschung/ dissertationsprojekte.html, letzter Zugriff 16.12.2016. 2) Spraul, Gunter: Der Franktireurkrieg 1914. Untersuchungen zum Verfall einer Wissenschaft und zum Umgang mit nationalen Mythen, Berlin 2016. 19 Abb. 2: Beispielseite aus einer Regimentsschrift. Das 3. badische Dragoner-Regiment Prinz Karl Nr. 22, Berlin 1934, S. 370-371. punkt bieten. Die Verfasser wollten die vermeintlichen „Großtaten der deutschen Waffen im Weltkriege“3 vor dem Vergessen bewahren, als die deutsche Armee aufgrund der Bedingungen des Versailler Vertrages auf eine Streitkraft von 100.000 Mann reduziert werden musste. Es ging neben dem Gedenken auch darum, eine bestimmte Deutung des Krieges durchzusetzen: Denn, wie einer der Initiatoren meinte, die Kriegsteilnehmer könnten „nicht dulden, daß unsere Kinder und Enkel ihre Kenntnisse über den Weltkrieg aus den Quellen unserer Feinde schöpfen“, schließlich verfolgten die Kriegsgegner das Ziel, „die Kriegsgeschichtsschreibung über den Weltkrieg in ihrem Sinne zu gestalten“.4 Aus den schmalen Heften der frühen 1920er Jahre entwickelten sich im Laufe der 1930er Jahre zunehmend umfangreiche Bände. Die chronologische Wiedergabe der Kriegstagebücher wurde um 3) Ebd., S. 70. 4) Ebd. 5) Murawski, Erich: Truppen-Geschichten alter und neuer Art, in: Wehrkunde, 8. (1959), S. 157-164, 212-218, hier S. 162. 6) Ebd., S. 158. persönliche Erinnerungen ergänzt und mit Fotos, Gefechtsskizzen und Kartenmaterial illustriert. Stellenbesetzungspläne und Gefallenenlisten liefern Informationen zu einzelnen Personen.5 Regimentsgeschichten wurden nach 1918 von verschiedenen deutschen Bibliotheken gesammelt. Auch die Weltkriegsbücherei, die heutige Bibliothek für Zeitgeschichte in der Württembergischen Landesbibliothek, legte eine umfangreiche Sammlung von Regimentsgeschichten des Ersten Weltkriegs an. Dieser Bestand war insbesondere für den deutschen Leihverkehr von Bedeutung. Nachgewiesen wurden die Werke unter anderem in der regelmäßig erscheinenden „Bücherschau der Weltkriegsbücherei“.6 Die nun begonnene Digitalisierung der Regimentsgeschichten erleichtert der Forschung die Nutzung dieser wiederentdeckten Quellengattung. Christian Westerhoff WLBforum Ausgabe 2017/1
https://openalex.org/W2899417071	http://ojs.sites.ufsc.br/index.php/mixsustentavel/article/download/2843/2211	English	null	THE INTEGRATION OF GREEN-BUILDING TASKS AND PROJECT-DESIGN PROCESSO - CASE STUDY	Mix Sustentável	2,018	cc-by	3,938	ARTIGOS ARTIGOS Alessandro Orsi, Ph.D. (Polytechnic University of Valencia); Eugenio Pellicer Armiñana, Ph.D. (Polytechnic University of Valencia) Key Words Green-building; Project management; Integrated process; LEAN;Process waste; LEED. ABSTRACT The development of a green-building project following a speciic reference standard such as LEED, brings new conditions and restrains for all subjects involved in the process. Such changes afect technicians, owners, bureaucracy and also the management tasks either during design or construction phases. Within this scope, the management of sustainability-related activities plays a key role for the optimization of the design-project development. This research analyzes the design process of two diferent case-study projects undergoing the same green-building certiication from the project management perspective. In both project sustainability-related activities were performed throughout the design process however, in one of them such activities were integrated to the building-design development and in the other they were not. The projects selected for the scope of the research is a new nursing home complex located in Northern Italy and an oice building project in Barcelona (Spain) currently pursuing the LEED certiication. A new me- thodology was created in order to analyze the project and evaluate the efects of detected project-management issues under three diferent points of view: costs, time and building sustainability. Such “issues” were identiied by researchers on the basis of the LEAN-deinition of “waste”. The scope of the research is to demonstrate a positive relationship be- tween integration of the process green-building tasks and successful development of green-building projects within the European construction environment. The results showed that integration of green-building tasks through the de- velopment of the design process can considerably afect the cost, schedule and sustainability of the project design. 105 Mix Sustentável \| Florianópolis \| v.4 \| n.2 \| p.105-112 \| abr. \| 2018 Key Words Green-building; Project management; Integrated process; LEAN;Process waste; LEED. y Green-building; Project management; Integrated process; LEAN;Process waste; LEED. 1. INTRODUCTION The intent is to develop, with future research, a deep hands-on analysis where speciic problems related to speciic activities and circumstances could be identi- ied and, possibly, prevented. 106 The concept of sustainability has been standardized internationally through the implementation of diferent protocols but the majority of the research studies have been developed on the basis of common project mana- gement processes that refer to the United States cons- truction industry [8] (Lopez & Sánchez – 2010). Recently, sustainability has become a key aspect of the construc- tion ield [9] (Enache, Pommer & Horman – 2009) and this includes also project management. However, despite their demonstrated beneits, green buildings are not yet perceived as attractive projects because most people as- sociate green features with expensive technologies that add cost [10] (Castro-Lacouture et al.). 1. INTRODUCTION The whole research is based on the comparison of real case-study projects and has been carried out through three diferent stages: The importance of sustainability within the construc- tion business has been increasing dramatically during the last decades [1] (P. Hansford et al. – 2013) and, as some research studies point out, “an increased emphasis must be placed on the processes and competencies required to deliver high-performance buildings” [2] (Horman et al. – 2006). Currently, many researchers focus on unders- tanding diferent aspects of delivering green-building projects in order to minimize waste, maximize value, and reduce cost. During the last years several research studies analyzed diferent project management issues related to green-building developments. Their main goal is to op- timize the project management process for developing green-building projects focusing on diferent aspects, such as, counterfactual analysis [3] (Klotz et al. - 2009), LEAN processes [4] (Lapinski et al. – 2006), piloting evalua- tion metrics [5] (Korkmaz et al. – 2010). • Data collection and process illustration; • Process Analysis and detection of project-manage- ment issues; • Estimate of the impact of project-management issues on project costs, schedule and sustainability. The projects selected for the case-study is a new nur- sing-home complex located in Trento, Northern Italy, and an oice building located in Barcelona, Spain, both certiied under the LEED protocol, with a total budget of approximately 1o Million Euros. The choice of these case- -study projects was made on the basis of the following statements: • Direct access to project information and contact with all technicians involved in the project; • Simultaneity between research and project design development; As Lenle points out in a recent study, “the links betwe- en studies devoted to project management and innovation management are complex and marked by a relative lack of communication between the two ields” [6] (Lenle – 2008). Moreover, during the last years project management prac- tice has evolved into a business process and got detached from the practical aspects of the job tasks [7] (Kerzer – 2013). The scope of this research is to develop a practical compa- rison between projects with diferent levels of integration for the development of green-building tasks. • Project sustainability referring to LEED credits as ben- chmark for evaluation. The choice of real case-study projects helped resear- chers testing a new methodology for the analysis of the project management issues in green-building develop- ments. Mix Sustentável \| Florianópolis \| v.4 \| n.2 \| p.105-112 \| abr. \| 2018 A integração das tarefas de sustentabilidade com o proceso de desenho do projeto. - Estudo de casos - A integração das tarefas de sustentabilidade com o proceso de desenho do projeto. - Estudo de casos - 2.1 Deliverables and potential benefits of the research. The present research has two main outputs: • The endorsement of the new methodology developed by researchers in previous investigations for analyzing sustainability-related issues within the development of a European green-building design project. • The evaluation of the impact of process integration for sustainability-related tasks within the design process in terms of costs, time and sustainability features. 3. METHODOLOGY No speci- ic clauses were introduced in order to determine how and for which compensation LEED-related services would have been performed. During the later design and construction phases the costs of such services were subject to luctuation on the basis of the cons- truction cost variation. • No appropriate clauses in bid documentation. No speci- ic clauses were introduced in order to determine how and for which compensation LEED-related services would have been performed. During the later design and construction phases the costs of such services were subject to luctuation on the basis of the cons- truction cost variation. • Systematic cuts to budget due to change-orders and de- lays. The delay of the project design phase brought to price increase and big deicits in the project budget which involved also the sustainability-related aspect. • Lack of knowledge about energy modelling role and pro- cess. The mechanical engineers in charge of the design development developed an energy model that could not be interfaced with the LEED-required software. Another energy modeler had then to be contracted in order to partially or totally redevelop the original mo- del however, the second energy modeler was brought in too late in order to have signiicant impact on the project because by the time the energy model was i- nished the inal design had already been inished and approved with little or no margin for modiication. 107 The results obtained from the data collection process allowed researchers to identify the project priorities or, in other words, the independent variables that had to be considered for the scope of this research. Such indepen- dent variables are: Lack of project manager supervising the whole project. A project manager for sustainability-related and LEED- related issues was contracted from the beginning but no general project manager was overviewing the whole process. This brought to a lack of coordination between subjects involved and consequent fragmen- tation of the process. Lack of project manager supervising the whole project. A project manager for sustainability-related and LEED- related issues was contracted from the beginning but no general project manager was overviewing the whole process. This brought to a lack of coordination between subjects involved and consequent fragmen- tation of the process. • Time deviation: intended as the delay sufered by all sustainability-related activities of the project impac- ted by any of the project management issues during the design-phase development. 3. METHODOLOGY The scope of this research is to analyze the efect of process integration for green-building design delivery wi- thin the European Community. This study focuses on the practical implementation of a methodology developed during a previous investi- gation where researchers analyzed a single case-study project and developed a method to estimate the entity of project management problems generated by the lack of process integration [11] (Orsi & Guillamón, 2016). Mix Sustentável \| Florianópolis \| v.4 \| n.2 \| p.105-112 \| abr. \| 2018 Alessandro Orsi e Eugenio Pellicer Armiñana Following the original scope, researchers focused on identifying project management “problems”, deined on the basis of the “waste” deinition of the LEAN philo- sophy. In simple words, any type of activity performed during the process that in spite of consuming resour- ces doesn’t bring added value to the inal product [12] (J. Liker - 2003). Five types of problems were considered for the purpose of this research: waiting (process delays), transportation (unnecessary displacement of people or materials), extra-processing (re-manufacturing and reite- ration), costs (unforeseen costs for project-related tasks), defects (project weaknesses that didn’t allow the achieve- ment of the expected LEED certiication). Project-related information were collected with two diferent methods: project documentation analysis and personal interviews. Project owners provided all project documentation such as technical reports and drawings and included all infor- mation related to each phase, activity and event afecting the project design phase from the early preliminary de- sign stage until the inal executive phase. Interviews were made by researchers personally to technicians and per- sonnel involved in the project. The interview process was standardized by using a common procedure for all inter- viewees. Each subject recognized all the problems they encountered during the design development and indica- ted them in the list of project activities developed before. • Misunderstanding of Commissioning Authority’s (CxA) tasks and process. Project designers and owner didn’t understand the role of the Commissioning Authority and in spite of the suggestions of the LEED consultant the design was carried out without the CxA help until the very last stage. • Misunderstanding of Commissioning Authority’s (CxA) tasks and process. Project designers and owner didn’t understand the role of the Commissioning Authority and in spite of the suggestions of the LEED consultant the design was carried out without the CxA help until the very last stage. • No appropriate clauses in bid documentation. 3. METHODOLOGY • Cost deviation: intended as all additional costs caused by project-management issues for the development of sustainability-related activities. Problems related to project schedule and therefore to time variance were analyzed and evaluated with the use of a project management software, Microsoft Project. The list of activities was used to create a Gantt diagram for the whole project. Problems were accounted as activities and identiied with diferent colors depending on their rela- tionship with time, costs or sustainability. For the purpose of the present research only issues related to green-buil- ding activities were taken into consideration. Duration of each activity was deined on the basis of the data collec- ted from the project documentation. • Sustainability deviation: intended as the loss of cer- tiication points, under the LEED reference standard, caused by project-management issues for the develo- pment of sustainability-related activities. Problems, as deined above, were identiied by all subjects involved and were gathered together in several “categories of issues” which represent the dependent va- riables researchers aimed to focus on. The categories of issues identiied for the purpose of the present research are listed below: Mix Sustentável \| Florianópolis \| v.4 \| n.2 \| p.105-112 \| abr. \| 2018 Cross-case analysis: sustainability. Cross-case analysis: sustainability. For all projects analyzed for the scope of this research sustainability was never considered as the priority. None of the project budgets was ever modiied for a sustainability- -related problem and this had severe consequences on the inal level of sustainability of the project. However, the re- searcher noticed a substantial diference between the way the LEED procedure was developed in the two projects. For the oice building project sustainability was an integrated aspect of the design that was constantly upgraded, modi- ied and adapted to the new schedule and budget needs. For the nursing home project however, sustainability was developed more as an outsider activity which had to be considered just once-in-a-while during comprehensive meetings with all technicians involved. This detachment of green-building activities from the design-development phase caused a growing gap between what should have been done and what could be done leading to a withdraw of many green-building features and tasks. 110 This leads to the second contribution of the present study: the relationship between project management and green-building projects which also supports the rela- tionship between sustainability and afordability. Green-building activities as critical tasks for the schedu- ling process. The analysis of diferent case studies led researchers to identify a parallelism between design activities, (in- cluding architectural, mechanical, structural design) and sustainability-related activities. As cited above, for the nursing home project such activities were not integrated in the design process on a frequent basis but were con- sidered only sporadically for global meetings. This didn’t happened for the second case study where results were sensibly diferent. Researchers saw that the schedule de- veloped for design-related tasks did not always coincide with the sustainability-related one and, being the LEED certiication a long process with no speciic deadlines, these activities were never considered in the global plan- ning procedure and therefore, even if behind schedu- le, were never considered critical. This was the cause of several problems, such as, the misunderstanding of the CxA role or the lack of use given to the energy model. A integração das tarefas de sustentabilidade com o proceso de desenho do projeto. - Estudo de casos - an international scale where subjects involved have dife- rent backgrounds, benchmark and procedures. project owner had a diferent order of priorities for each of the three independent variables. For the nursing-home project the most important was always the “cost” variable mainly because, as explained above, it depended on a pu- blic funding which had already been approved and could not be changed. However, this project also had “time” as the least important variable and, according to the analysis, these two variables are heavily related one to the other. Most of the issues that generated the cost variance depen- ded on delays which imposed change orders, project rema- nufacturing tasks and other expensive activities. Therefore, is important to notice that cost variance and time variance depend one from the other or, said in other words, from the project management perspective, also during the design phase of a green-building project, time is money. This leads to the irst contribution of the present stu- dy: the quality of the project sustainability features could be improved by enhancing the integration between sub- jects involved in the design process. Positive relationship between green-building features and project management. Researcher demonstrate the existence of a relationship between the level of project integration and sustainabili- ty for the development of green-building projects. The cross-case analysis showed that both ields are mutually linked and that the eicacy of one can impact the success of the other. Following the literature review researchers focused also on the strong relationship between project management and process integration (Jainendrakumar, 2015). The relationship between project management and green-building development can also be seen as the relationship between two subjects which goal is to opti- mize the use of available resources. Let these resources be mainly time and costs for project management and water, energy and others for sustainability. Finally, all resources can be spent and both project management and sustai- nability focus on spending them the best possible way. 6. CONCLUSIONS Importance of process integration for the development of green-building projects. Cost Analysis: Cost Analysis: Indirect costs estimate was often ambiguous because could not be linked to written documents nor to any spe- ciic project activity. Information related to indirect costs were collected through interviews to subjects who some- times could not identify project management wastes. [5] Korkmaz et al.; High-Performance Green Building Design Process Modeling and Integrated Use of Visualization Tools; Journal of Architectural Engineering; 2010. [5] Korkmaz et al.; High-Performance Green Building Design Process Modeling and Integrated Use of Visualization Tools; Journal of Architectural Engineering; 2010. [6] Sylvain Lenle; Exploration and Project Management; International Journal of Project Management; 2008. Researchers only analyzed the cost of the problems they had related information of, there might have been other extra costs that couldn’t be estimate because nobo- dy appointed them as problems and so researchers didn’t even know the existence of. 111 [7] Harold Kerzer; Project Management, a systems approach to planning, scheduling and controlling; 11th Edition; 2013. [7] Harold Kerzer; Project Management, a systems approach to planning, scheduling and controlling; 11th Edition; 2013. REFERENCE LIST Considering sustainability-related tasks as critical activi- ties within the global design planning and goal may pre- vent future issues for both sustainability features, budget and time spent. This is the third contribution of this study which follows the original idea of Horman related to the priority of sustainability-related tasks (Horman, 2006). [1] P. Hansford, D. Bower, M. Clare et al.; Executive Summary – Construction 2025; HM Government Oicial Document; Crown Editor; 2013. [2] Horman M., Riley D., Lapinski A., et al.; Delivering Green Buildings – Process Improvements for Sustainable Construction; Journal of Green Building; 2006. Time analysis: Time analysis: [3] Klotz L., Johnson P., Leopard T., Maruszewski S., Hormann M., Riley D.; Campus Construction as a Research Laboratory: A Model for Intra-Campus Collaboration; Journal of Professional Issues in Engineering Education and Practice, Vol. 135, No. 4; October 2009. Estimating the delay of single activities resulted so- metimes diicult because depended from tasks which dependency could not be calculated. Therefore, for the purpose of this research activities with undeined sche- duling features were considered not individually but as part of groups of activities (milestones) whose start and ending point could be determined univocally. [4] Lapinski A., Horman M., Riley D.; LEAN Processes for Sustainable Project Delivery; Journal of Construction Engineering and Management, 2006. [4] Lapinski A., Horman M., Riley D.; LEAN Processes for Sustainable Project Delivery; Journal of Construction Engineering and Management, 2006. Importance of process integration for the development of green-building projects. The analysis of the results highlighted the positive re- lationship between process integration and development of green-building projects which has to be perceived from a broad perspective. Integration intended as physi- cal integration, in which each component can physically interact with each other, and timely integration where te- chnicians involved interact on a frequent basis with each other. Promoting this broad concept of integration in rela- tionship with the development of green-building projects has a great potential impact on the business especially at Mix Sustentável \| Florianópolis \| v.4 \| n.2 \| p.105-112 \| abr. \| 2018 Alessandro Orsi e Eugenio Pellicer Armiñana David Riley, Amy Grommes, Corinne Thatcher; Teaching Sustainability in Building Design and Engineering; Journal of Green Building; 2007. David Riley, Amy Grommes, Corinne Thatcher; Teaching Sustainability in Building Design and Engineering; Journal of Green Building; 2007. [12] Jefrey Liker; The Toyota Way: 14 Management Principles from the World’s Greatest Manufacturer; 3rd edition; McGraw-Hill Education; 2003. [13] Harris R.B.; Precedence and Arrow Networking Techniques for Construction, John Wiley and Sons; John Wiley & Sons Inc; 1978. Mastroianni R. Abdelhamid T.; The Challenge: The Impetus For Change To Lean Project Delivery; 11th Annual Conference for Lean Construction, Blacksburg, Virginia; 2003. Enache-Pommer et al.; A uniied process approach to healthcare project delivery: Synergies between greening strategies, lean principles and BIM; Conference Proceeding Paper – Construction Research Congress; 2010. Matt Stevens; The Construction MBA; 1st Edition; McGraw Hill; 2012. Kimberly A. Neuendorf; The Content Analysis Guidebook; 2nd Edition; Sage Publications, California; 2002. Sustainability Analysis [8] Lopez R., Sanchez F.; Sustainability in construction projects: analysis of a building with two sustainable as- sessment tool; Proceedings from the 13th International Congress on Project Engineering, Badajoz, 2010. [8] Lopez R., Sanchez F.; Sustainability in construction projects: analysis of a building with two sustainable as- sessment tool; Proceedings from the 13th International Congress on Project Engineering, Badajoz, 2010. For the purpose of the present work researchers took into consideration only a single green-building protocol, LEED. This protocol represents only a fraction of the gre- en-building construction market and therefore results of the present research have to be considered partially valid. Finally, as a general limitation for the work, resear- chers specify that avoiding the causes that determined the problems mentioned above is a necessary condition but maybe not suicient to avoid the waste. The problems listed above have been calculated with reference to an optimum and ideal situation characterized by zero waste in terms of time, costs and sustainability. Researchers do not have evidence that such waste can be fully avoided. In order to validate this thesis, researchers would need to analyze other projects where appropriate means and resources are implemented in order to prevent wastes listed above. This, along with other ideas listed below, re- presents one possible ield for the development of future research works. [9] Enache-Pommer E., Horman M.; Key Processes in the Building Delivery of Green Hospitals; Proceeding from the Construction Research Congress 2009. [10] Daniel Castro-Lacouture, Jorge A. Sefair, Laura Flórez, Andrés L. Medaglia; Optimization model for the selection of materials using a LEED-based green building rating system in Colombia; Building and Environment, Volume 44, Issue 6; June 2009 [11] Orsi A., Guillamón I.; Optimization of green-buil- ding design processes – Case Study; Mix Sustentável, n. 03/2016. 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https://openalex.org/W2128289824	https://europepmc.org/articles/pmc4064301?pdf=render	English	null	Who mixes with whom among men who have sex with men? Implications for modelling the HIV epidemic in southern India	Journal of theoretical biology	2,014	cc-by	13,269	Who mixes with whom among men who have sex with men? Implications for modelling the HIV epidemic in southern India K.M. Mitchell a,n, A.M. Foss a, H.J. Prudden a, Z. Mukandavire a, M. Pickles a,b, J.R. Williams b, H.C. Johnson a, B.M. Ramesh c,d, R. Washington c,e, S. Isac c, S. Ra A.E. Phillips b, J. Bradley f,g, M. Alary g,h,i, S. Moses d, C.M. Lowndes g,j, C.H. Wat M.-C. Boily b, P. Vickerman k,a K.M. Mitchell a,n, A.M. Foss a, H.J. Prudden a, Z. Mukandavire a, M. Pickles a,b, J.R. Williams b, H.C. Johnson a, B.M. Ramesh c,d, R. Washington c,e, S. Isac c, S. Rajaram f A.E. Phillips b, J. Bradley f,g, M. Alary g,h,i, S. Moses d, C.M. Lowndes g,j, C.H. Watts a, M.-C. Boily b, P. Vickerman k,a a London School of Hygiene and Tropical Medicine, London, UK b Imperial College London, London, UK c Karnataka Health Promotion Trust, Bangalore, India d University of Manitoba, Winnipeg, MB, Canada e St. John’s Research Institute, Bangalore, India f CHARME-India Project, Bangalore, India g Centre de recherche du CHU de Québec, Québec, QC, Canada h Département de medicine sociale et preventive, Université laval, Québec, QC, Canada i Institut national de santé publique du Québec, Québec, QC, Canada j Public Health England, London, UK k University of Bristol, Bristol, UK a London School of Hygiene and Tropical Medicine, London, UK b Imperial College London, London, UK c Karnataka Health Promotion Trust, Bangalore, India d University of Manitoba, Winnipeg, MB, Canada e St. John’s Research Institute, Bangalore, India f CHARME-India Project, Bangalore, India g Centre de recherche du CHU de Québec, Québec, QC, Canada h Département de medicine sociale et preventive, Université laval, Québec, QC, Canada i Institut national de santé publique du Québec, Québec, QC, Canada j Public Health England, London, UK k University of Bristol, Bristol, UK H I G H L I G H T S Different mixing scenarios are explored for 3 groups of role-segregated MSM. Models show that the mixing scenario affects both R0 and endemic HIV prevalence. When models are ﬁt to data, predicted HIV trends are unaffected by mixing. Impact of targeted (but not non-targeted) interventions can be affected by mixing. n Correspondence to: Department of Global Health and Development, London School of Hygiene and Tropical Medicine, London WC1H 9SH, UK. Tel.: þ44 207 927 2760; fax: þ44 20 7637 5391. E-mail addresses: Kate.Mitchell@lshtm.ac.uk (K.M. Mitchell), Anna.Foss@lshtm.ac.uk (A.M. Foss), Holly.Prudden@lshtm.ac.uk (H.J. Prudden), Zindoga.Mukandavire@lshtm.ac.uk (Z. Mukandavire), M.Pickles@imperial.ac.uk (M. Pickles), JR.Williams@imperial.ac.uk (J.R. Williams), Helen.Johnson@lshtm.ac.uk (H.C. Johnson), bmramesh@khpt.org (B.M. Ramesh), reynold@khpt.org (R. Washington), shajyisac@khpt.org (S. Isac), rajaram@khpt.org (S. Rajaram), a.phillips05@imperial.ac.uk (A.E. Phillips), jbradley360@shaw.ca (J. Bradley), malary@uresp.ulaval.ca (M. Alary), Stephen.Moses@med.umanitoba.ca (S. Moses), Catherine.lowndes@phe.gov.uk (C.M. Lowndes), Charlotte.Watts@lshtm.ac.uk (C.H. Watts), mc.boily@imperial.ac.uk (M.-C. Boily), Peter.Vickerman@lshtm.ac.uk (P. Vickerman). Contents lists available at ScienceDirect Contents lists available at ScienceDirect http://dx.doi.org/10.1016/j.jtbi.2014.04.005 0022-5193/& 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). journal homepage: www.elsevier.com/locate/yjtbi Who mixes with whom among men who have sex with men? Implications for modelling the HIV epidemic in southern India Journal of Theoretical Biology 355 (2014) 140–150 Journal of Theoretical Biology 355 (2014) 140–150 ors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/ http://dx.doi.org/10.1016/j.jtbi.2014.04.005 0022-5193/& 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creative 1. Introduction mixing in models including MSM role segregation is more com- plex and a variety of methods have been used for different settings (Alam et al., 2010; Goodreau and Golden, 2007; Goodreau et al., 2005; Lou et al., 2009; Sullivan et al., 2012). These previous models have all divided the population into three groups: exclusively insertive, exclusively receptive, and versatile; none have allowed for occasional receptive acts by insertive MSM or any insertive acts by mainly receptive MSM. For men who have sex with men (MSM), taking the receptive role in anal sex carries a higher risk of acquiring HIV from an infected partner than taking the insertive role (Jin et al., 2010; Pinkerton et al., 2000; Vittinghoff et al., 1999). Mathematical models have shown that role segregation among MSM (adopting a ﬁxed role – insertive or receptive – during all anal sex acts) can reduce the size of an HIV epidemic (Goodreau and Golden, 2007; Goodreau et al., 2005; Punyacharoensin et al., 2011; Van Druten et al., 1992; Wiley and Herschkorn, 1989). This is because when there is high role segrega- tion, men consistently taking the receptive role have a high risk of acquiring infection but a low risk of transmitting, whereas when there is little role segregation, individuals acquiring HIV through receptive acts are more likely to transmit infection through taking the insertive role in some subsequent sexual encounters (Goodreau et al., 2005; Trichopoulos et al., 1988). Similarly, if individuals ﬂuctuate between taking the insertive and receptive roles, this can increase HIV prevalence (Alam et al., 2010). In this paper, we develop several new mixing matrices (which determine the probabilities of mixing between different MSM groups), that all ensure balancing of insertive and receptive anal sex acts, for three MSM groups with different levels of insertive and receptive anal sex (note that in contrast to previous models, none of these groups have exclusively insertive or receptive sex). We parameterise these matrices with data from Bangalore in southern India, and explore how different mixing scenarios within this framework affect the level of overall mixing between groups, the R0 for HIV, and the size of an HIV epidemic. We also explore the implications of these different mixing scenarios for estimating the impact of a targeted HIV intervention after ﬁtting a dynamic HIV transmission model incorporating these mixing assumptions to data from Bangalore. a b s t r a c t Article history: Received 4 January 2013 Received in revised form 1 April 2014 Accepted 2 April 2014 Available online 13 April 2014 Keywords: Mathematical model Mixing matrix Sexually transmitted infection Disassortative mixing Pre-exposure prophylaxis Article history: Received 4 January 2013 Received in revised form 1 April 2014 Accepted 2 April 2014 Available online 13 April 2014 In India, the identity of men who have sex with men (MSM) is closely related to the role taken in anal sex (insertive, receptive or both), but little is known about sexual mixing between identity groups. Both role segregation (taking only the insertive or receptive role) and the extent of assortative (within-group) mixing are known to affect HIV epidemic size in other settings and populations. This study explores how different possible mixing scenarios, consistent with behavioural data collected in Bangalore, south India, affect both the HIV epidemic, and the impact of a targeted intervention. Deterministic models describing HIV transmission between three MSM identity groups (mostly insertive Panthis/Bisexuals, mostly receptive Kothis/Hijras and versatile Double Deckers), were parameterised with behavioural data from Bangalore. We extended previous models of MSM role segregation to allow each of the identity groups to have both insertive and receptive acts, in differing ratios, in line with ﬁeld data. The models were used to explore four different mixing scenarios ranging from assortative (maximising within-group mixing) to disassortative (minimising within-group mixing). A simple model was used to obtain insights into the relationship between the degree of within-group mixing, R0 and equilibrium HIV prevalence under different mixing scenarios. A more complex, extended version of the model was used to compare the http://dx.doi.org/10.1016/j.jtbi.2014.04.005 0022-5193/& 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). K.M. Mitchell et al. / Journal of Theoretical Biology 355 (2014) 140–150 141 predicted HIV prevalence trends and impact of an HIV intervention when ﬁtted to data from Bangalore. With the simple model, mixing scenarios with increased amounts of assortative (within-group) mixing tended to give rise to a higher R0 and increased the likelihood that an epidemic would occur. a b s t r a c t When the complex model was ﬁt to HIV prevalence data, large differences in the level of assortative mixing were seen between the ﬁts identiﬁed using different mixing scenarios, but little difference was projected in future HIV prevalence trends. An oral pre-exposure prophylaxis (PrEP) intervention was modelled, targeted at the different identity groups. For intervention strategies targeting the receptive or receptive and versatile MSM together, the overall impact was very similar for different mixing patterns. However, for PrEP scenarios targeting insertive or versatile MSM alone, the overall impact varied considerably for different mixing scenarios; more impact was achieved with greater levels of disassortative mixing. & 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license & 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). 1. Introduction The intervention explored is pre-exposure prophylaxis (PrEP), an oral antiretroviral medication which when taken daily by uninfected MSM has been shown to reduce their risk of acquiring HIV (Grant et al., 2011, 2010). Since PrEP is only delivered to, and directly affects, HIV negative individuals, it can be independently targeted at different population groups. In some countries, MSM sexual identity is related to the role taken in anal sex (Goodreau et al., 2005), and this is seen in India, where Kothis and Hijras tend to take the receptive role, Panthis and Bisexuals usually take the insertive role, and Double Deckers are versatile, taking both insertive and receptive roles (Asthana and Oostvogels, 2001; Boyce, 2007; Phillips et al., 2008). Hence it is likely that considerable role segregation occurs among MSM in India. Reported behaviour does not map exactly onto identity groups however, with some insertive acts reported by Kothis and some receptive acts reported by Bisexuals and Panthis (Phillips et al., 2008). Limited data are currently available on mixing between these different identity groups in India. It is important to explore the implications of different possible mixing patterns between MSM groups in the southern Indian setting, since there is an extensive literature showing that mixing between different groups (stratiﬁed by age, race or levels of sexual activity) can have a large impact on HIV prevalence (Boily and Anderson, 1991; Garnett and Anderson, 1994; Hallett et al., 2007; Turner et al., 2004). 2.1. Data sources The size of the KH population in Bangalore was estimated from mapping surveys carried out by non-governmental organisations (Bill & Melinda Gates Foundation, 2008), and the size of the DD population estimated from their frequency relative to the numbers of KH in the IBBA surveys. Total MSM population size was estimated from conﬁdential Polling Booth Surveys (PBS) conducted among the general population in neighbouring districts (Bellary, Belgaum and Mysore (Lowndes et al., 2012)). The size of the PB population could thus be estimated by subtracting the sizes of the KH and DD populations from the total population size. Note that there is considerable uncertainty in these metrics, particularly the popula- tion sizes, which is taken into account in the analyses. mixing matrix is presented in Supplementary materials, together with details on how the number of sex acts and overall mixing probabilities were calculated. 2006 (Phillips et al., 2008) were used to calculate the percentage of anal sex acts that are insertive for each group. The size of the KH population in Bangalore was estimated from mapping surveys carried out by non-governmental organisations (Bill & Melinda Gates Foundation, 2008), and the size of the DD population estimated from their frequency relative to the numbers of KH in the IBBA surveys. Total MSM population size was estimated from conﬁdential Polling Booth Surveys (PBS) conducted among the general population in neighbouring districts (Bellary, Belgaum and Mysore (Lowndes et al., 2012)). The size of the PB population could thus be estimated by subtracting the sizes of the KH and DD populations from the total population size. Note that there is considerable uncertainty in these metrics, particularly the popula- tion sizes, which is taken into account in the analyses. 2.5.1. Description of the simple model InsKH þInsPB þInsDD ¼ RecKH þRecPB þRecDD ð3Þ Substituting (1) and (2) into (3) and re-arranging gives NPB ¼ NKHcKHð12xKHÞþNDDcDDð12xDDÞ cPBð2xPB 1Þ ð4Þ InsKH þInsPB þInsDD ¼ RecKH þRecPB þRecDD ð3Þ InsKH þInsPB þInsDD ¼ RecKH þRecPB þRecDD A deterministic compartmental SI model was developed to describe the transmission of HIV between and amongst the three MSM groups (KH, DD and PB). The MSM are further divided into susceptible and infectious compartments, denoted by Si and Ii (where i¼KH, DD or PB), and the total size of each identity group, Ni, is given by Si þIi. Susceptible MSM in each identity group (Si), become infected at a per-person time dependent rate λi, and move into the infected compartment (Ii). The force of infection, λi, is a function of the number of anal sex acts per month per man (ci), the proportion of acts which are insertive (xi), the probability that HIV is acquired from an infectious partner per receptive act (βrec), the relative risk of acquiring HIV from an insertive compared with a receptive sex act (κ), the mixing matrices (ρIns and ρRec), and the prevalence of HIV in each of the groups they are mixing with (given by Ij=Nj). MSM in both susceptible and infected compart- ments cease sexual activity with other men for non-HIV related reasons and leave the MSM population at a constant per-person rate μi. Additionally, MSM in the infected compartments leave the population as they develop AIDS, at which point they are assumed to cease sexual activity, at a constant per-person rate α. New sexually active MSM join the population at a constant rate μiN0 i where N0 i is assumed to be the initial size of the MSM in group i, prior to the introduction of HIV into the population. Substituting (1) and (2) into (3) and re-arranging gives NPB ¼ NKHcKHð12xKHÞþNDDcDDð12xDDÞ cPBð2xPB 1Þ ð4Þ Substituting (1) and (2) into (3) and re-arranging gives NPB ¼ NKHcKHð12xKHÞþNDDcDDð12xDDÞ cPBð2xPB 1Þ ð4Þ Substituting (1) and (2) into (3) and re-arranging gives ð4Þ 2.3. Mixing matrices 2.2. Balancing overall number of sex acts 2.2. Balancing overall number of sex acts Mixing matrices were developed describing the mixing between three MSM groups: KH, PB and DD. Mixing was determined within the constraints of group-speciﬁc data on group size, frequency of anal sex, and proportion of acts which are insertive. 2.5. Analyses using simple, unﬁtted model The overall numbers of insertive (Insi) and receptive (Reci) acts for each identity group were calculated as: A simple model (which does not include acute or late-stage HIV, nor any condom use trends) was developed and used to calculate the basic reproductive number (R0) and equilibrium prevalence of HIV for the different mixing scenarios, and these were compared with each other and with Q (the degree of mixing) for multiple parameter combinations. The proportion of new infections from each group when HIV reached endemic equili- brium prevalence was also calculated to estimate the contribution of each group to HIV transmission. For these initial analyses, the model was not ﬁt to HIV prevalence data. This allowed direct comparisons to be made between the four mixing scenarios for each parameter combination. Insi ¼ Nicixi ð1Þ Reci ¼ Nicið1xiÞ ð2Þ ð1Þ Insi ¼ Nicixi Reci ¼ Nicið1xiÞ ð2Þ where Ni ¼population size, ci ¼number of anal sex acts per month per man, and xi ¼proportion of acts which are insertive, for each identity group (i¼KH, PB, DD). where Ni ¼population size, ci ¼number of anal sex acts per month per man, and xi ¼proportion of acts which are insertive, for each identity group (i¼KH, PB, DD). Balancing was achieved by altering the PB population size (for which no data are available) to give an equal number of insertive and receptive acts across the whole population. If the total numbers of insertive and receptive acts in the whole population balance, then: 2.4. Calculating the degree of overall mixing (Q) For each mixing scenario, and for each parameter combination considered, the overall degree of mixing was calculated from the overall mixing matrix (Supplementary materials) using Q (Gupta et al., 1989), which assesses within-group mixing. For a matrix with n groups, given the probabilities (ρi;j) that group i mixes with group j, Q is calculated as: Q ¼ ∑i ¼ jρ i;j 1 n1 A Q of 1 indicates completely assortative mixing, 0 indicates proportionate mixing, and a negative Q value indicates disassor- tative mixing. When three groups are used (n¼3), a Q of 0.5 indicates completely disassortative mixing. 2.1. Data sources The data were collected from MSM in Bangalore for evaluation of the Avahan intervention, an HIV prevention program targeted at vulnerable groups in six high prevalence states in India (Chandrasekaran et al., 2008). MSM were analysed in three groups, by the role taken in anal sex: Kothis/Hijras (KH; predominantly receptive), Panthis/Bisexuals (PB; predominantly insertive) and Double Deckers (DD; versatile). Data from Integrated Biological and Behavioural Assessment (IBBA) surveys in 2006 (Brahmam et al., 2008) and 2009 were used to calculate group-speciﬁc HIV prevalence, frequency of anal sex and condom use. Data from the 2006 IBBA survey on the identity of main MSM partners (for the subset of MSM who reported having a main partner) was used to inform the structure of the ‘setting plausible’ mixing matrix (Section 2.3). Historical condom use trends were based upon a statistical analysis of data from the 2006 IBBA survey on how long MSM had been having sex with men and how long they had always used condoms with male sex partners, which was used to recon- struct the trend in consistent condom use over time (Lowndes et al., 2010). Data from a special behavioural survey (SBS) carried out in Disassortative mixing is deﬁned as preferentially mixing with other groups rather than with your own group. For example, in heterosexual models, 100% disassortative mixing by gender is usually assumed, where men only have sex with women and vice versa. Disassortative mixing is also expected in role-segregated MSM populations, since those taking the receptive role must mix with those taking the insertive role. When there is disassortative mixing, there is usually a discrepancy in the number of sex acts reported by different groups (e.g. men usually report having more sex acts than women), so for modelling, the data have to be adjusted to allow the number of acts between different groups to ‘balance’. In models of heterosexual STI transmission, simple and transparent methods for balancing sex acts have been developed (Garnett and Anderson, 1994; Gupta et al., 1989; Hallett et al., 2007; Turner et al., 2004). However, balancing and determining K.M. Mitchell et al. / Journal of Theoretical Biology 355 (2014) 140–150 142 2006 (Phillips et al., 2008) were used to calculate the percentage of anal sex acts that are insertive for each group. 2.3. Mixing matrices Having balanced the total number of insertive and receptive acts, we then distributed the insertive and receptive acts between the different groups within data constraints, using one of four different mixing scenarios: (i) maximum assortative (maximising within- group mixing while maintaining the calculated numbers of inser- tive and receptive acts for each group, which prevents fully assortative mixing); (ii) setting plausible (in which DD have max- imum within-group mixing while the other groups mix disassorta- tively; this scenario was developed following discussions with local MSM and analysis of data on the reported identity of main MSM partners); (iii) proportionate (in which receptive acts are distributed in proportion to the number of insertive acts offered by each group; note that this does not give rise to overall proportionate (random) mixing in the population due to the different groups having differing levels of insertive and receptive sex), and (iv) disassorta- tive (minimising within-group mixing while maintaining the calcu- lated numbers of insertive and receptive acts for each group, which prevent fully disassortative mixing). Note that the name of each matrix refers to the rules used to distribute the sex acts within the data constraints—it does not reﬂect the resulting overall pattern of mixing, which is also inﬂuenced by the relative amounts of insertive and receptive sex that each group has. The derivation of each The rate of change of the number of susceptible (Si) and HIV-infected (Ii) MSM in each group (i¼KH, DD, PB) is given by the following system of differential equations dSi dt ¼ μiN0 i λiSi μiSi dIi dt ¼ λiSi μiIi αIi 8 < : K.M. Mitchell et al. / Journal of Theoretical Biology 355 (2014) 140–150 143 The force of infection is given by The force of infection is given by of the next generation matrix (ρ denotes the spectral radius). Here, the structure of the next generation matrix hinders the derivation of an analytic expression for R0. Therefore we calculate R0 numerically using code in Cþ þ which makes use of the Unsym- meig routine from Press et al. (2007), computing the eigenvalues of a real nonsymmetric matrix by reduction to Hessenberg form, followed by QR iteration. Local stability of the disease-free equili- brium for the simple model when R0 o1 follows from Theorem 2 in van den Driessche and Watmough (2002). 2.5.3. Calculating the equilibrium prevalence of HIV for the simple model 2.5.3. Calculating the equilibrium prevalence of HIV for the simple model The mixing matrix is also re-calculated at each time step, using the methods described in Supplementary materials, using current population sizes and contact rates for the three groups. The endemic equilibrium for simple model in terms of the group force of infection (λn i ) is given by We repeated analyses with the simple model making an alternative assumption that the sizes of the total MSM population and each MSM group (KH, DD and PB) remain constant over time, by replacing all of those ceasing sexual activity and leaving the population (due to AIDS or other reasons) with sexually active MSM of the same identity group newly entering the susceptible population. 2.5.2. Calculating the basic reproductive number (R0) for the simple model 2.5.2. Calculating the basic reproductive number (R0) for the simple model The basic reproductive number (R0) is the average number of secondary HIV cases arising from the introduction of a single infectious individual into a completely susceptible population, and is mathema- tically deﬁned as the spectral radius of the next generation operator (Diekmann et al., 1990; van den Driessche and Watmough, 2002). Using an approach in van den Driessche and Watmough (2002) to calculate R0 for the simple model, at the disease-free equili- brium (ξ0 ¼ ðS0 KH; I0 KH; S0 DD; I0 DD; S0 PB; I0 PB; Þ ¼ ðN0 KH; 0; N0 DD; 0; N0 PB; 0ÞÞ, the F matrix for the rate of new infections coming into each compartment, and the V matrix for all other transitions into and out of compart- ments are given by Due to the complexity of the model, it was not possible to explicitly derive algebraically tractable solutions for the endemic equilibrium. We used numerical simulations to demonstrate the existence and uniqueness of the endemic equilibrium. The equili- brium HIV prevalence was calculated for each mixing matrix using numerical simulation of the deterministic transmission model, starting with 1% of each group in the infected compartment (Ii) and the remainder in the susceptible compartment (Si). For each parameter set, and each mixing scenario in turn, the equations were solved numerically using an Euler algorithm and a time step of 0.001 years, which continued until HIV prevalence reached N0 KHcKHxKHκβRρIns;KH;DD þcKHð1xKHÞβRρRec;KH;DD N0 DD N0 KHcKHxKHκβRρIns;KH;PB þcKH 1xKH ð ÞβRρRec;KH;PB N0 PB cDDxDDκβRρIns;DD;DD þcDDð1xDDÞβRρRec;DD;DD N0 DDcDDxDDκβRρInc;DD;PB þcDDð1xDDÞβRρRec;DD;PB N0 PB N0 PBcPBxPBκβRρInc;PB;DD þ cPBð1xPBÞβRρRec;PB;DD N0 DD cPBxκβRρInc;PB;PB þcPBð1xPBÞβRρRec;PB;PB 1 C C C C C A N0 KHcKHxKHκβRρIns;KH;DD þcKHð1xKHÞβRρRec;KH;DD N0 DD N0 KHcKHxKHκβRρIns;KH;PB þcKH 1xKH ð ÞβRρRec;KH;PB N0 PB cDDxDDκβRρIns;DD;DD þcDDð1xDDÞβRρRec;DD;DD N0 DDcDDxDDκβRρInc;DD;PB þcDDð1xDDÞβRρRec;DD;PB N0 PB N0 PBcPBxPBκβRρInc;PB;DD þ cPBð1xPBÞβRρRec;PB;DD N0 DD cPBxκβRρInc;PB;PB þcPBð1xPBÞβRρRec;PB;PB 1 C C C C C A F ¼ cKHxiKHκβRρIns;KH;KH þcKHð1xKHÞβRρRec;KH;KH N0 DDcDDxDDκβRρIns;DD;KH þ cDDð1 xDDÞβRρRec;DD;KH N0 KH N0 PBcPBxPBκβRρInc;PB;KH þcPBð1 xPBÞβRρRec;PB;KH N0 KH 0 B B B B B @ V ¼ αþμKH 0 0 0 αþμDD 0 0 0 αþμPB 0 B @ 1 C A: The next generation matrix is given by 2.3. Mixing matrices λiðtÞ ¼ ∑ n j ¼ 1 cixiρIns;i;jκβrec Ij Nj þ cið1xiÞρRec;i;jβrec Ij Nj where n is the number of groups in the model (n¼3 for all analyses here). In order to maintain balancing of sex acts over time, cPB is recalculated at each time step using the following equation, which is re-arranged from Eq. (4) cPB ¼ NKHcKHð12xKHÞþNDDcDDð12xDDÞ NPBð2xPB 1Þ 2.5.3. Calculating the equilibrium prevalence of HIV for the simple model We repeated analyses with the simple model making an alternative assumption that the sizes of the total MSM population and each MSM group (KH, DD and PB) remain constant over time, ξn ¼ Sn KH ¼ μKHN0 KH λn KH þμKH; In KH ¼ μKHN0 KHλn KH ðα þμKHÞðλn KH þμKHÞ Sn DD ¼ μDDN0 DD λn DD þμDD; In DD ¼ μDDN0 DDλn DD ðα þ μDDÞðλn DD þ μDDÞ Sn PB ¼ μPBN0 PB λn PB þμPB; In PB ¼ μPBN0 PBλn PB ðα þ μPBÞðλn PB þμPBÞ 8 > > > > > < > > > > > : ξn ¼ Sn KH ¼ μKHN0 KH λn KH þμKH; In KH ¼ μKHN0 KHλn KH ðα þμKHÞðλn KH þμKHÞ Sn DD ¼ μDDN0 DD λn DD þμDD; In DD ¼ μDDN0 DDλn DD ðα þ μDDÞðλn DD þ μDDÞ Sn PB ¼ μPBN0 PB λn PB þμPB; In PB ¼ μPBN0 PBλn PB ðα þ μPBÞðλn PB þμPBÞ 8 > > > > > < > > > > > : ξn ¼ Sn KH ¼ μKHN0 KH λn KH þμKH; In KH ¼ μKHN0 KHλn KH ðα þμKHÞðλn KH þμKHÞ Sn DD ¼ μDDN0 DD λn DD þμDD; In DD ¼ μDDN0 DDλn DD ðα þ μDDÞðλn DD þ μDDÞ Sn PB ¼ μPBN0 PB λn PB þμPB; In PB ¼ μPBN0 PBλn PB ðα þ μPBÞðλn PB þμPBÞ 8 > > > > > < > > > > > : by replacing all of those ceasing sexual activity and leaving the population (due to AIDS or other reasons) with sexually active MSM of the same identity group newly entering the susceptible population. where Sn i is the number of susceptible MSM, and In i the number of infected MSM, in group i at endemic equilibrium. The next generation matrix is given by The rates of change of the number of MSM who are susceptible (Si), have acute (Ii;1), chronic (Ii;2) or pre-AIDS (Ii;3) HIV infection in each MSM group (i ¼KH, DD, PB) are given by the following system of equations acute HIV infection compartment (Ii;1), where they remain for 1/γ1 years before moving into the chronic infection stage (Ii;2). MSM stay in the chronic infection stage for on average 1/γ2 years before moving into the pre-AIDS stage (Ii;3), which they leave at a rate α as they develop AIDS and cease sexual activity. As in the simple model, MSM leave all compartments at a constant per-person rate μi as they cease sexual activity with other men for non-HIV related reasons, and are recruited into the population at a constant rate μiN0 i . As in the simple model, the contact rate for the PB group and the mixing parameters were re-calculated at each timestep to ensure balancing of sex acts between the different groups. The rates of change of the number of MSM who are susceptible (Si), have acute (Ii;1), chronic (Ii;2) or pre-AIDS (Ii;3) HIV infection in each MSM group (i ¼KH, DD, PB) are given by the following system of equations To see the contribution of each MSM group to the spread of HIV, we calculated the proportion of new infections which are generated by each group (i.e. proportion of newly infected who are infected by members of that group) when HIV was at endemic equilibrium. At the endemic equilibrium in the numerical simula- tions, the proportion of new HIV infections coming from each group was calculated as follows: infected by members of that group) when HIV was at endemic equilibrium. At the endemic equilibrium in the numerical simula- tions, the proportion of new HIV infections coming from each group was calculated as follows: ∑3 i ¼ 1 cixiκβrecρInc;i;j þcið1xiÞβrecρRec;i;j Sn i ∑3 j ¼ 1 ∑3 i ¼ 1 cixiκβrecρIns;i;j þcið1xiÞβrecρRec;i;j Sn i where Sn i is the proportion of susceptible MSM in group i at endemic equilibrium. 2.5.5. Model parameters and initial analyses using simple model 2.5.5. The next generation matrix is given by Model parameters and initial analyses using simple model Uncertainty ranges for behavioural parameters were obtained from data for Bangalore or neighbouring areas if data for Banga- lore were not available (Section 2.1), and biological parameters were obtained from the literature (all given in Supplementary Table S1). For analyses involving the simple model, all behavioural and biological parameters were varied within the ranges shown in Supplementary Table S1. Parameters were sampled from their ranges 1000 times using Latin Hypercube sampling (Blower and Dowlatabadi, 1994), assuming a uniform distribution for each parameter. To check whether the assumed distribution made a difference, parameters were then resampled 1000 times, ﬁrst assuming a triangular distribution for each parameter (using the mode and range given in Supplementary Table S1), and then assuming a normal distribution for each parameter (using the mean and standard deviation given in Supplementary Table S1). Parameter sets which gave rise to a data-plausible overall MSM population size (between 8501 and 122,600) were used to calcu- late Q, R0 and the equilibrium HIV prevalence using each of the different mixing scenarios, using the calculations in Sections 2.4, 2.5.2 and 2.5.3. R0 was estimated using data at the start of the epidemic, and Q was calculated at equilibrium prevalence. All of these analyses were repeated using the alternative model assump- tion of population sizes remaining constant over time. dSi dt ¼ μiN0 i Siðλi þμiÞ dIi;1 dt ¼ λiSi Ii;1ðμi þγ1Þ dIi;2 dt ¼ γ1Ii;1 Ii;2ðμi þγ2Þ dIi;3 dt ¼ γ2Ii;2 Ii;3ðμi þαÞ: 8 > > > > > > < > > > > > > : The force of infection is given by, λiðtÞ ¼ ∑ n j ¼ 1 ð1eCf CðtÞÞ cixiρIns;i;jκ βrec;1 Ij;1 Nj þβrec;2 Ij;2 Nj þβrec;3 Ij;3 Nj þ cið1xiÞρRec;i;j βrec;1 Ij;1 Nj þβrec;2 Ij;2 Nj þβrec;3 Ij;3 Nj where eC is condom efﬁcacy against HIV, f CðtÞ is the proportion of sex acts in which condoms are used correctly and βrec;j is the per- act transmission from an individual in infection stage Ii;j. Based on statistical analyses reconstructing historical condom use trends (Lowndes et al., 2010), condom use was assumed to change over time in a linear piecewise manner according to the following equation f CðtÞ ¼ π1; tZ2006 ðtt0ÞΔc; t0 oto2006; π4π0 π0; otherwise 8 > < > : otherwise otherwise 2.6. Analyses using more complex model, ﬁtted to data where π1 is the proportion of sex acts in which condoms are used correctly in the year 2006 (from 2006 IBBA survey data (Brahmam et al., 2008)), t0 is the year in which condom use would have been zero when reconstructed condom use trends are extrapolated backwards in time, Δc is the yearly increase in the proportion of sex acts in which condoms are used correctly (Lowndes et al., 2010) and π0 is the initial baseline level of condom use assumed in the population prior to the recently recorded increase. where π1 is the proportion of sex acts in which condoms are used correctly in the year 2006 (from 2006 IBBA survey data (Brahmam et al., 2008)), t0 is the year in which condom use would have been zero when reconstructed condom use trends are extrapolated backwards in time, Δc is the yearly increase in the proportion of sex acts in which condoms are used correctly (Lowndes et al., 2010) and π0 is the initial baseline level of condom use assumed in the population prior to the recently recorded increase. A more complex model was developed which included three stages of HIV infection (to account for the increased transmission probabilities associated with early and late-stage HIV infection) and time-varying condom use. This model was ﬁtted to HIV prevalence data, and model ﬁts were used to compare the differences in mixing patterns and HIV prevalence trends for different mixing scenarios, the impact of a targeted PrEP inter- vention from 2015, and the effective reproductive number (R) with and without PrEP. We repeated analyses with the complex model making the alternative assumption that the sizes of the total MSM population and each MSM group remain constant over time, with all those leaving the population, due to developing AIDS or other causes, being replaced with new susceptible MSM of the same identity group. The next generation matrix is given by N0 KHcKHxκβRρIns;KH;PB þcKHð1xKHÞβRρRec;KH;PB N0 PBðα þμPBÞ N0 DDcDDxDDκβRρIns;DD;PB þ cDDð1xDDÞβRρRec;DD;PB N0 PBðα þμPBÞ cPBxκβRρIns;PB;PB þcPBð1xPBÞβRρRec;PB;PB α þμPB ¼ RKH;KH RKH;DD RKH;PB RDD;KH RDD;DD RDD;PB RPB;KH RPB;DD RPB;PB 0 B @ 1 C A: FV 1 ¼ cKHxKHκβRρIns;KH;KH þcKHð1xKHÞβRρRec;KH;KH α þμKH N0 KHcKHxKHκβRρIns;KH;DD þ cKHð1 xKHÞβRρRec;KH;DD N0 DDðα þμDDÞ N0 DDcDDxDDκβRρIns;DD;KH þ cDDð1xDDÞβRρRec;DD;KH N0 KHðα þμKHÞ cDDxDDκβRρIns;DD;DD þcDDð1xDDÞβRρRec;DD;DD α þμDD N0 PBcPBxκβRρIns;PB;KH þcPBð1xPBÞβRρRec;PB;KH N0 KHðα þμKHÞ N0 PBcPBxκβRρIns;PB;DD þcPBð1xPBÞβRρRec;PB;DD N0 DDðα þμDDÞ FV 1 ¼ cKHxKHκβRρIns;KH;KH þcKHð1xKHÞβRρRec;KH;KH α þμKH N0 DDcDDxDDκβRρIns;DD;KH þ cDDð1xDDÞβRρRec;DD;KH N0 KHðα þμKHÞ N0 PBcPBxκβRρIns;PB;KH þcPBð1xPBÞβRρRec;PB;KH N0 KHðα þμKHÞ endemic equilibrium (deﬁned as changing by less than 0.01% per year). The solution proﬁle for the infected population using various initial conditions for R0 o1 and R0 41 for all mixing scenarios shows convergence to the disease-free and a unique endemic equilibrium respectively (Supplementary Fig. S1), suggesting that a unique endemic equilibrium exists for this model. Here, Ri;j for (i; j)¼KH, PB and DD are the expected number of group i individuals infected by group j individuals when a single newly infected individual in group j enters the disease-free population at equilibrium. It follows that the basic reproductive number for the simple model is given by R0 ¼ ρðFV 1Þ, which is the dominant eigenvalue K.M. Mitchell et al. / Journal of Theoretical Biology 355 (2014) 140–150 144 2.5.4. Contribution of each group to incidence at equilibrium acute HIV infection compartment (Ii;1), where they remain for 1/γ1 years before moving into the chronic infection stage (Ii;2). MSM stay in the chronic infection stage for on average 1/γ2 years before moving into the pre-AIDS stage (Ii;3), which they leave at a rate α as they develop AIDS and cease sexual activity. As in the simple model, MSM leave all compartments at a constant per-person rate μi as they cease sexual activity with other men for non-HIV related reasons, and are recruited into the population at a constant rate μiN0 i . As in the simple model, the contact rate for the PB group and the mixing parameters were re-calculated at each timestep to ensure balancing of sex acts between the different groups. 3.1. Analyses using simple, unﬁtted model where ~pi is the proportion of MSM in group i who are using PrEP and eP is the effectiveness of PrEP in preventing HIV infection. Out of the 1000 parameter sets selected from uniform para- meter ranges, 595 gave a plausible total MSM population size when receptive and insertive sex acts were balanced. These 595 parameter sets were used in Sections 3.1.1–3.1.4, where all comparisons are between matched runs which differ only in the mixing scenario used. Analyses using parameters drawn from triangular or normal distributions gave qualitatively similar results—differences are noted where relevant. Analyses using the assumption of a constant population size also gave qualitatively similar results. PrEP effectiveness – which combines PrEP efﬁcacy and adher- ence – was assumed to be 42%, as measured in the iPrEx trial (Grant et al., 2011, 2010). For an optimistic high-adherence strategy, PrEP effectiveness was assumed to be 92%, based on trial ﬁndings for individuals with detectable treatment drug in their blood (Grant et al., 2011). Coverage of the target group(s) (i.e. the percentage of sex acts of susceptible MSM covered by the PrEP intervention) was assumed to be 30% or 60% when using a realistic effectiveness of 42%. For the optimistic high-adherence strategy, 90% coverage was assumed. The optimistic strategy was included to see whether differences emerged between the predictions for the different mixing scenarios under high intervention impact, assuming that any such differences would be greater and more easily observed with greater impact. PrEP was targeted separately at each of the three MSM identity groups, at the KH and DD groups together (because these groups are more likely to be in contact with current MSM-speciﬁc interventions than PB), and then at all three groups simultaneously. 3.1.1. Effect of mixing scenario upon overall mixing (Q) 3.1.1. Effect of mixing scenario upon overall mixing (Q) Q is always negative for the ‘proportionate’ mixing scenario, meaning that the overall mixing is disassortative (Supplementary Fig. S2). For the disassortative mixing scenario, the combination of groups which are given priority affects the value of Q (Supplementary Fig. S2). Combination (b), where PB and DD are given priority, shows the most even reductions in Q (when compared with ‘proportionate’ mixing) across the range explored here, and is used for disassortative mixing in all subsequent reported results and ﬁgures (i.e. priority is given to minimising within-group mixing among PB and DD in setting mixing patterns). 2.6.3. Comparing the impact of a targeted PrEP intervention 2.6.3. Comparing the impact of a targeted PrEP intervention 2.6.3. Comparing the impact of a targeted PrEP intervention In order to test whether the choice of mixing scenario affects the predicted impact of a targeted intervention, we looked at a hypothetical PrEP intervention. PrEP was modelled against a background of reduced condom use to ensure that any differences in the effect of a PrEP intervention would be clearly seen. Condom use was assumed to drop linearly between 2010 and 2015, down to a level 20% (absolute difference) below the 2010 level, remaining constant thereafter. This fall in condom use might occur due to intervention fatigue or reduced perception of risk as HIV preva- lence falls, but has not been observed in this setting to date. Here, a1 ¼ cKHðecf c 1Þðep ~pKHÞ , a2 ¼ cDDðecf c 1Þðep ~pDDÞ, a3 ¼ cPBðecf c 1Þðep ~pPBÞ, b1 ¼ βrec;1 þðγ1ðβrec;3γ2 þβrec;2ðαþμKHÞÞ=ðαþμKHÞ ðγ2 þμKHÞÞ, b2 ¼ βrec;1 þðγ1ðβrec;3γ2 þβrec;2ðαþμDDÞÞ=ðαþμDDÞðγ2 þμDDÞÞ, b3 ¼ βrec;1 þðγ1ðβrec;3γ2 þβrec;2ðαþμPBÞÞ=ðαþμPBÞðγ2 þμPBÞÞ, d1 ¼ xKK ðκρIns;KH;KH ρRec;KH;KHÞþρRec;KH;KH, d2 ¼ xKKðκρIns;KH;DD ρRec;KH;DDÞþ ρRec;KH;DD, d3 ¼ xKKðκρIns;KH;PB ρRec;KH;PBÞþρRec;KH;PB, d4 ¼ xDD ðκρIns;DD;KH ρRec;DD;KHÞþρRec;DD;KH, d5 ¼ xDDðκρIns;DD;DD ρRec;DD;DDÞþ ρRec;DD;DD, d6 ¼ xDDðκρIns;DD;PB ρRec;DD;PBÞþρRec;DD;PB, d7 ¼ xPBðκρIns;PB;KH ρRec;PB;KHÞþρRec;PB;KH, d8 ¼ xPBðκρIns;PB;DD ρRec;PB;DDÞþρRec;PB;DD and d9 ¼ xPBðκρIns;PB;PB ρRec;PB;PBÞþρRec;PB;PB. PrEP was introduced in the model in 2015 without any gradual rollout phase. Incorporating PrEP in the model results in the following force of infection As before, the effective reproductive number is given by R ¼ ρðFV 1Þ and is calculated using a numerical algorithm coded in Cþ þ, for scenarios with condom use and with or without PrEP included. Local stability of the disease-free equilibrium for the complex model system when Ro1 also follows from van den Driessche and Watmough (2002). λiðtÞ ¼ ∑ n j ¼ 1 ð~pið1eCf CðtÞÞð1ePÞþð1 ~piÞð1eCf CðtÞÞÞ cixiρIns;i;jκ βrec;1 Ij;1 Nj þβrec;2 Ij;2 Nj þβrec;3 Ij;3 Nj þ cið1xiÞρRec;i;j βrec;1 Ij;1 Nj þβrec;2 Ij;2 Nj þβrec;3 Ij;3 Nj 3.1.2. Effect of mixing scenario upon R0 R0 tends to be similar for the propo 3.1.2. Effect of mixing scenario upon R0 R0 tends to be similar for the proportionate and disassortative mixing scenarios (Fig. 1). R0 is very similar for the maximum assortative and setting plausible mixing scenarios (Fig. 1). R0 tends to be greater for maximum assortative and setting plausible mixing than for either proportionate or disassortative mixing, although a few exceptions are seen. A smaller range in R0 was seen when parameters were drawn from triangular or normal distributions due to less frequent sampling of extreme parameter values (maximum R0 10 for normal distributions, 12 for 3. Results 3.1. Analyses using simple, unﬁtted model 2.6.1. Complex model structure 2.6.1. Complex model structure The complex model is an extension of the simple HIV model which includes three stages of HIV infection, acute, chronic and pre-AIDS, where HIV-positive MSM in the acute and pre-AIDS stages are more highly infectious than those in the chronic stage. As in the simple model, susceptible MSM (Si), become infected at a rate λi, which is determined by the number and type (insertive or receptive) of sex acts they have, the per-act probabilities of acquiring HIV from an HIV-infected partner associated with receptive and insertive sex acts, mixing patterns and HIV pre- valence of partners in different groups. Additionally in this model, infection rates are affected by time-varying condom use (which is assumed to be the same across all groups), and by the stage of HIV infection of HIV-positive partners. Once infected, MSM enter the 2.6.2. Model ﬁtting and analyses of model ﬁts The complex model was ﬁtted to HIV prevalence data for the three MSM groups from 2006 and 2009, using each mixing scenario in turn. All of the behavioural and biological parameters (detailed in Supplementary Table S1) were sampled from their ranges 1 million times using Latin Hypercube sampling, assuming a uniform distribution for each parameter. All parameter sets producing HIV prevalences within the range for each group in both 2006 and 2009, and population sizes for the KH and DD groups and the total MSM population within the ranges estimated K.M. Mitchell et al. / Journal of Theoretical Biology 355 (2014) 140–150 145 simpliﬁed next generation matrix is given as follows: in 2011 were retained as model ﬁts. For these ﬁts Q, within-group mixing, and overall and group-speciﬁc HIV prevalence over the period 1985–2030 were recorded. FV 1 ¼ a1b1d1 γ1 þμKH N0 KH KHa1b2d2 N0 DDðγ1 þ μDDÞ N0 KHa1b3d3 N0 PBðγ1 þμPBÞ N0 DDa2b1d4 N0 KHðγ1 þμKHÞ a2b2d5 γ1 þμDD N0 DDa2b3d6 N0 PBðγ1 þμPBÞ N0 PBa3b1d7 N0 KHðγ1 þμKHÞ N0 PBa3b2d8 N0 DDðγ1 þ μDDÞ a3b3d9 γ1 þ μPB 0 B B B B B @ 1 C C C C C A 3.1. Analyses using simple, unﬁtted model For each individual parameter combination, Q always progressively decreases for the mixing patterns in the following order: maximum assortative, setting plausible, proportion- ate, disassortative (data not shown). Intervention impact was measured by summing up the total number of new infections occurring between 2015 and 2030 with and without the PrEP intervention included, and taking the difference to calculate the number of infections averted by the PrEP intervention. This was expressed as a percentage of the number of infections that would have occurred in the absence of PrEP (percentage of infections averted). 2.6.4. Calculating the effective reproductive number (R) for the complex model Note that only one form of disassortative mixing is shown— giving priority to the PB and DD groups. The diagonal line shows equivalence. triangular and 19 for uniform distributions), but the patterns observed remained the same. the equilibrium prevalence and R0, with equilibrium prevalence 0 when R0 is less than 1 and rising to a plateau as R0 increases above 1 (Supplementary Fig. S3 column 3). Interestingly, this broadly follows the relationship between R0 and endemic prevalence (p¼11/R0) that is found for simple epidemic models. 3.1.3. Effect of mixing scenario upon equilibrium HIV prevalence We noted that the solution proﬁles for the infected population for different initial conditions converge to a unique endemic equilibrium when R0 41 and the disease-free equilibrium when R0 o1 (showing the existence of a forward bifurcation). Although we did not analytically derive conditions for the existence of the endemic equilibrium, the solution proﬁles for the infected popula- tion using different initial conditions show that the model system has a unique endemic equilibrium when R0 41. 3.1.5. Contribution of each group to incidence at equilibrium The proportion of new infections generated by each group when HIV is at endemic equilibrium varies substantially across the different parameter sets. Over all the parameter sets and mixing scenarios, PB tend to cause the greatest proportion of infections (median 41%, 95% credible interval 13–79%), followed by DD (median 33%, 95% CrI 7–71%), with KH responsible for the fewest (median 20%, 95% CrI 5–54%). This differs somewhat from the relative sizes of each group: at equilibrium HIV prevalence, PB make up on average 68% of the MSM population, DD 20% and KH 12%. This pattern remains true for each mixing scenario, and there is no clear relationship between mixing scenario and the proportion of infections coming from each group. For all mixing scenarios, as R0 increases, the proportion of new cases coming from PB increases on average, and the proportion coming from DD decreases; the proportion coming from KH decreases except when setting plausible mixing is used, when it tends to increase with higher R0. Equilibrium HIV prevalence is similar between mixing scenar- ios at higher HIV prevalences (425%) (data not shown). At lower prevalences, maximum assortative or setting plausible mixing tend to raise the equilibrium prevalence. With maximum assorta- tive or setting plausible mixing, epidemics sometimes occur that would not in the proportionate or disassortative scenarios. 2.6.4. Calculating the effective reproductive number (R) for the complex model 2.6.4. Calculating the effective reproductive number (R) for the complex model We deﬁne the effective reproductive number, R, as the number of secondary cases arising from a single index case in a completely susceptible population in the presence of an intervention (here, the intervention is condom use and/or PrEP). Proceeding in the same way as we calculated R0 for the simple model, R was calculated at the disease-free equilibrium using the approach in van den Driessche and Watmough (2002). The K.M. Mitchell et al. / Journal of Theoretical Biology 355 (2014) 140–150 146 R0 for proportionate mixing R0 for maximum assortative mixing R0 for proportionate mixing R0 for setting plausible mixing R0 for proportionate mixing R0 for disassortative mixing (b) R0 for maximum assortative mixing R0 for setting plausible mixing R0 for maximum assortative mixing R0 for disassortative mixing (b) R0 for setting plausible mixing R0 for disassortative mixing (b) 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 Fig. 1. R0 for the different mixing scenarios. R0 for each scenario is plotted against each other scenario in turn. Note that only one form of disassortative mixing is shown— giving priority to the PB and DD groups. The diagonal line shows equivalence. R0 for proportionate mixing R0 for maximum assortative mixing 0 5 10 15 20 0 5 10 15 20 R0 for proportionate mixing R0 for setting plausible mixing 20 0 5 10 15 20 0 5 10 15 20 R0 for setting plausible i i R0 for setting plausible Fig. 1. R0 for the different mixing scenarios. R0 for each scenario is plotted against each other scenario in turn. Note that only on giving priority to the PB and DD groups. The diagonal line shows equivalence. cenarios. R0 for each scenario is plotted against each other scenario in turn. Note that only one form of disassortative mixing is show ups. The diagonal line shows equivalence. Fig. 1. R0 for the different mixing scenarios. R0 for each scenario is plotted against each other scenario in turn. 2.6.4. Calculating the effective reproductive number (R) for the complex model More rarely, an HIV epidemic does not occur for maximum assortative or setting plausible mixing when it does for the proportionate or disassortative scenarios (data not shown). 3.1.4. Relationships between R0, Q and equilibrium HIV prevalence Across all of the different mixing scenarios, R0 showed a weak positive association with Q (Supplementary Fig. S3 column 1), which was only statistically signiﬁcant for proportionate and setting plausible mixing. Weak but statistically signiﬁcant positive associations were seen between equilibrium prevalence and Q (Supplementary Fig. S3 column 2), with the strongest associations seen for the proportionate mixing scenario. A distinctive and strong relationship is seen between 3.2. Analyses using more complex model, ﬁtted to data 3.2. Analyses using more complex model, ﬁtted to data 3.2.1. Implications of the mixing scenario for patterns of mixing and HIV prevalence for ﬁts to an HIV epidemic model When separate model ﬁts were found using each mixing scenario, more ﬁts were found with increasing levels of disassortative mixing K.M. Mitchell et al. / Journal of Theoretical Biology 355 (2014) 140–150 147 different mixing patterns gave somewhat different estimates for the group-speciﬁc impact for all three groups, particularly the DD and PB, but the overall impact in the whole MSM population was very similar (Fig. 4; Table 1). The estimated percentage of infec- tions averted after 15 years of PrEP for this intervention strategy ranged from a median of 25.4% (IQR 20.6–28.5%) for setting plausible mixing to 27.5% (IQR 21.3–30.1%) for assortative mixing. For this same targeting strategy, no great difference in the predicted overall impact was seen for lower (30%) PrEP coverage or for an optimistic PrEP strategy (92% effectiveness, 90% coverage; Table 1). Targeting only KH gave similar results—the overall impact was similar for the different mixing scenarios, for all of the PrEP strategies (Table 1). When only DD were targeted, substantial differences were seen in the overall impact for each of the intervention strategies, most dramatically for the optimistic inter- vention strategy (92% effectiveness, 90% coverage; Fig. 5, Table 1)— the overall impact ranged from 28.3% of infections averted for setting plausible mixing (IQR 14.6–35.2%) to 43.1% for disassorta- tive mixing (IQR 28.7–54.0%). If only PB were targeted, differences (maximum assortative scenario: 105 ﬁts; setting plausible: 172; proportionate: 305; disassortative: 550). Some differences were seen between the posterior parameter distributions of the ﬁts for the different mixing scenarios, particularly in the DD population size and the number of sex acts for each identity group (Supplementary Fig. S4). different mixing patterns gave somewhat different estimates for the group-speciﬁc impact for all three groups, particularly the DD and PB, but the overall impact in the whole MSM population was very similar (Fig. 4; Table 1). The estimated percentage of infec- tions averted after 15 years of PrEP for this intervention strategy ranged from a median of 25.4% (IQR 20.6–28.5%) for setting plausible mixing to 27.5% (IQR 21.3–30.1%) for assortative mixing. 3.2.2. Implications of mixing scenario for predicted impact of a targeted PrEP intervention 3.2.2. Implications of mixing scenario for predicted impact of a targeted PrEP intervention When a plausible PrEP intervention was modelled – 42% effectiveness, 60% coverage, targeted at KH and DD – using 1.0 maximum assortative setting plausible 100 Q 0.5 'proportionate' disassortative 60 80 0.0 % of acts within group 20 40 -0.5 MSM group 0 KH DD PB Fig. 2. (a) Overall mixing (Q) and (b) degree of mixing within groups, for ﬁts found using different mixing scenarios. Q and degree of mixing within groups are shown at their 2006 values. The heavy line in the middle of each box indicates the median, the limits of the box the 25th and 75th percentiles, and the whiskers the 2.5th and 97.5th percentiles. Mixing scenarios presented in decreasing order of assortativeness. Number of ﬁts: assortative 105, setting plausible 171, proportionate 305, disassortative 550. 100 60 80 % of acts within group 20 40 MSM group 0 KH DD PB 1.0 maximum assortative setting plausible Q 0.5 'proportionate' disassortative 0.0 -0.5 Fig. 2. (a) Overall mixing (Q) and (b) degree of mixing within groups, for ﬁts found using different mixing scenarios. Q and degree of mixing within groups are shown at their 2006 values. The heavy line in the middle of each box indicates the median, the limits of the box the 25th and 75th percentiles, and the whiskers the 2.5th and 97.5th percentiles. Mixing scenarios presented in decreasing order of assortativeness. Number of ﬁts: assortative 105, setting plausible 171, proportionate 305, disassortative 550. 15 30 maximum assortative setting plausible 10 20 25 sett g p aus b e 'proportionate' disassortative 5 5 10 15 MSM group HIV prevalence in 2020 (%) 0 MSM group HIV prevalence in 2006 (%) 0 KH DD PB KH DD PB Fig. 3. HIV prevalence in (a) 2006 and (b) 2020, for different groups for ﬁts found using different mixing scenarios. The heavy line in the middle of each box indicates the median, the limits of the box the 25th and 75th percentiles, and the whiskers the 2.5th and 97.5th percentiles. Mixing scenarios presented in decreasing order of assortativeness. Number of ﬁts: assortative 105, setting plausible 171, proportionate 305, disassortative 550. The ﬁtting bounds for HIV prevalence in 2006 are shown (horizontal lines). Note the different y-axis scales for (a) and (b). 3.2. Analyses using more complex model, ﬁtted to data For this same targeting strategy, no great difference in the predicted overall impact was seen for lower (30%) PrEP coverage or for an optimistic PrEP strategy (92% effectiveness, 90% coverage; Table 1). Targeting only KH gave similar results—the overall impact was similar for the different mixing scenarios, for all of the PrEP strategies (Table 1). When only DD were targeted, substantial differences were seen in the overall impact for each of the intervention strategies, most dramatically for the optimistic inter- vention strategy (92% effectiveness, 90% coverage; Fig. 5, Table 1)— the overall impact ranged from 28.3% of infections averted for setting plausible mixing (IQR 14.6–35.2%) to 43.1% for disassorta- tive mixing (IQR 28.7–54.0%). If only PB were targeted, differences Comparing the ﬁts obtained using the different mixing scenar- ios, Q and the group-speciﬁc level of within-group mixing both varied substantially between scenarios (Fig. 2). Despite these differences, the ﬁtted HIV prevalence for each group was very similar for the different mixing scenarios, both at the ﬁtted time points (shown for 2006, Fig. 3a) and for future predictions (shown for 2020, Fig. 3b). 3.2.2. Implications of mixing scenario for predicted impact of a targeted PrEP intervention 4. Discussion The heavy line in the middle of each box indicates the median, the limits of the box the 25th and 75th percentiles, and the whiskers the 2.5th and 97.5th percentiles. Mixing scenarios presented in decreasing order of assortativeness. Number of ﬁts: assortative 105, setting plausible 171, proportionate 305, disassortative 550. Table 1 Median percentage of infections averted in whole MSM population for different PrEP intervention strategies. Eff.¼effectiveness (efﬁcacy adherence). Text in bold highlights where the absolute difference in impact between the mixing scenarios is 10% or more or where the relative difference in impact ((max–min)/min) is 20% or greater. Intervention Mixing scenario Greatest difference in impact between scenarios Target group Eff. Coverage Maximum assortative Setting plausible Proportionate Disassortative Absolute Relative KH 42 30 6.7 7.2 6.4 6.3 0.9 14.9 KH 42 60 13.3 14.3 12.8 12.5 1.8 14.6 KH 92 90 38.9 44.0 40.4 39.5 5.1 13.0 DD 42 30 6.6 6.1 7.4 7.0 1.3 24.7 DD 42 60 12.5 11.4 14.5 13.9 3.0 26.4 DD 92 90 31.4 28.3 42.2 43.1 14.8 52.4 KHþDD 42 30 14 3 13 0 14 1 13 2 1 2 9 5 MSM group % infections averted 0 20 40 60 80 100 KH DD PB all max. assortative setting plausible proportionate disassort Fig. 5. Percentage of infections averted during a 15-year PrEP intervention with 92% effectiveness and 90% coverage of DD group alone (no treatment of KH or PB groups), for different mixing scenarios. The heavy line in the middle of each box indicates the median, the limits of the box the 25th and 75th percentiles, and the whiskers the 2.5th and 97.5th percentiles. Mixing scenarios presented in decreasing order of assortativeness. Number of ﬁts: assortative 105, setting plausible 171, proportionate 305, disassortative 550. MSM group % infections averted 0 10 20 30 40 50 KH DD PB all max. assortative setting plausible proportionate disassort MSM group % infections averted 0 20 40 60 80 100 KH DD PB all max. assortative setting plausible proportionate disassort MSM group % infections averted 0 20 40 60 80 100 KH DD PB all max. assortative setting plausible proportionate disassort Fig. 5. Percentage of infections averted during a 15-year PrEP intervention with 92% effectiveness and 90% coverage of DD group alone (no treatment of KH or PB groups), for different mixing scenarios. 3.2.3. Effective reproductive number for PrEP interventions h ff i d i b i d i The effective reproductive number, R, varied considerably across the different parameter sets. Just before the introduction of PrEP (with condom use at 20% below its peak level), R was around 1 (median R¼1.02, IQR 0.92–1.14 for proportionate mix- ing), and this was very similar for the different mixing scenarios. Increasingly effective PrEP interventions reduced R further: when PrEP was given to all MSM groups, R was a median 0.89 for a 42% effective PrEP intervention with 30% coverage, reduced to 0.76 with 60% coverage, and to 0.17 for a 92% effective PrEP 4. Discussion The heavy line in the middle of each box indicates the median, the limits of the box the 25th and 75th percentiles, and the whiskers the 2.5th and 97.5th percentiles. Mixing scenarios presented in decreasing order of assortativeness. Number of ﬁts: assortative 105, setting plausible 171, proportionate 305, disassortative 550. Fig. 5. Percentage of infections averted during a 15-year PrEP intervention with 92% effectiveness and 90% coverage of DD group alone (no treatment of KH or PB groups), for different mixing scenarios. The heavy line in the middle of each box indicates the median, the limits of the box the 25th and 75th percentiles, and the whiskers the 2.5th and 97.5th percentiles. Mixing scenarios presented in decreasing order of assortativeness. Number of ﬁts: assortative 105, setting plausible 171, proportionate 305, disassortative 550. Fig. 4. Percentage of infections averted during a 15-year PrEP intervention with 42% effectiveness and 60% coverage of the KH and DD groups (no treatment of PB group), for different mixing scenarios. The heavy line in the middle of each box indicates the median, the limits of the box the 25th and 75th percentiles, and the whiskers the 2.5th and 97.5th percentiles. Mixing scenarios presented in decreasing order of assortativeness. Number of ﬁts: assortative 105, setting plausible 171, proportionate 305, disassortative 550. 3.2.2. Implications of mixing scenario for predicted impact of a targeted PrEP intervention 30 maximum assortative setting plausible 20 25 sett g p aus b e 'proportionate' disassortative 5 10 15 MSM group HIV prevalence in 2006 (%) 0 KH DD PB 15 10 5 MSM group HIV prevalence in 2020 (%) 0 KH DD PB Fig. 3. HIV prevalence in (a) 2006 and (b) 2020, for different groups for ﬁts found using different mixing scenarios. The heavy line in the middle of each box indicates the median, the limits of the box the 25th and 75th percentiles, and the whiskers the 2.5th and 97.5th percentiles. Mixing scenarios presented in decreasing order of assortativeness. Number of ﬁts: assortative 105, setting plausible 171, proportionate 305, disassortative 550. The ﬁtting bounds for HIV prevalence in 2006 are shown (horizontal lines). Note the different y-axis scales for (a) and (b). K.M. Mitchell et al. / Journal of Theoretical Biology 355 (2014) 140–150 148 intervention with 90% coverage, and was not affected by the mixing scenario. R was higher when only a single group was targeted, and varied somewhat between different mixing patterns for the high coverage PrEP intervention. were also seen in the overall impact under the optimistic inter- vention strategy (Table 1)—the overall impact ranged from 50.7% of infections averted for assortative mixing to 63.9% for disassor- tative. When PrEP was given at the same coverage to all three groups, very little difference was seen in either the group-speciﬁc or the total population impact. 3.2.4. Analyses using model with constant population size Analyses using the assumption of a constant population size gave similar results for the variability in Q and prevalence values (although it predicted higher levels of prevalence – around 2.3-fold higher – in 2020 than the model with variable population size). The effective reproduction number and impact of a targeted PrEP intervention were all of a similar magnitude for both versions of the model. 4. Discussion This analysis has shown that a range of different mixing patterns are consistent with observed behavioural data from MSM group % infections averted 0 10 20 30 40 50 KH DD PB all max. assortative setting plausible proportionate disassort Fig. 4. Percentage of infections averted during a 15-year PrEP intervention with 42% effectiveness and 60% coverage of the KH and DD groups (no treatment of PB group), for different mixing scenarios. The heavy line in the middle of each box indicates the median, the limits of the box the 25th and 75th percentiles, and the whiskers the 2.5th and 97.5th percentiles. Mixing scenarios presented in decreasing order of assortativeness. Number of ﬁts: assortative 105, setting plausible 171, proportionate 305, disassortative 550. MSM group % infections averted 0 20 40 60 80 100 KH DD PB all max. assortative setting plausible proportionate disassort Fig. 5. Percentage of infections averted during a 15-year PrEP intervention with 92% effectiveness and 90% coverage of DD group alone (no treatment of KH or PB groups), for different mixing scenarios. The heavy line in the middle of each box indicates the median, the limits of the box the 25th and 75th percentiles, and the whiskers the 2.5th and 97.5th percentiles. Mixing scenarios presented in decreasing order of assortativeness. Number of ﬁts: assortative 105, setting plausible 171, proportionate 305, disassortative 550. MSM group % infections averted 0 10 20 30 40 50 KH DD PB all max. assortative setting plausible proportionate disassort Fig. 4. Percentage of infections averted during a 15-year PrEP intervention with 42% effectiveness and 60% coverage of the KH and DD groups (no treatment of PB group), for different mixing scenarios. The heavy line in the middle of each box indicates the median, the limits of the box the 25th and 75th percentiles, and the whiskers the 2.5th and 97.5th percentiles. Mixing scenarios presented in decreasing order of assortativeness. Number of ﬁts: assortative 105, setting plausible 171, proportionate 305, disassortative 550. MSM group % infections averted 0 10 20 30 40 50 KH DD PB all max. assortative setting plausible proportionate disassort Fig. 4. Percentage of infections averted during a 15-year PrEP intervention with 42% effectiveness and 60% coverage of the KH and DD groups (no treatment of PB group), for different mixing scenarios. Table 1 / Journal of Theoretical Biology 355 (2014) 140–150 149 2008; Newman, 2003), and its lower bound varies with the number of groups considered (Doherty et al., 2009). However, as our mixing matrices here all contained the same number of groups, the Q metric can be used to make direct comparisons between matrices. MSM in Bangalore, and that these different mixing patterns can have a large effect on epidemic potential for scenarios that otherwise have the same risk behaviour. However, despite these effects, all possible mixing patterns are consistent with available HIV prevalence trends from Bangalore, and have little effect on projected future HIV prevalence trends. In addition, the different mixing patterns have little effect upon the predicted impact of an untargeted PrEP intervention, but can affect the impact of a targeted PrEP interven- tion, especially if targeted at groups with versatile behaviour (rather than those mainly taking a ﬁxed role in anal sex). MSM in Bangalore, and that these different mixing patterns can have a large effect on epidemic potential for scenarios that otherwise have the same risk behaviour. However, despite these effects, all possible mixing patterns are consistent with available HIV prevalence trends from Bangalore, and have little effect on projected future HIV prevalence trends. In addition, the different mixing patterns have little effect upon the predicted impact of an untargeted PrEP intervention, but can affect the impact of a targeted PrEP interven- tion, especially if targeted at groups with versatile behaviour (rather than those mainly taking a ﬁxed role in anal sex). This analysis highlights the importance of collecting MSM sexual mixing data in this setting, especially for the versatile DD, to improve our understanding of the likely impact of targeted interventions and to better guide resource allocation, and for identifying the most suitable mixing matrix for future modelling of this population. Until such data become available, this analysis suggests that any of the mixing scenarios used here would be suitable for use in MSM models which are used for estimating prevalence trends or the impact of non-targeted interventions, but caution should be exercised if the models are being used to predict the impact of targeted interventions in these or similar MSM populations. Table 1 Table 1 Median percentage of infections averted in whole MSM population for different PrEP intervention strategies. Eff.¼effectiveness (efﬁcacy adherence). Text in bold highlights where the absolute difference in impact between the mixing scenarios is 10% or more or where the relative difference in impact ((max–min)/min) is 20% or greater. Table 1 Median percentage of infections averted in whole MSM population for different PrEP intervention strategies. Eff.¼effectiveness (efﬁcacy adherence). Text in bold highlights where the absolute difference in impact between the mixing scenarios is 10% or more or where the relative difference in impact ((max–min)/min) is 20% or greater. Intervention Mixing scenario Greatest difference in impact between scenarios Target group Eff. Coverage Maximum assortative Setting plausible Proportionate Disassortative Absolute Relative KH 42 30 6.7 7.2 6.4 6.3 0.9 14.9 KH 42 60 13.3 14.3 12.8 12.5 1.8 14.6 KH 92 90 38.9 44.0 40.4 39.5 5.1 13.0 DD 42 30 6.6 6.1 7.4 7.0 1.3 24.7 DD 42 60 12.5 11.4 14.5 13.9 3.0 26.4 DD 92 90 31.4 28.3 42.2 43.1 14.8 52.4 KHþDD 42 30 14.3 13.0 14.1 13.2 1.2 9.5 KHþDD 42 60 27.5 25.4 27.1 25.8 2.1 8.5 KHþDD 92 90 73.3 70.6 73.6 73.5 2.9 4.2 PB 42 30 9.6 11.4 10.1 11.5 1.8 19.1 PB 42 60 18.4 21.9 19.5 22.4 4.0 21.9 PB 92 90 50.7 58.6 54.4 63.9 13.2 26.1 KHþDDþPB 42 30 22.9 22.9 23.0 23.0 0.1 0.6 KHþDDþPB 42 60 41.9 41.8 42.0 41.8 0.2 0.5 KHþDDþPB 92 90 92.2 92.3 92.3 92.2 0.1 0.1 Table 1 Median percentage of infections averted in whole MSM population for different PrEP intervention strategies. Eff.¼effectiveness (efﬁcacy adherence). Text in bold highlights where the absolute difference in impact between the mixing scenarios is 10% or more or where the relative difference in impact ((max–min)/min) is 20% or greater. d in whole MSM population for different PrEP intervention strategies. Eff.¼effectiveness (efﬁcacy adherence). Text in bold n impact between the mixing scenarios is 10% or more or where the relative difference in impact ((max–min)/min) is 20% or greater. edian percentage of infections averted in whole MSM population for different PrEP intervention strategies. Eff.¼effectivene ghlights where the absolute difference in impact between the mixing scenarios is 10% or more or where the relative difference in imp K.M. Mitchell et al. References Alam, S.J., Romero-Severson, E., Kim, J.H., Emond, G., Koopman, J.S., 2010. Dynamic sex roles among men who have sex with men and transmissions from primary HIV infection. Epidemiology 21, 669–675. Asthana, S., Oostvogels, R., 2001. The social construction of male ‘homosexuality’ in India: implications for HIV transmission and prevention. Soc. Sci. Med. 52, 707–721. Bill & Melinda Gates Foundation, 2008. Use it or Lose it: How Avahan Used Data to Shape its HIV Prevention Efforts in India. New Delhi. Blower, S.M., Dowlatabadi, H., 1994. Sensitivity and uncertainty analysis of complex models of disease transmission—an HIV model, as an example. Int. Stat. Rev. 62, 229–243, http://dx.doi.org/10.2307/1403510. Boily, M.C., Anderson, R.M., 1991. Sexual contact patterns between men and women and the spread of HIV-1 in urban centers in Africa. IMA J. Math. Appl. Med. Biol. 8, 221–247. Boyce, P., 2007. ‘Conceiving kothis’: men who have sex with men in India and the cultural subject of HIV prevention. Med. Anthropol. 26, 175–203, http://dx.doi. org/10.1080/01459740701285582. Brahmam, G.N., Kodavalla, V., Rajkumar, H., Rachakulla, H.K., Kallam, S., Myakala, S.P., Paranjape, R.S., Gupte, M.D., Ramakrishnan, L., Kohli, A., Ramesh, B.M., for the IBBA Study Team, 2008. Sexual practices, HIV and sexually transmitted infections among self-identiﬁed men who have sex with men in four high HIV prevalence states of India. AIDS 22, S45–S57, http://dx.doi.org/10.1097/01.aids.00003 43763.54831.15. Chandrasekaran, P., Dallabetta, G., Loo, V., Mills, S., Saidel, T., Adhikary, R., Alary, M., Lowndes, C.M., Boily, M.C., Moore, J., for the Avahan Evaluation Partners, 2008. Evaluation design for large-scale HIV prevention programmes: the case of Avahan, the India AIDS initiative. AIDS 22, S1–S15. PrEP interventions targeting DD had the greatest variation in predicted impact by mixing scenario, due to DD having the greatest variation in within-group mixing across the different mixing scenarios. These differences occur because DD are versatile in their sexual role behaviour, having large quantities of both receptive and insertive sex, and so they can either have a large proportion of acts with other DD under the more assortative mixing scenarios, or many acts with members of other MSM groups for the more disassortative mixing scenarios. Diekmann, O., Heesterbeek, J.A.P., Metz, J.A.J., 1990. On the deﬁnition and the computation of the basic reproduction ratio R0 in models for infectious diseases in heterogeneous populations. J. Math. Biol. 28, 365–382. Doherty, I.A., Schoenbach, V.J., Adimora, A.A., 2009. Sexual mixing patterns and heterosexual HIV transmission among African Americans in the southeastern United States. Table 1 It is difﬁcult from this analysis to draw conclusions about the ‘true’ mixing pattern for this population, since very different scenar- ios, particularly with regard to the extent of mixing within the versatile group, are compatible with group-speciﬁc HIV prevalence data. The proportionate mixing matrix considered here tends to give rise to a partially disassortative mixing pattern (median of Q 0.16, IQR 0.22 to 0.11), with the insertive group having the most within-group mixing, and very little within-group mixing amongst versatile MSM; the extent of within-group mixing in this matrix is heavily impacted by the relative sizes of the different groups as well as their preference for insertive and receptive sex, and the insertive group is considerably larger than the other groups. In contrast, the ‘setting plausible’ matrix by design gives considerable within-group mixing for versatile MSM, and tends to result in more assortative population-level mixing (median of Q 0.00, IQR 0.07 to 0.11). Acknowledgements This work was supported by the Wellcome Trust (086431/Z/08/Z). Funding for the data collection came from the Bill & Melinda Gates Foundation (33978). The views expressed herein are those of the authors and do not necessarily reﬂect the ofﬁcial policy or position of the Wellcome Trust or the Bill & Melinda Gates Foundation. We would like to thank three anonymous referees for their helpful comments on earlier versions of this manuscript. In line with epidemiological theory, the mixing scenarios which gave the most assortative mixing tended to give a higher R0 and were more likely to give rise to an HIV epidemic (Garnett et al., 1992; Vynnycky and White, 2010). However, using the different mixing scenarios in a model which was then ﬁt to HIV prevalence data showed that although they give rise to considerably different mixing patterns, each can be used to ﬁt the model to available HIV prevalence data, and all give very similar projections for future HIV prevalence trends (although posterior parameter distributions differ substantially). Additionally, if these ﬁtted models are used to project the impact of an intervention given to all groups, then the mixing scenario has little effect on the intervention impact. If the intervention is targeted at particular identity groups, how- ever, the mixing scenario may affect the predicted impact. KH and DD are the most likely groups to be targeted in this setting, since they are more easily identiﬁed (and more likely to self-identify) as MSM. PB are less likely to be targeted, since they tend to be harder to reach, and are a larger, lower-risk and more ‘hidden’ population than either the KH or the DD (Prudden et al., 2012), so that although the model predicts differences in the overall impact between the different mixing scenarios when PB alone are targeted, this is unlikely to be a targeting strategy that is considered in this setting. For the most likely targeting strategies in this setting, where KH or KH and DD together are targeted, the overall impact of the intervention is unaffected by the mixing scenario. However, when DD alone are targeted, differences in the overall impact are predicted, and so if this strategy is considered, our modelling suggests that MSM mixing patterns could have a large effect on the intervention impact. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jtbi.2014.04.005. 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Lancet 2, 965–966. g/ / Jin, F.Y., Jansson, J., Law, M., Prestage, G.P., Zablotska, I., Imrie, J.C.G., Kippax, S.C., Kaldor, J.M., Grulich, A.E., Wilson, D.P., 2010. References J. Acquir. Immune Deﬁc. Syndr. 52, 114–120. van den Driessche, P., Watmough, J., 2002. Reproduction numbers and sub- threshold endemic equilibria for compartmental models of disease transmis- sion. Math. Biosci. 180, 29–48. The Q metric which we have used to measure the extent of overall mixing has some limitations: it only takes into account within-group mixing, weights the different groups equally (Keeling and Rohani, Garnett, G.P., Anderson, R.M., 1994. Balancing sexual partnerships in an age and activity stratiﬁed model of HIV transmission in heterosexual populations. IMA J. Math. Appl. Med. Biol. 11, 161–192. K.M. Mitchell et al. / Journal of Theoretical Biology 355 (2014) 140–150 150 Garnett, G.P., Swinton, J., Brunham, R.C., Anderson, R.M., 1992. 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https://openalex.org/W4293101511	https://www.researchsquare.com/article/rs-1408412/latest.pdf	English	null	Interpeduncular cistern intrathecal targeted drug delivery for Intractable Postherpetic Neuralgia: a case report	Research Square (Research Square)	2,022	cc-by	2,557	Interpeduncular cistern intrathecal targeted drug delivery for Intractable Postherpetic Neuralgia: a case report Feng Fu Characteristic Medical Center of PAP Xianfeng Jiang Characteristic Medical Center of PAP Lei Gong Characteristic Medical Center of PAP Chen Yun Characteristic Medical Center of PAP Fengwu Tang (  doctang@126.com ) Characteristic Medical Center of PAP Case presentation Herein, we describe a 69-year-old male had a one-year history of PHN after developing a vesicular rash in the ophthalmic division of cranial nerve V (trigeminal nerve) distribution. The pain was rated 7-8 at rest and 9-10 of breakthrough pain(BTP) on an numeric rating scale(NRS). Despite receiving aggressive multimodal therapies including large doses of oral analgesics and Sphenopalatine Ganglion Block (SGB), with no relief of his pain, and subsequently the patient elected to pursue an implantable IDDS with the catheter tip placed at the interpeduncular cistern. The frequency of BTP episodes was also decreased. The patient’s continuous daily dose was adjusted to 0.032 mg/day after 3 months of follow-up and stop 5 months later. Conclusion The use of interpeduncular cistern intrathecal infusion with low dose of opioids by IDDS may present an effective alternative in the management of severe PHN and other forms of neuropathic-associated craniofacial pain. Background Intractable Postherpetic Neuralgia(PHN) can be difficult to manage though receiving aggressive multimodal therapies. Patients who experience uncontrolled refractory PHN on head despite conservative treatment may benefit greatly from intrathecal drug delivery system （IDDS）. For craniofacial neuropathic pain，the traditional approach has been to place the intrathecal catheter tip usually below the level of cranial nerve root entry zones, which may lead to an insufficient analgesic effect. Case Report Posted Date: March 21st, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1408412/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. d ll License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/7 Introduction Postherpetic neuralgia (PHN) is defined as persistent pain with dermatomal distribution in patients who have recovered from herpes zoster (HZ). Additionally, patients with PHN frequently experience physical and mental pain and sleep disorders, severely affect a patient’s quality of life. Generally, approximately 20% of patients with HZ will experience PHN and more than 50% of PHN occurs in patients who are ≥ 60 years of age[1, 2]. Similarly, a cross-sectional study conducted in 24 hospitals in seven cities in China, found that 29.8% of HZ patients developed PHN and that 66.3% of those patients were > 60 years of age[3]. Currently, first-stage treatments for PHN is medication, followed by the interventional therapies such as subcutaneous injection, ganglion block, pulsed radiofrequency, nerve or spinal cord stimulation, as second- stage therapies. However, patients who experience uncontrolled refractory pain despite conservative treatment may benefit greatly from intrathecal drug delivery system (IDDS), which is referred to as third-stage therapy by the Polyanalgesic Consensus Conference (PACC) [4]. Page 2/7 Page 2/7 Page 2/7 The traditional teaching of many Best Practices and Guidelines has reasonably recommended that the catheter tip optimal placement nearest the site of pain[4]. However, for craniofacial neuropathic pain, the traditional approach has been to place the intrathecal catheter tip usually below the level of cranial nerve root entry zones, which may lead to an insufficient analgesic effect[5]. Limited data exist as to appropriate and best catheter tip placement. To provide reference for clinical treatment, the present manuscript aims to briefly describe a case of intrathecal targeted drug delivery by placing the catheter tip near the interpeduncular cistern for the treatment of PHN patient with refractory pain. Case Presentation A 69-year-old male had a one-year history of PHN after developing a vesicular rash in the ophthalmic division of cranial nerve V (trigeminal nerve) distribution. The patient was originally treated with antiviral medication, and the rash subsequently resolved. The patient, however, develop PHN with sharp, burning, and electrical type of pain in the distribution of his prior rash. The pain was rated 7–8 at rest and 9–10 of breakthrough pain(BTP) on an numeric rating scale(NRS), which at first responded to gabapentin, oxycodone, and lidocaine patches, but then became refractory to these treatments. The patient also tried twice Sphenopalatine Ganglion Block (SGB), with no relief of his pain. The patient with anxiety and insomnia were getting worse due to the severity of pain, and the patient agreed to proceed with the IDDS implantation procedure. After the success of general anesthesia, the patient was placed in a left lateral decubitus position. Intraoperative fluoroscopy was mandatory to confirm access to the C3-4 intrathecal space and the catheter tip(SP-10210, Soph-A-Port, Sophysa, France) was carefully placed within the interpeduncular cistern (Fig. 1, A and B). The intrathecal catheter was then tunneled to the upper chest subcutaneous pocket where the subcutaneous port was implanted (Fig. 1, C and D). An external drug delivery system was connected to the port using a nondestructive needle. No procedural complications were encountered while inserting the pump. A continuous daily hydromorphone hydrochloride(Yichang Human -well Pharmaceutical Co.,Ltd, China ) dose was started at 0.064mg/day and gradually titrated up to 0.128mg/day for improved pain control. For more efficient control of BTP, a bolus was added with 0.0003 mg each time and a 1 hour lockout interval. The patient developed nausea and vomiting at the beginning of medication, which was gradually controlled after drug treatment. No sedation, confusion, nor respiratory depression occurred. He was discharged from the hospital with pain reduction had been well maintained at the NRS pain score of 3/10 at rest and 5/10 at worst during breakthrough pain. The frequency of BTP episodes was also decreased. The patient’s continuous daily dose was adjusted to 0.032 mg/day after 3 months of follow-up and stop 5 months later. Discussion Page 3/7 Page 3/7 With the increasing understanding of pain pathophysiology and intrathecal analgesia, implanted IDDS has been widely recognized worldwide for the treatment of various types of chronic intractable pain. IDDS can directly inject drugs into the cerebrospinal fluid, which has the advantages of higher selectivity, significantly lesser doses of drugs and fewer adverse effects (AEs) [6]. However, in order to take advantage of this, proper patient selection and careful application of the therapy needs to be exercised. With the increasing understanding of pain pathophysiology and intrathecal analgesia, implanted IDDS has been widely recognized worldwide for the treatment of various types of chronic intractable pain. IDDS can directly inject drugs into the cerebrospinal fluid, which has the advantages of higher selectivity, significantly lesser doses of drugs and fewer adverse effects (AEs) [6]. However, in order to take advantage of this, proper patient selection and careful application of the therapy needs to be exercised. This patient had severe unrelenting PHN in the cranial nerve V (trigeminal nerve) distribution, which greatly impacted his quality of life. Despite receiving aggressive multimodal therapies including large doses of oral analgesics and SGB nerve blocks, with no relief of his pain, and subsequently the patient elected to pursue an implantable IDDS with the catheter tip placed at the interpeduncular cistern. After pump placement and initiation of intrathecal hydromorphone, his pain was significantly improved, demonstrating interpeduncular cistern intrathecal targeted drug delivery may be an effective method of treating a plethora of PHN in the trigeminal nerve area. This case proves that it is particularly important to place the catheter tip in the corresponding position according to the patient's pain level in order to achieve better analgesic effect. Current concepts of Cerebrospinal Fluid (CSF) flow dynamics emphasize pulsatile flow with oscillatory bidirectional craniocaudad CSF movement[7, 8]. Pulsatile flow patterns of CSF have been shown to imposes significant limitations on intrathecal targeted medications delivery via an IDDS, which involves a continuous low- flow intrathecal infusion through a small catheter, such that drugs was limited to a few centimeters of the catheter tip (2–3 vertebrae)[9, 10]. The trigeminal nerve is a mixed nerve, which contains general somatosensory and special visceral movement of two kinds of fibers. Sensory fibers into the pons continue forward to the sensory nucleus, spinal trigeminal nucleus or midbrain nucleus[11–12]. Acknowledgments We are very grateful to Xiao Hong Li for editing the English text of a draft of this manuscript. Availability of data and method The data used and/or method during the current case is available from the corresponding author upon reasonable request. Funding This study was funded by the Key project of Chinese Academy of Sciences (No. ZDRW-ZS-2016-2-1). Availability of data and method This study was funded by the Key project of Chinese Academy of Sciences (No. ZDRW-ZS-2016-2-1). Discussion According to clinical experience, the spinal trigeminal nucleus is second order neuron to transmits the pain signal centrally. In spite of this, the catheter tip is always placed far away from the trigeminal nerve root entry zone (where the target receptors are located) in the traditional way, thus possibly leading to insufficient analgesia. Its effect is attributed to the dense concentration of opioid receptors surrounding the brainstem and the CSF flow dynamics theory, the interpeduncular cistern intrathecal targeted drug delivery method for intractable PHN in the trigeminal nerve area could be more effective compared with the traditional approach. Based on our previous experience, it is difficult to reach the high cervical region by entering the catheter from the relatively safe L2 level due to the lack of a suitable catheter, let alone the intracranial region. Cisterna magna puncture was considered because of its safety, but was abandoned result of difficulty in fixation.Therefore, we chose to puncture through C3-4 and place the catheter tip slowly upwards. The catheter must be advanced slowly into cervical subarachnoid space under continuous X-ray to avoid damage to the tissues. Angle adjustment is very important in the placement process. If the catheter encountered resistance, it was withdrawn and adjust the angle to attempted again. The catheter tip must be exceptionally slow and careful to pass through the C1 level, after which the catheter was easily advanced to this level in our case. Page 4/7 The difficulties of the case mentioned above highlights the need of advancing in intrathecal catheter design, access techniques, imaging, and greater understanding of pain pathway. Technical innovations include new catheters, its tip is soft and the body is strong for easier placement. Ultrasound-guided puncture appears to be a safety technique for the puncture of catheter, while providing better visualization and no radiation exposure.We anticipate that this will occur in several concurrent phases, which will usher in an era where the intracisternal space is recognized as a highly valued therapeutic target. Conclusion In conclusion, interpeduncular cistern intrathecal infusion with low dose of opioids by IDDS is an effective and safe way to alleviate severe PHN and other forms of neuropathic-associated craniofacial pain with few adverse effects. Authors’ contributions Design of the work-F F, XF J, FW T; data collection and analysis-F F, L G; interpretation of data-F F, C Y, FW T; drafting and revision of the manuscript-F F, XF J, C Y, FW T. All authors approved the final version of the manuscript. Ethics approval and consent to participate We confirm that we have read the journal’s position on issues involved in ethical publication and affirm that this report is consistent with these guidelines. As this is a case report describing clinical observations, ethics approval was waived. References 1. Johnson RW, Rice ASC. Clinical practice postherpetic neuralgia. N Engl J Med 2014;371(16):1526– 33. 1. Johnson RW, Rice ASC. Clinical practice postherpetic neuralgia. N Engl J Med 2014;371(16):1526– 33. 1. Johnson RW, Rice ASC. Clinical practice postherpetic neuralgia. N Engl J Med 2014;371(16):1526– 33. 2. Massengill JS, Kittredge JL. Practical considerations in the pharmacological treatment of postherpetic neuralgia for the primary care provider. J Pain Res 2014;7:125–32. 3. Yang F, Yu S, Fan B, Liu Y, Chen YX, Kudel I, Concialdi K, DiBonaventura M, Hopps M, Hlavacek P, Cappelleri JC, Sadosky A, Parsons B, Udall M. The Epidemiology of Herpes Zoster and Postherpetic Neuralgia in China: Results from a Cross-Sectional Study. Pain Ther 2019;8(2):249–259. 4. The Polyanalgesic Consensus Conference (PACC): Recommendations on Intrathecal Drug Infusion Systems Best Practices and Guidelines. Neuromodulation 2017 Jun;20(4):405–406. 4. The Polyanalgesic Consensus Conference (PACC): Recommendations on Intrathecal Drug Infusion Systems Best Practices and Guidelines. Neuromodulation 2017 Jun;20(4):405–406. 5. Zou D, Zhang W, Wang Y. Prepontine Cistern Intrathecal Targeted Drug Delivery for Cancer-Related Craniofacial Pain. Pain Med 2021;22(12):3112–3114. 5. Zou D, Zhang W, Wang Y. Prepontine Cistern Intrathecal Targeted Drug Delivery for Cancer-Related Craniofacial Pain. Pain Med 2021;22(12):3112–3114. 6. Hayek SM, Hanes MC. Intrathecal therapy for chronic pain: current trends and future needs. Curr Pain Headache Rep 2014;18(1):388. 6. Hayek SM, Hanes MC. Intrathecal therapy for chronic pain: current trends and future needs. Curr Pain Headache Rep 2014;18(1):388. 7. Henry-Feugeas MC, Idy-Peretti I, Baledent O, Poncelet-Didon A, Zannoli G, Bittoun J, Schouman- Claeys E. Origin of subarachnoid cerebrospinal fluid pulsations:a phase-contrast MR analysis. Magn Reson Imaging 2000;18:387–95. 7. Henry-Feugeas MC, Idy-Peretti I, Baledent O, Poncelet-Didon A, Zannoli G, Bittoun J, Schouman- Claeys E. Origin of subarachnoid cerebrospinal fluid pulsations:a phase-contrast MR analysis. Magn Reson Imaging 2000;18:387–95. 8. Friese S, Hamhaber U, Erb M, Kueker W, Klose U. The influence of pulse and respiration on spinal cerebrospinal fluid pulsation. Invest Radiol 2004;39:120–30. 8. Friese S, Hamhaber U, Erb M, Kueker W, Klose U. The influence of pulse and respiration on spinal cerebrospinal fluid pulsation. Invest Radiol 2004;39:120–30. 9. Bernards CM. Cerebrospinal fluid and spinal cord distribution of baclofen and bupivacaine during slow intrathecal infusion in pigs. Anesthesiology 2006;105:169–78. 9. Bernards CM. Cerebrospinal fluid and spinal cord distribution of baclofen and bupivacaine during slow intrathecal infusion in pigs. Anesthesiology 2006;105:169–78. 10. Flack SH, Anderson CM, Bernards C. Competing interests All authors declare that they have no conflict of interests in this study. All authors declare that they have no conflict of interests in this study. Consent for publication Consent for publication Page 5/7 Page 5/7 Written informed consent was obtained from the patient for publication of this case report and accompanying images. References Morphine distribution in the spinal cord after chronic infusion in pigs[J]. Anesth Analg 2011;112(2):460–4. 10. Flack SH, Anderson CM, Bernards C. Morphine distribution in the spinal cord after chronic infusion in pigs[J]. Anesth Analg 2011;112(2):460–4. 11. Okada S, Katagiri A, Saito H, Lee J, Ohara K, Iinuma T, Bereiter DA, Iwata K. Differential activation of ascending noxious pathways associated with trigeminal nerve injury. Pain 2019;160(6):1342–1360. 11. Okada S, Katagiri A, Saito H, Lee J, Ohara K, Iinuma T, Bereiter DA, Iwata K. Differential activation of ascending noxious pathways associated with trigeminal nerve injury. Pain 2019;160(6):1342–1360. 12. Reske-Nielsen E, Oster S, Pedersen B. Herpes zoster ophthalmicus and the mesencephalic nucleus: A neuropathological Study[J]. Acta Pathol Microbiol Immunol Scand A 1986;94(4):263–269. 12. Reske-Nielsen E, Oster S, Pedersen B. Herpes zoster ophthalmicus and the mesencephalic nucleus: A neuropathological Study[J]. Acta Pathol Microbiol Immunol Scand A 1986;94(4):263–269. Figures Page 6/7 Figure 1 Head CT and 3D reconstruction taken after the procedure. The arrowhead indicates the cathete the arrow indicates the pump. Horizontal bitmap (A), Sagittal bitmap (B) and 3D reconstruction Figure 1 Head CT and 3D reconstruction taken after the procedure. The arrowhead indicates the catheter tip the arrow indicates the pump. Horizontal bitmap (A), Sagittal bitmap (B) and 3D reconstruction (C, Figure 1 Head CT and 3D reconstruction taken after the procedure. The arrowhead indicates the catheter tip, and the arrow indicates the pump. Horizontal bitmap (A), Sagittal bitmap (B) and 3D reconstruction (C,D). Page 7/7 Page 7/7
https://openalex.org/W2030854959	https://zenodo.org/records/2466951/files/article.pdf	English	null	A Proximate Analysis of the Seed of the Common Pigweed, <i>Amaranthus</i> <i>Retroflexus</i>	The journal of industrial and engineering chemistry/Journal of industrial and engineering chemistry	1,918	public-domain	2,540	AKALYSIS g After completing a determination the capsule may be lifted out of the apparatus by means of a wire hook, and the spent acid may be drawn out with a pipette. The apparatus is then ready for the next determination without even refilling with COZ. MOISTURE IN 20- AND 7 2-MESH SAMPLES-O~ gram samples were dried in an electric drying oven at exactly 100’ C. Preliminary tests showed that 14 and 4 hrs., respectively, were required to bring the 20- and 72-mesh samples to constant weight. -20-Mesh- -72-Mesh- RESULTS Argillaceous Calcite Limestone NanCOa aq. Per cent COz Per cent COz Per cent COz 43.53 24.71 34.74 43.67 24.34 34.40 43.40 24.67 By absorption method 43.52 24.36 34.59 24.59 34.30 - Av., 43.51 - By ignition Av. 24.53 43.59 By absorption method 24.47(a) Moisture content, grams., .... 0.1127 0.1129 0.0860 0.0860 0.0860 Average percentage of moisture 11.28 8.60 20 Mesh 72 Mesh I I1 I I1 I11 Moisture content, grams., .... 0.1127 0.1129 0.0860 0.0860 0.0860 Average percentage of moisture 11.28 8.60 I I1 I I1 I11 Moisture content, grams., .... 0.1127 0.1129 0.0860 0.0860 0.0860 Average percentage of moisture 11.28 8.60 AsH-Ash was determined on one-gram samples in an electric muffle at a temperature of 620’ C. I I1 I I1 I11 I I1 I11 Ash content in grams., ....................... 0.0445 0.0448 0.0446 Average percentage of ash.. ......... ..... 4.46 AsH-Ash was determined on one-gram samples AsH-Ash was determined on one-gram samples in an electric muffle at a temperature of 620’ C. I I1 I11 in an electric muffle at a temperature of 620’ C. I I1 I11 Ash content in grams., ....................... 0.0445 0.0448 0.0446 Average percentage of ash.. ......... ..... 4.46 I I1 I11 Ash content in grams., ....................... 0.0445 0.0448 0.0446 Average percentage of ash.. ......... ..... 4.46 pletely to an ash- over a Bunsen burner. It was very difficult to burn the substance com- (a) Average of 100 determinations varying from 24.1 per cent to 24.8 per cent. SEVERANCE CHEMICAL LABORATORY OBERLIN COLLEGE, OBERLIN, OHIO one-gram portions of the material, dried respectively to constant weight in an air oven and in a vacuum sulfuric acid desiccator, were extracted in Soxhlet extractors with anhydrous, alcohol-free ethyl ether to completion, which required 16 hrs. DETERMINATIOK O F OIL (ETHER EXTRACT)-AbOUt SEVERANCE CHEMICAL LABORATOR OBERLIN COLLEGE, OBERLIN, OHIO SEVERANCE CHEMICAL LABORATOR OBERLIN COLLEGE, OBERLIN, OHIO ISachse, Chem. Zenlr., 1877, 732; Bureau of Chemistry, U. S. Dept. 2 Browne, J . Am. Chem. Soc., 1906, 439. of Agriculture, Bull. 107. T H E JOCR,VAL OF I N D C S T R I A L A N D ENGI-VEERING C H E M I S T R Y July, 1918 July, 1918 July, 1918 529 By EVERHART P. HARDINC AND WALTER A. EOGE Received August 3, 1917 I11 IV I I1 Oven dried, grams oil.. ............... 0.0798 0.0793 0.0788 0.0791 Desiccator dried, grams oil. ...... 0.0814 0.0788 0.0801 .... Oven dried, average percentage of oil.. . Desiccator dried, average percentage of oil 7.92 8.46 It was thought by the authors of this paper that the partially carbonized bracts of the seeds of this common plant might make a good filtering medium €or decolorizing sugar and other colored solutions. This suggested other possible uses of the seeds which led to their proximate analysis. The dried oil dissolved in cold sulfuric ether, but not in cold petroleum ether. PRoTEIK-Protein was determined by Gunning’s modification of Kjeldahl’s method, using one-gram samples and the nitrogen conversion factor 6.2 j. I I1 I11 IV 19.13 Proteincontent, grams.. ............... 0.1880 0.1873 0.1867 0.1880 Average percentage of protein. . , , . . , . . , PRoTEIK-Protein was determined by Gunning’s modification of Kjeldahl’s method, using one-gram samples and the nitrogen conversion factor 6.2 j. p. 37. 1 Minnesota Agricultural Experiment Station, Bull. 129, March 1913, Vr X 2 7 3 X 0.001965 - 760 The seeds were stripped from plants growing in Waseca County in the southern part of Minnesota. They were cleaned by removing foreign matter and chaff (bracts). The separation and removal of the bracts was difficult and tedious. Approximately 75 per cent of the seeds were black and fully matured, the rest were red, showing varying degrees of maturity. The sample was rapidly ground to 20-mesh size and the moisture determined on a portion of this size to represent total moisture in the seeds. The rest was air-dried for 7 days and then ground to a 7a-mesh size. Moisture and all other determinations were made on this size sample. and call this the constant for the apparatus, K. We then have : K P Wt. of coz = ~ 2 7 3 + t The percentage of COZ in the carbonate then follows, thus IOO K P Percentage of COz = ( 2 7 3 + t ) X Wt. of sample‘ By use of logarithms the necessary calculation may be made in less than 2 min. The total time con- sumed in making a determination, including the weigh- ing and calculation, need not be over I j min. A PROXIMATE ANALYSIS OF THE SEED OF THE COMMON PIGWEED, AMARANTHUS RETROFLEXUS L By EVERHART P. HARDINC AND WALTER A. EOGE Received August 3, 1917 By EVERHART P. HARDINC AND WALTER A. EOGE By EVERHART P. HARDINC AND WALTER A. EOGE Received August 3, 1917 N A L OF I N D U S T R I A L A N D E N G I N E E R I N G C B E M I S T R Y Vol. IO, ?Jo. 7 -I---. 7 1 1 - 7-111-----. 1 2 1 2 1 2 CueO, mg .......... 299.6 298.0 297.5 298.4 300.1 299.4 Dextrose, mg ....... 137.0 136.2 135.95 136.40 137.25 136.90 Percentage of starch 41.09 40.91 40.98 Starch, mg ......... 123.3 122.6 122.4 122.7 123.5 123.2 Average percentage of starch.. . . . . . . . . . . .40.96 DETERMINATION OF CRUDE FIBER-This determina- tion was made on 2-g. samples containing 8.57 per cent of moisture. Kennedy’s modification’ of Sweeney’s method was used and modified by filtering and ig- niting in alundum crucibles. g I I1 I11 IV 0.2585 0.2650 0.2590 0.2580 0.0358 0.0380 0.0398 0.0382 0.2127 0.2220 0.2192 0.2198 Crude fiber and ash, grams.. , . . . . 10.63 11.1100 10.96 10.99 10.92 STARCH D E T E R M I N A T I O N (DIASTASE METHOD)-Three- gram samples were used and the diastase conversion carried out as outlined in Leach’s “Food Inspection and Analysis,” p. 284. The Munson-Walker method was used for determining the amount of dextrose. The reductions were determined on an aliquot part of a definite volume equivalent to 0.240 g. of material. For the conversion 0.60 g. of commercial diastase with a reduction equivalent of 33.1 mg. of cuprous oxide was used. TANNIr;-Qualitative tests showed tannin. SUMMARY -PERCENTAGES ON- F 20-Mesh 72-Mesh Oven- Desiccator- CONSTITUENTS as Received Air-Dried Dried Dried Moisture. ................. 11.28 8.60 0.00 .... .......... 4.33 4.46 4.88 .... Oil (ether extract). . . . . . . . . . 7.03 7.24 7.92 8.46 Protein. 19.13 .... . . . . . . . . 20.93 Starch (diastase). 33.39 36.52 40.98 44.83 .... ---PERCENTAGES ON-- 20-h.Iesh 72-Mesh Oven- CONSTITU~NTS as Received Air-Dried Dried Hemicellulose (starch by acid conversion . . . . . . 7.37 7.59 8.31. trace trace trace . . . . . . Sugar (after inversion). . . . . . . . . . . . . . . . . . 2.32 2.39 2.31 Crudefiber ............................ 10.59 10.92 11.92 Tannin and other undetermined substance by difference 6.35 6.52 7.17 Sugar (after inversion computed to cane sugar) ....................... 2.08 2.15 2.35 ......................... Th i l i h h h d ld oxide was used. 7 1 7 ----II----. 7 - 1 1 1 7 1 2 1 2 1 2 CuzO, mg ........ 233.9 234.1 232.8 233.3 232.8 232.0 Dextrose, mg ..... N A L OF I N D U S T R I A L A N D E N G I N E E R I N G C B E M I S T R Y Vol. IO, ?Jo. 7 89.40 89.50 88.85 89.10 88.85 88.46 .. 80.46 80.55 79.96 80.19 79.96 79.61 starch ......... 33.53 33.56 33.32 33.41 33.32 33.17 Average percentage of starch ... 33.39 determination of sugar was difficult on account of the colloidal condition of the sugar extract. This difficulty was finally overcome by keeping the solution just slightly alkaline, which seemed to settle the col- loids. Filtering was avoided as far as possible by increasing the volume of the solution and pipetting an aliquot volume. DETERMINATION O F SUGAR ( R E D U C I N G SUGARS)-The The proximate analysis shows that the seeds would make a good component part of a stock food and as seeds of related species have been found to contain considerable amounts of potassium nitrate, the rather high protein content would suggest that they might. be valuable as a chicken or bird food. q Five-gram samples were boiled in 150 cc. of 50 per cent neutral alcohol for an hour on a steam bath, with reflux condenser. The solution was cooled to room temperature and the volume made up to 500 cc. with 95 per cent alcohol made just alkaline. After thor- oughly mixing and settling over night, 400 cc. were pipetted off with continuous suction and evaporated on a water bath to 20 cc. This volume was made up to 2 5 0 cc., using 2 cc. of lead acetate to clarify. After clarifying, 2 0 0 cc. were pipetted with continuous suction into a beaker and the excess of lead precipi- tated with anhydrous sodium carbonate. The solu- tion was filtered and jo cc. of the filtrate used for determining the sugar by the Munson-Walker method. From an aliquot part of the solution equivalent to 0.80 g. of material only a mere trace of CuzO was formed. CHEMICAL LABORATORY UNIVBRSITY OF MINNESOTA CHEMICAL LABORATORY MINNEAPOLIS UNIVBRSITY OF MINNESOTA CHEMICAL LABORATORY MINNEAPOLIS UNIVBRSITY OF MINNESOTA CHEMICAL LABORATORY MINNEAPOLIS UNIVBRSITY OF MINNESOTA THE DETECTION OF VEGETABLE GUMS IN FOOD PRODUCTS THE DETECTION OF VEGETABLE GUMS IN FOOD PRODUCTS By A. A. COOK AND A. G. WOODM The use of gums in food products is dependent mainly on their physical properties, the most note- worthy of which is their colloidal nature. This property enables the gum substance to hold within itself relatively large quantities of water and still impart a decided “body” t o the mixture. Their use is specifically, then, as thickeners and binders in such food products as marshmallow preparations, ice cream, custards, pie fillings, egg substitutes, and flavoring emulsions. The gums ordinarily employed are gum arabic, gum tragacanth, Indian gum, agar-agar, and commercial dextrin. Gelatin, egg albumin, a n d . commercial glucose, as well as starch, are also used for the same purpose. DETERMINATION OF S U G A R (AFTER INVERSION)- Fifty cubic centimeters of the solution in the pre- ceding determination from which the excess lead was precipitated were pipetted into a 100-cc. graduated flask, 5 cc. of concentrated hydrochloric acid added, the volume made up to I O O cc. with distilled water, and allowed to remain over night at about zoo C. DESCRIPTION O F PL.4NT’ This variety of pigweed is commonly called “red root,” “rough pigweed,” “green amaranthus” and “Chinaman’s greens.” It is an annual weed which grows from a well-formed and fairly deep-rooted tap root. The root is generally red. The plant grows from I to 3 ft. high and is branched, the branches coming obliquely from the stem. The stem and leaves are rough. The plant flowers fron; July to September. These flowers are very inconspicuous, appearing in the angle formed by the stem and leaf stalk. The seeds are oval, black and shiny, and ripen during August, or before. The weed occurs in all parts of the State of Minnesota and thrives in all kinds of soil, but prefers a rich loam. It is common in gardens and waste places and does most injury by crowding out crop plants. I I1 I11 IV 19.13 Proteincontent, grams.. ............... 0.1880 0.1873 0.1867 0.1880 Average percentage of protein. . , , . . , . . , s T A R c H DE T E R MI N A TI o N ( ACI D c o NVE R SI o N ME TH OD) -Three-gram samples were used. The usual acid conversion1 of starch into dextrose was made and the amount of dextrose determined by the Munson- Walker method.z The seeds occupied a volume of 2.10 cc., for which allowance was made. Aliquot parts of the solution equivalent to 0.30 g. of substance were used in the reductions and the starch conversion factor of 0.90 was used. The following amounts of reduced cuprous oxide and the corresponding weights of dextrose and starch were found. p. 37. 1 Minnesota Agricultural Experiment Station, Bull. 129, March 1913, T H E J O U R N A L OF I N D U S T R I A L A N D E N G I N E E R I N G C B E M I S T R Y Vol. IO, ?Jo. 7 530 T H E J O U R N A L OF I N D U S T R I A L A N D E N G I N E E R I N G C B E M I S T R Y Vol. IO, ?Jo. 7 T H E J O U R N A L OF I N D U S T R I A L A N D E N G I N E E R I N G C B E M I S T R Y Vol. IO, ?Jo. 7 T H E J O U R N A L OF I N D U S T R I A L A N D E N G I N E E R I N G C B E M I S T R Y V p EXISTING METHODS EXISTING METHODS The methods which have been proposed for the de- tection of this class of materials are based for the most part on isolated reactions for a particular gum, depending on some color or solubility test of the crude gum itself , and having no -reference to the detection of small amounts of the gum in a complex food mixture. Of the few that are more general perhaps the best THISJOURNAL4 (1912) 600 CuzO, mg .............................. 21.00 21.40 21.90 21.10 Dextrose, mg.. ......................... 9.40 9.56 9.76 9.44 --I---. c---II--. 1 2 1 2 Percentage of dextrose,. , , . , , , , , . , , , . , , . Average percentage of dextrose.. ......... Percentage calculated as cane sugar.. . . . . . 2.35 2.39 2.44 2.36 2.39 2.15 The acid was nearly neutralized and the sugar de- termined in an aliquot part of the solution, equivalent to 0.40 g. of material, by the Munson-Walker method. 1 THIS JOURNAL, 4 (1912), 600.
https://openalex.org/W4200388377	https://www.frontiersin.org/articles/10.3389/fcell.2021.793428/pdf	English	null	Interplay Among Metabolism, Epigenetic Modifications, and Gene Expression in Cancer	Frontiers in cell and developmental biology	2,021	cc-by	18,017	Edited by: Lutao Du, Second Hospital of Shandong University, China Edited by: Lutao Du, Second Hospital of Shandong University, China Edited by: Lutao Du, Second Hospital of Shandong University, China Reviewed by: Marco Sciacovelli, University of Cambridge, United Kingdom Jian Zhang, Fourth Military Medical University, China Lei Chen, Eastern Hepatobiliary Surgery Hospital, China Reviewed by: Marco Sciacovelli, University of Cambridge, United Kingdom Jian Zhang, Fourth Military Medical University, China Lei Chen, Eastern Hepatobiliary Surgery Hospital, China Correspondence: Wei Huang weihuang@ccmu.edu.cn Yan Wang yanwang@cicams.ac.cn †These authors have contributed equally to this work and share ﬁrst authorship Correspondence: Wei Huang weihuang@ccmu.edu.cn Yan Wang yanwang@cicams.ac.cn †These authors have contributed equally to this work and share ﬁrst authorship Keywords: epigenetic modiﬁcations, metabolic reprogramming, metabolic enzymes, gut microbiota, clinical trails Specialty section: This article was submitted to Epigenomics and Epigenetics, a section of the journal Frontiers in Cell and Developmental Biology Received: 12 October 2021 Accepted: 01 December 2021 Published: 24 December 2021 Citation: Huo M, Zhang J, Huang W and Wang Y (2021) Interplay Among Metabolism, Epigenetic Modiﬁcations, and Gene Expression in Cancer. Front. Cell Dev. Biol. 9:793428. doi: 10.3389/fcell.2021.793428 Specialty section: This article was submitted to Epigenomics and Epigenetics, a section of the journal Frontiers in Cell and Developmental Biology REVIEW published: 24 December 2021 doi: 10.3389/fcell.2021.793428 INTRODUCTION Cancer is a disease with high morbidity and mortality and is a serious threat to human health (Bray et al., 2021). Genome instability and mutations contribute to the extraordinary diversity of cancer, and tumors acquire multiple hallmarks during their multistep development, including the reprogramming of energy metabolism (Hanahan and Weinberg, 2011; Sung et al., 2021). Over the past few decades, researchers have found that the metabolic characteristics of tumor cells are signiﬁcantly different from those of normal cells. Tumor cells have high nutrient and energy requirements, based on their characteristics of sustaining proliferative signaling, evading growth suppressors, and deregulating cellular energetics (Hanahan and Weinberg, 2011). The metabolic reprogramming of tumor cells enables them to obtain essential nutrients from a nutrition-deﬁcient environment to maintain continuous cell growth and proliferation (Pavlova and Thompson, 2016). On one hand, the activation of oncogenes and the deﬁciency of tumor suppressor genes promote the metabolic reprogramming of tumors, to achieve stronger nutrient-utilization ability and provide material and Received: 12 October 2021 Accepted: 01 December 2021 Published: 24 December 2021 Epigenetic modiﬁcations and metabolism are two fundamental biological processes. During tumorigenesis and cancer development both epigenetic and metabolic alterations occur and are often intertwined together. Epigenetic modiﬁcations contribute to metabolic reprogramming by modifying the transcriptional regulation of metabolic enzymes, which is crucial for glucose metabolism, lipid metabolism, and amino acid metabolism. Metabolites provide substrates for epigenetic modiﬁcations, including histone modiﬁcation (methylation, acetylation, and phosphorylation), DNA and RNA methylation and non-coding RNAs. Simultaneously, some metabolites can also serve as substrates for nonhistone post-translational modiﬁcations that have an impact on the development of tumors. And metabolic enzymes also regulate epigenetic modiﬁcations independent of their metabolites. In addition, metabolites produced by gut microbiota inﬂuence host metabolism. Understanding the crosstalk among metabolism, epigenetic modiﬁcations, and gene expression in cancer may help researchers explore the mechanisms of carcinogenesis and progression to metastasis, thereby provide strategies for the prevention and therapy of cancer. In this review, we summarize the progress in the understanding of the interactions between cancer metabolism and epigenetics. EPIGENETIC MODIFICATIONS Increasing evidence suggests that cancer is a metabolic disease. Epigenetic regulation plays a crucial role in metabolic regulation and tumorigenesis (Li et al., 2018; Bates, 2020). To maintain homeostasis and ensure cell survival, cells must dynamically respond to changes in the environment and reprogram their metabolic state. Similarly, the harmonization of gene expression is required to ensure normal cell function. Epigenetic modiﬁcations provide an ideal mechanism for the regulation of gene expression and metabolic reprogramming. Speciﬁcally, changes in histone modiﬁcation are fast and reversible and rely on metabolic intermediates as cofactors for modiﬁcation. Therefore, it is of great signiﬁcance to understand the relationship among the metabolic environment, epigenetic modiﬁcations, and the expression of genes that play a role in many diseases, particularly cancer (Li et al., 2018). Citation: Altered metabolic proﬁles mainly occur with respect to the uptake and metabolism of glucose and amino acids and the synthesis of lipids. For example, the Warburg effect, observed in cancer, suggests that even under sufﬁcient oxygen conditions, malignant tumor cells are active in anaerobic glycolysis, which yields lactic acid, instead of oxidative phosphorylation like that in normal differentiated cells, characterized by a high glucose uptake rate and active glycolysis; moreover, a high level of the metabolite lactic acid correlates with poor tumor prognosis (Warburg, 1956; Som et al., 1980; Vander Heiden et al., 2009). Although aerobic glycolysis is an inefﬁcient way to generate adenosine 5′-triphosphate (ATP), it meets the nutrient needs for cancer cell proliferation via the incorporation of metabolized nutrients into biomass. This reveals a link between cellular metabolism and cell growth control (Vander Heiden et al., 2009). As a highly versatile nutrient, glutamine is also important for tumor cell growth. Cancer cells take up glutamine via glutamine transporter (ASCT2), also known as solute carrier family 1 member 5 (SLC1A5) (Wang et al., 2015). Further, cancer cells are addicted to glutamine through oncogene-dependent pathways involving c-MYC, AKT (Serine/Threonine Kinase 1), and p53 (Tumor Protein P53) (Wettersten et al., 2017). Likewise, as a principal growth-supporting substrate, glutamine provides nitrogen for the biosynthesis of purine and pyrimidine nucleotides, glucosamine-6-phosphate, and nonessential amino acids (Pavlova and Thompson, 2016). Additionally, a process known as glutaminolysis can divert abundant glutamine to replenish the tricarboxylic acid (TCA) cycle (Wong et al., 2017). Lifestyle-related factors, especially diet and nutrition, have a profound effect on human health. Moreover, the gut microbiota plays an important role in this by further metabolizing nutrients from the diet and producing a variety of chromatin-modifying compounds, ultimately regulating histone methylation and acetylation by modulating the intracellular pools of metabolites (Dai et al., 2020). Epigenetic modiﬁcatios and metabolism are two fundamental biological processes. Epigenetic alterations and metabolic reprogramming in cancer are highly interrelated. Oncogene- driven metabolic reprogramming alters the epigenetic landscape by regulating DNA and histone modiﬁcation-related enzyme activity. However, the expression of metabolic genes is regulated by epigenetic mechanisms, thus, altering the metabolome. Therefore, the crosstalk between epigenetics and metabolism plays a crucial role in carcinogenesis and cancer progression through the proliferation, metastasis, and heterogeneity of cancer cells. Citation: December 2021 \| Volume 9 \| Article 793428 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 1 Huo et al. Metabolism Crosstalk with Epigenetic Modiﬁcations energy for biosynthesis. At the same time, a lack of nutrition in solid tumors also requires malignant tumor cells to possess metabolic ﬂexibility to maintain growth and survival (Boroughs and DeBerardinis, 2015). Altered metabolic proﬁles mainly occur with respect to the uptake and metabolism of glucose and amino acids and the synthesis of lipids. For example, the Warburg effect, observed in cancer, suggests that even under sufﬁcient oxygen conditions, malignant tumor cells are active in anaerobic glycolysis, which yields lactic acid, instead of oxidative phosphorylation like that in normal differentiated cells, characterized by a high glucose uptake rate and active glycolysis; moreover, a high level of the metabolite lactic acid correlates with poor tumor prognosis (Warburg, 1956; Som et al., 1980; Vander Heiden et al., 2009). Although aerobic glycolysis is an inefﬁcient way to generate adenosine 5′-triphosphate (ATP), it meets the nutrient needs for cancer cell proliferation via the incorporation of metabolized nutrients into biomass. This reveals a link between cellular metabolism and cell growth control (Vander Heiden et al., 2009). As a highly versatile nutrient, glutamine is also important for tumor cell growth. Cancer cells take up glutamine via glutamine transporter (ASCT2), also known as solute carrier family 1 member 5 (SLC1A5) (Wang et al., 2015). Further, cancer cells are addicted to glutamine through oncogene-dependent pathways involving c-MYC, AKT (Serine/Threonine Kinase 1), and p53 (Tumor Protein P53) (Wettersten et al., 2017). Likewise, as a principal growth-supporting substrate, glutamine provides nitrogen for the biosynthesis of purine and pyrimidine nucleotides, glucosamine-6-phosphate, and nonessential amino acids (Pavlova and Thompson, 2016). Additionally, a process known as glutaminolysis can divert abundant glutamine to replenish the tricarboxylic acid (TCA) cycle (Wong et al., 2017). Lifestyle-related factors, especially diet and nutrition, have a profound effect on human health. Moreover, the gut microbiota plays an important role in this by further metabolizing nutrients from the diet and producing a variety of chromatin-modifying compounds, ultimately regulating histone methylation and acetylation by modulating the intracellular pools of metabolites (Dai et al., 2020). energy for biosynthesis. At the same time, a lack of nutrition in solid tumors also requires malignant tumor cells to possess metabolic ﬂexibility to maintain growth and survival (Boroughs and DeBerardinis, 2015). Deoxyribonucleic Acid and Ribonucleic Acid Methylation y Many studies have shown that DNA and RNA methylation plays an important role in regulating cancer metabolism (Saghaﬁnia et al., 2018; Uddin et al., 2021). Abnormal methylation in the promoter region of tumor suppressor genes is key to tumorigenesis and cancer development (Nishiyama and Nakanishi, 2021). Zinc ﬁnger DHHC- Type containing 1 (ZDHHC1), a recently discovered tumor suppressor gene, is silenced in a variety of cancers through abnormal hypermethylation to inhibit glucose metabolism and the pentose phosphate pathway (Le et al., 2020). The transcription factor brother of the regulator of imprinted sites (BORIS) regulates the methylation of pyruvate kinase M1/2 (PKM) exons and the alternative splicing of PKM mRNA to mediate the Warburg effect and promote breast cancer (Singh et al., 2017). In colon cancer, hypermethylation of the derlin3 (DERL3) promoter region promotes high expression of solute carrier family 2 member 1 (SLC2A1) to enhance the Warburg effect (Lopez-Serra et al., 2014). Methyltransferase 3, N6- adenosine-methyltransferase complex catalytic subunit 3 (METTL3) attaches m6A-IGF2BP2/3 to stabilize the expression of hexokinase 2 (HK2), resulting in the expression of phosphorylated glucose hexokinases, and SLC2A1, resulting in the expression of glucose transporter (GLUT1), in colon cancer to activate the glycolytic pathway (Shen et al., 2020) (Figure 1). Heritable changes in gene expression that do not include changes to the DNA sequence itself are termed epigenetic changes. Epigenetics mainly are manifested as DNA methylation, histone post-translational modiﬁcations (PTMs), such as acetylation, methylation, phosphorylation, ubiquitination, glycosylation, lactylation, succinylation, and other acyl modiﬁcations, including O-linked N-acetylglucosamine modiﬁcation (O-GlcNAcylation) (Esteller, 2008; Tan et al., 2011; Suva et al., 2013; Huang et al., 2014; Tessarz and Kouzarides, 2014; Dai et al., 2020), as well as RNA methylation, including N6-methyladenosine (m6A) and 5-methylcytosine (m5C) (Han et al., 2020). Epigenetic plasticity in the process of tumorigenesis and development facilitates the acquisition of hallmark characteristics of cancer (Esteller, 2008; Hanahan and Weinberg, 2011). Many metabolic intermediates serve as epigenetic modiﬁcation substrates or cofactors, and the various epigenetic processes are primarily governed by the concentrations of the involved reactants (Simithy et al., 2017; Wagner et al., 2017; Thakur and Chen, 2019). December 2021 \| Volume 9 \| Article 793428 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 2 Huo et al. Huo et al. Deoxyribonucleic Acid and Ribonucleic Acid Methylation Metabolism Crosstalk with Epigenetic Modiﬁcations FIGURE 1 \| Regulation of glycolysis and gluconeogenesis enzymes by epigenetic modiﬁcatio epigenetic regulation of the key proteins, GLUT1, HK2, PKM, ENO2, LDHA, IDH, and the key FIGURE 1 \| Regulation of glycolysis and gluconeogenesis enzymes by epigenetic modiﬁcations. Epigenetic regulatory enzymes, lncRNA, and circRNA regulate the epigenetic regulation of the key proteins, GLUT1, HK2, PKM, ENO2, LDHA, IDH, and the key enzymes FBP and PCK in the process of gluconeogenesis. GLUT1, glucose transporter type 1; HK2, hexokinase 2; PKM, pyruvate kinase M1/2; ENO2, enolase 2; LDHA, lactate dehydrogenase A; IDH, isocitrate dehydrogenase (NADP (+)); FBP, fructose-bisphosphatase; PCK, phosphoenolpyruvate carboxykinase. FIGURE 1 \| Regulation of glycolysis and gluconeogenesis enzymes by epigenetic modiﬁcations. Epigenetic regulatory enzymes, lncRNA, and circRNA regulate the epigenetic regulation of the key proteins, GLUT1, HK2, PKM, ENO2, LDHA, IDH, and the key enzymes FBP and PCK in the process of gluconeogenesis. GLUT1, glucose transporter type 1; HK2, hexokinase 2; PKM, pyruvate kinase M1/2; ENO2, enolase 2; LDHA, lactate dehydrogenase A; IDH, isocitrate dehydrogenase (NADP (+)); FBP, fructose-bisphosphatase; PCK, phosphoenolpyruvate carboxykinase. December 2021 \| Volume 9 \| Article 793428 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 3 Metabolism Crosstalk with Epigenetic Modiﬁcations Huo et al. hypoxic conditions, contributing to the Warburg effect (Yang et al., 2014). HIFAL (the anti-sense lncRNA of HIF1α) recruits PHD3 to PKM2 to promote the hydroxylation of its proline residues, and the PKM2/PHD3 complex is then guided by heterogeneous nuclear ribonucleoprotein F (hnRNPF) into the nucleus to enhance the transcriptional activation of HIF1α and promote glycolysis (Zheng et al., 2021). Meanwhile, HIF-1α- stabilizing long noncoding RNA (HISLA) inhibits the interaction between prolyl hydroxylase domain-containing protein 2 (PHD2) and HIF1α to prevent the degradation of HIF1α and promote glycolysis, hindering breast cell apoptosis (Chen et al., 2019). Many genes involved in glucose metabolism are transcriptionally activated by HIF1α (Semenza, 2003) (Figure 2). The reduced transcription of fructose bisphosphates 1 (FBP1), a rate-limiting enzyme in gluconeogenesis, mediates disruptions to gluconeogenesis and increased glycolytic activity, causing tumor progression and poor prognosis (Hirata et al., 2016). Under hypoxic conditions, HIF1a promotes the methylation of FBP1 by upregulating the expression of glycogen-branching enzyme 1 (GBE1) in lung adenocarcinoma (Li L. et al., 2020). The promoter region of fructose-bisphosphatase 2 (FBP2) in gastric cancer is densely methylated to promote glucose metabolism (Li et al., 2013) (Figure 1). Non-Coding Ribonucleic Acids Non Coding Ribonucleic Acids Recent studies have shown that non-coding RNAs regulate the metabolic remodeling of tumors, including sugar metabolism, lipid metabolism, cholesterol metabolism, and amino acid metabolism (Xu et al., 2021c; Sellitto et al., 2021). However, the mechanism by which long non-coding RNAs regulate tumor metabolism is still unclear. Many studies have explored the relationship between metabolism and non-coding RNAs. Urothelial cancer associated 1 (UCA1) has been shown to increase the activation of HK2 by inhibiting miR203 and to regulate glucose metabolism by increasing glucose uptake and lactic acid production, promoting the proliferation and metastasis of esophageal cancer (Liu H.-E. et al., 2020). CircCDKN2B-AS1, the cyclic structure of the long non-coding RNA CDKN2B-AS1, can combine with IMP U3 small nucleolar ribonucleoprotein 3 (IMP3) to stabilize the transcription of HK2 and promote aerobic glycolysis and the malignant phenotype in cervical cancer (Zhang Y. et al., 2020). Prostate cancer associated 3 (PCA3) targets miR- 132–3P and weakens its interaction with SREBP1, leading to lipid metabolism disorders caused by antimony exposure in prostate cancer (Guo et al., 2021) (Figure 1). Deoxyribonucleic Acid and Ribonucleic Acid Methylation g Peroxisome proliferator activated receptor alpha (PPARα), a nuclear receptor that regulates lipid homeostasis, inhibits DNA methyltransferase 1 (DNMT1)-mediated cyclin dependent kinase inhibitor 1A (CDKN1A) and PRMT6-mediated cyclin dependent kinase inhibitor 1b (CDKN1B) to promote colon cancer (Luo et al., 2019). YTH n6-methyladenosine RNA binding protein 2 (YTHDF2) is downstream of epidermal growth factor receptor/ src proto-oncogene, non-receptor tyrosine kinase/extracellular signal-regulated kinase (EGFR/SRC/ERK) and plays an important role in the proliferation and invasion of glioma cells. Studies have shown that YTHDF2 mediates the downregulation of liver X receptor-alpha (LXRA) mRNA through m6A to inﬂuence glioblastoma (GBM) cholesterol homeostasis (Fang et al., 2021). Argininosuccinate synthase 1 (ASS1) in cisplatin-resistant bladder cancer cells is hypermethylated, resulting in greatly downregulated expression, suppressing the apoptotic effects of cisplatin (Yeon et al., 2018). Recent research on microRNAs has led to increasing evidence on the pivotal role of miRNAs in all stages of tumor development. MiR-9-1 targets HK2 to inhibit glycolysis, reduce the formation of lactic acid, and promote metabolic reprogramming in nasopharyngeal carcinoma cells (Xu Q. L. et al., 2021). Meanwhile, miR-542–3p elevates HK2 to induce glycolysis in GBM (Kim et al., 2021). miRNA-1185-2-3p inhibits Golgi phosphoprotein 3 (GOLPH3L) and affects central carbon metabolism regulated by GOLPH3L (Xu et al., 2021b). MiR- 16–5p and 15b-5p coregulate ALDH1A3, which can inhibit the ubiquitination of PKM2 and regulate glycolysis to exert anti- cancer effects (Huang X. et al., 2021). In ovarian cancer, miR- 424–5p inhibits mitochondrial elongation factor 2 (MIEF2), which regulates mitochondrial ﬁssion, inhibits glucose metabolism from oxidative phosphorylation to glycolysis, and inhibits tumor growth (Zhao et al., 2020). LIX1-like protein (LIX1L) promotes miR-21–3p, inhibits FBP1, reduces lactic acid production, and affects sugar metabolism to inhibit tumor growth (Zou et al., 2021) (Figure 1). MiR-15a-5p inhibits acetate uptake and acyl-CoA synthetase short chain family member 2 (ACSS2) and H4 acetylation in the nucleus under hypoxia, inhibiting fatty acid synthesis in lung cancer cells and further suppressing malignancy in lung cancer (Ni et al., 2020). Histone Modiﬁcations Meanwhile, the interaction between LSD1 and snail family transcriptional repressor 2 (SNAIL2, also known as SLUG, an important epigenetic regulator of de novo adipose tissue) mediates the demethylation of H3K9 and stimulates FASN expression and lipogenesis (Liu Y. et al., 2020; Manuel and Haeusler, 2020). Protein arginine methyltransferase 5 (PRMT5) regulates fat cells by promoting the expression of fatty acid synthase (FASN; lipid synthesis gene) by methylating sterol regulatory element binding transcription factor 1 (SREBP1) (Jia et al., 2020). (Rausch et al., 2021). Enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), a key epigenetic enhancer, suppresses gene transcription by promoting H3K27me3 (Zhang T. et al., 2020). Recently, EZH2 was shown to affect tumor cell metabolism, including carbohydrate metabolism, amino acid metabolism, and lipid metabolism. EZH2 regulates the Warburg effect to promote tumorigenesis and cancer development. EZH2-mediated histone H3 lysine 27 trimethylation mediates the downregulation of LINC00261 to promote glycolysis in pancreatic cancer (Zhai et al., 2021). EZH2 is enriched in the WNT promoter region and regulates H3K27me3 to suppress the transcription of WNTs, including WNT-1, 6, 10a, and 10b genes, which are involved in the process of adipogenesis (Wang et al., 2010). In lung cancer, lysine methyltransferase 2D (KMT2D) is highly expressed and regulates the super enhancers H3K4me1 and H3K27ac of period circadian regulator 2 (PER2; circadian inhibitory factor), affecting the expression of PER2 and its downstream glycolytic genes (Alam et al., 2020). As a H3K27me2 reader, PHD Finger Protein 20 Like 1(PHF20L1) plays important roles in promoting the Warburg effect via many glycolysis-related genes (GRGs) in breast cancer (Hou et al., 2020). Lysine-speciﬁc histone demethylase 1 (LSD1) also plays an important role in the metabolic regulation of hepatocellular carcinoma. LSD1 mediates the methylation of H3K4 to inhibit the expression of PPARgamma coactivator 1alpha (PGC1α), thereby, affecting mitochondrial oxidative respiration (Sakamoto et al., 2015). Meanwhile, the interaction between LSD1 and snail family transcriptional repressor 2 (SNAIL2, also known as SLUG, an important epigenetic regulator of de novo adipose tissue) mediates the demethylation of H3K9 and stimulates FASN expression and lipogenesis (Liu Y. et al., 2020; Manuel and Haeusler, 2020). Protein arginine methyltransferase 5 (PRMT5) regulates fat cells by promoting the expression of fatty acid synthase (FASN; lipid synthesis gene) by methylating sterol regulatory element binding transcription factor 1 (SREBP1) (Jia et al., 2020). Histone Modiﬁcations Various PTMs act on histones. Most histone modiﬁcation sites are at the N-terminal end of the nucleosome tail of H3 and H4 histones. Common histone modiﬁcations include methylation, acetylation, ubiquitination, SUMOylation, and phosphorylation (Ahringer and Gasser, 2018). However, in recent years, new histone modiﬁcations have been discovered, including crotonylation, GlcNAcylation, and citrullination (Blakey and Litt, 2015; Zhang M. et al., 2020). Different histone modiﬁcations are associated with different chromatin states—speciﬁcally, the methylation of H3K4 activates transcription, trimethylation at H3K27 is associated with transcription inhibition, acetylation of the histone tail generally promotes transcription, and deacetylation of histones inhibits transcription (Carter and Zhao, 2021). Epigenetic changes control the expression of many metabolic genes, which are important in cancer metabolism (Sun et al., 2021). Increasing evidence shows that histone methylation and metabolic variations in cancer cells are highly correlated HIF1α upregulates the transcription of genes encoding glucose transporters and glycolytic enzymes to regulate tumor glucose metabolism. In recent years, several studies have shown that long non-coding RNAs (lncRNAs) play key roles in regulating the HIF1α pathway (Tan Y. T. et al., 2021). LncRNA-p21 is a hypoxia-reactive lncRNA that can bind HIF1α to inhibit the ubiquitination of HIF1α and then promote glycolysis under December 2021 \| Volume 9 \| Article 793428 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 4 Huo et al. Huo et al. Metabolism Crosstalk with Epigenetic Modiﬁcations FIGURE 2 \| Epigenetic regulation of HIF1A regulates glycolytic enzymes. HIFAL recruits PHD3 to PKM2, and the PKM2/PHD3 complex is then guided by HNRNPF into the nucleus to enhance the transcriptional activation of HIF1α to promote the expression of genes related to glucose metabolism. LncRNA-P21 protects HIF1A stability. PHD3, prolyl hydroxylase domain-containing protein 3; HNRNPF, heterogeneous nuclear ribonucleoprotein F; HIFAL, anti-sense lncRNA of HIF1A; PKM2, pyruvate kinase M2. FIGURE 2 \| Epigenetic regulation of HIF1A regulates glycolytic enzymes. HIFAL recruits PHD3 to PKM2, and the PKM2/PHD3 complex is then guided by HNRNPF into the nucleus to enhance the transcriptional activation of HIF1α to promote the expression of genes related to glucose metabolism. LncRNA-P21 protects HIF1A stability. PHD3, prolyl hydroxylase domain-containing protein 3; HNRNPF, heterogeneous nuclear ribonucleoprotein F; HIFAL, anti-sense lncRNA of HIF1A; PKM2, pyruvate kinase M2. Histone Modiﬁcations Acetylation and deacetylation can also affect the transcriptional output of metabolic genes in various working models. EZH2-deﬁcient cells show increased H3K27 acetylation, indicating that acetylation and trimethylation of H3K27 have a repulsive effect in regulating WNT expression (Wang et al., 2010). In contrast, EZH2 can also inhibit the expression of apolipoprotein E (APOE) in adipocytes to promote lipoprotein-dependent lipid accumulation (Yiew et al., 2019). Histone deacetylase 11 (HDAC11) inhibits histone acetylation of serine/threonine kinase 11(STK11) promoter to inhibit its transcription, thereby, promoting the glycolysis pathway and leading to tumor stemness (Bi et al., 2021). Sirtuin 6 (SIRT6) blocks the expression of hypoxia inducible factor 1 subunit alpha (HIF1α) to regulate glucose homeostasis by regulating the deacetylation of H3K9 (Zhong et al., 2010). Sirtuin 4 (SIRT4) inhibits the expression of sirtuin 1 (SIRT1) by inhibiting glutamine metabolism, and SIRT1 promotes the deacetylation December 2021 \| Volume 9 \| Article 793428 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 5 Huo et al. Metabolism Crosstalk with Epigenetic Modiﬁcations of H4K16 to regulate the stemness of breast cancer cells (Du et al., 2020). Ubiquitously transcribed TPR gene on the X chromosome (UTX) is enriched in the uncoupling protein 1 (UCP1) and PPARG coactivator 1 Alpha (PGC1α) promoters and mediates the demethylation of H3K27me3, which interacts with the histone acetyltransferase (HAT) protein CREB binding protein (CBP), leading to the acetylation of H3K27 (H3K27ac). UTX mediates H3K27me3 demethylation and H3K27ac and converts the target gene from a transcriptionally repressed state to a transcriptionally activated state to positively regulate the thermogenesis of brown adipocytes (Zha et al., 2015). Under hypoxia, acetate increases the acetylation of H3K9, H3K27 and H3K56 in the promoter regions of ACACA and FASN to activate de novo lipid synthesis (Gao et al., 2016). E1A binding protein P300/CREB-binding protein (P300/CBP) mediates histone acetylation of H3K18 and H3K27 in hepatocellular carcinoma to regulate the expression of glycolysis-related metabolic enzymes. In addition, the p300 inhibitor B029-2 effectively blocks the metabolic reprogramming of hepatocellular carcinoma (Cai et al., 2021). Histone lactation is also an emerging epigenetic modiﬁcation of histone lysine residues (Liberti and Locasale, 2020). It depends on tumor protein P53–E1A-binding protein P300 (p53–P300) to promote gene transcription and promote M2-like features in the late phase of M1 macrophage division (Zhang et al., 2019). EPIGENETIC REGULATION BY METABOLITES AND METABOLIC ENZYMES Metabolism is an umbrella term for a variety of different orderly chemical reactions that occur in organisms to maintain life and serve cellular demands, including energy generation and protein biosynthesis, thereby, maintaining biological structure and functions and responding to the environment. Metabolism can be regarded as a process of continuous material and energy exchange. Intermediates produced by metabolism often participate in epigenetic regulation by serving as substrates or cofactors for epigenome-modifying enzymes. During environmental disturbances, cellular metabolism transmits regulatory signals to the genome via epigenetic modiﬁcations (Reid et al., 2017; Cavalli and Heard, 2019) (Table 1). Although many studies have focused on the modiﬁcation of histones, many reports have shown that these histone modiﬁcation enzymes regulate the modiﬁcation of non-histone proteins, such as the sirtuin (SIRT) family, the deacetylase histone deacetylase (HDAC) family, the protein arginine n-methyltransferase (PRMT) family (Eom and Kook, 2014; Biggar and Li, 2015; Rodríguez-Paredes and Lyko, 2019; Navas and Carnero, 2021). These proteins, by regulating metabolic enzymes, play an important role in the occurrence and development of tumors. The SIRT family is an NAD+-dependent type III deacetylase. Many members of this family have recently been shown to regulate tumor metabolism through acetylation (Navas and Carnero, 2021). Sirtuin 2 (SIRT2) mediates the deacetylation of isocitrate dehydrogenase (NADP (+)) 1 (IDH1), thereby, promoting IDH1 activity and the production of α-ketoglutarate (α-KG), inhibiting liver metastasis of colon cancer and improving prognosis (Wang et al., 2020). SIRT3 inhibits the acetylation of IDH2 at K413 and promotes isocitrate dehydrogenase (NADP (+)) 2 (IDH2) activity by promoting IDH2 dimerization to inhibit glycolysis (Zou et al., 2017). Sirtuin 7 (SIRT7) in the liver increases fatty acid uptake and triglyceride synthesis and storage. SIRT7 inhibits the degradation of testicular nuclear receptor 4 (TR4) via the ddb1-and cul4-associated factor 1/damage speciﬁc DNA binding protein 1/cullin 4b (DCAF1/DDB1/CUL4B) complex (Yoshizawa et al., 2014) (Figure 1). Histone Modiﬁcations enzyme PEPCK1 in gluconeogenesis to increase gluconeogenesis and inhibit the Warburg effect in hepatocellular cancer (HCC) to prevent the development of this disease. However, PEPCK1 after SUMOylation becomes unstable and the interaction weakens, inhibiting gluconeogenesis and promoting glycolysis (Bian et al., 2017). In addition, metastasis associated 1 (MTA1) interacts with the transcription factor myc proto-oncogene protein (MYC) to regulate its transcription on the lactate dehydrogenase A (LDHA; an enzyme that catalyzes the production of lactic acid from pyruvate) via its promoter (Guddeti et al., 2019) and MTA1 upregulates HIF1α under hypoxic conditions by stimulating HIFα transcription (Huang W. et al., 2021). Six homeobox 1 (SIX1) is a key transcription factor that regulates glycolysis- related enzymes (GLUT1 and HK2). SIX1 increases the level of O-GlcNAcylation, and its O-GlcNAcylation enhances the stability of SIX1, coordinates glucose metabolism, and promotes the proliferation of HCC (Chu et al., 2020) (Figure 1). Metabolites Serve as Substrates for Epigenetic Modiﬁcations As a carrier of epigenetic information, chromatin plays an important role in regulating gene silencing and activation and permits stable inheritance through reversible DNA and histone modiﬁcation to maintain the biological functions of proteins (Allis and Jenuwein, 2016). In chromatin, DNA and histones are the substrates that are mainly modiﬁed. The most widely studied of all modiﬁcations are methylation-demethylation, acetylation-deacetylation, phosphorylation, ubiquitination, and ADP-ribosylation. As discussed earlier, changes in the metabolite pool caused by cancer metabolic reprogramming also affect the state of epigenetic modiﬁcations. Consequently, metabolic alterations in cancer have an effect on malignant phenotypes of cancer cells by manipulating epigenetic modiﬁcations (Figure3). The acetylation of K394 (regulated by the deacetylase P300/ CBP-associated factor (PCAF) and histone deacetylase 3 (HDAC3) of enolase 2 (ENO2), a key enzyme of glycolysis) inhibits ENO2 activity and glycolysis (Zheng et al., 2020). Gluconeogenesis is an important metabolic process in liver cell homeostasis; however, it is signiﬁcantly reduced in liver cancer. The tumor suppressor Nur77 interacts with the rate-limiting Acetyl-CoA Acetyl-CoA is a core metabolite and a substrate for further metabolism and acetylation. A variety of metabolic processes in cells participate in the formation of the acetyl-CoA pool. Glucose metabolism intermediates, such as acetate, citrate, and pyruvate, under the catalytic action of acetyl-CoA synthetase short-chain family member (ACSS), ATP citrate lyase (ACLY), and the pyruvate dehydrogenase complex (PDC), respectively, produce acetyl-CoA. In addition to glucose metabolism, the metabolism of amino acids and fatty acids also generate acetyl-CoA (Zaidi et al., 2012; Nagaraj et al., 2017; Sivanand et al., 2018). Acetylation occurs widely in histones and some non- histone proteins and plays a regulatory role in tumorigenesis and development. Histone acetylation is regulated by the intracellular acetyl- CoA concentration. Acetyl-CoA serves as an acetyl group donor in acetylation reactions. This process is catalyzed by HATs (Lee et al., 2014). Histone acetylation is determined by the ratio of acetyl-CoA/coenzyme A and is generally deregulated in cancer. In human gliomas and prostate tumors, Histone acetylation marks are signiﬁcantly regulated by pAkt (Ser473) levels, which modulates the metabolic enzyme ATP-citrate lyase, a key determinant of acetyl-CoA metabolism (Lee et al., 2014). Tumor cell surface GRP78 of activated α2-macroglobulin signals regulate tumor cell proliferation by inducing and activating ACLY and ACSS1 expression to generate acetyl- CoA (Gopal and Pizzo, 2017). Acetyl-CoA also serves as nonhistone acetylation modiﬁcation substrate to produce effects on the development of tumors. PHD ﬁnger-like domain-containing protein 5A (PHF5A) is acetylated at lysine 29 by p300 to promote cancer cell capacity for stress resistance and consequently, contributes to colon carcinogenesis. PHF5A K29 hyperacetylation induces the alternative splicing of KDM3A mRNA, which enhances its stability and promotes its expression (Wang H.-Y. et al., 2019; Wang Z. et al., 2019). Under low oxygen states or hypoxia, lysine acetyltransferase and CBP rely on acetate-dependent acetyl CoA synthetase 2 (ACSS2) to acetylate HIF-2, which contributes to cancer cell proliferation, migration, and invasion (Chen et al., 2015). α-Ketoglutarate, Succinyl-CoA, Fumarate Intermediates of the TCA cycle, α-KG, succinyl-CoA, fumarate, and citrate, regulate epigenetic modiﬁcations through their concentration in the metabolite pool. α-KG is a key cofactor for jumonji-domain-containing histone demethylases (JHDMs), which synergistically catalyze histone demethylation (Klose et al., 2006). Glucose Metabolism Glycolysis involves the breakdown of glucose or glycogen into pyruvate and nicotinamide adenine dinucleotide (NADH), December 2021 \| Volume 9 \| Article 793428 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 6 Metabolism Crosstalk with Epigenetic Modiﬁcations Huo et al. (Houtkooper et al., 2012; Moreira et al., 2016; Katsyuba et al., 2020). Under genotoxic stress or nutrient restriction conditions, upregulation of the NAD+ biosynthetic enzyme NAMPT protects cells against death via the deacetylation activity of SIRT3 and SIRT4 (Yang et al., 2007). In cancer, NAD+ is intended to reprogram metabolism to enable tumor progression, development, and survival (Hanahan and Weinberg, 2011). As an NAD+-consuming enzyme, SIRT1 acts as a tumor promoter and is upregulated in several human cancers. In HCC, the inhibition of NAD+ metabolism causes DNA damage in the early stages of tumorigenesis because of the inactivation of NAD+-consuming enzymes such as SIRT1. Genomic stability can be improved by NAD+ supplementation to prevent tumorigenesis (Tummala et al., 2014). Apart from deacetylase activity, sirtuins have multiple NAD+-dependent catalytic functions, such as desuccinylase, demalonylase, demyristoylase, depalmitoylase, and/or mono-ADP-ribosyltransferase activities. In ovarian cancers, nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2), which mediates the synthesis of NAD+ is highly upregulated. Knockdown of NMNAT2 signiﬁcantly decreases NAD+ in cytoplasm but increases NAD+ in the nucleus and consequently supports the catalytic activity of the mono (ADP-ribosyl) transferase (MART) PARP- 16, resulting in ribosome mono-ADP-ribosylation (MARylation). There is a signiﬁcant positive correlation between NMNAT-2 and MARylation levels in the samples of ovarian cancer patients, and a high level of MARylation will lead to poor prognosis with respect to progression free survival (Challa et al., 2021). In addition, mitochondrial dysfunction also affects the level of acetylation. Mitochondrial dysfunction causes a decreased NAD+/NADH ratio and increased reactive oxygen species (ROS), resulting in senescence (Wiley and Campisi, 2016). STAT3 deﬁciency induces senescence, mitochondrial dysfunction, and a lower NAD+/NADH ratio (Igelmann et al., 2021). The dynamic changes of NAD+/NADH ratio also has an effect on nonhistone deacetylation of lysine residues of sirtuins. In breast cancer, NAMPT causes p53 deacetylation and SIRT1 activation by increasing the NAD+ pool (Behrouzfar et al., 2017). accompanied by the production of a small amount of ATP. Under anoxic conditions, pyruvate can accept hydrogen from triose phosphate and gets reduced to lactate under the catalytic action of lactate dehydrogenase. Under aerobic conditions, pyruvate can be further oxidized and decomposed to form acetyl-CoA and enter the TCA cycle. Glucose Metabolism Intermediates and metabolites, such as acetyl- CoA, α-KG, succinate, fumarate and citrate, produced from glucose metabolism, have diverse effects on epigenetic modiﬁcations. Acetyl-CoA In Acute Myeloid Leukemia (AML) stem cells, the α-KG level is restricted by the branched-chain amino acid transaminase 1 (BCAT1), which is overexpressed in leukemia stem cells and transfers α-amino groups from BCAAs to α-KG, resulting in α-KG instability to maintain leukemia stem-cell function (Raffel et al., 2017). The α-KG dehydrogenase complex (α-KGDC) is the hub enzyme of various metabolic pathways involved in mitochondrial function and is a modulator of α-KG (Vatrinet et al., 2017). Succinyl-CoA is a substrate for succinylation (Zhang et al., 2011). Succinylation, as a post-translational modiﬁcation of proteins, can convert the side chain of cationic lysine residues into anionic ones and then affect Nicotinamide Adenine Dinucleotide (NAD+) The metabolite NAD+ serves as a catalytic cofactor for sirtuins, class III HDACs, and is essential for the deacetylation of lysine residues of sirtuins. NAD+ homeostasis is related to many diseases, including neurodegeneration, diabetes, and cancer. The ratio of NAD+/NADH regulates sirtuins activity, which is found to be higher in cancer cells than in noncancerous cells December 2021 \| Volume 9 \| Article 793428 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 7 Metabolism Crosstalk with Epigenetic Modiﬁcations Huo et al. TABLE 1 \| Regulation of epigenetic modiﬁcations by metabolites and metabolic enzymes. Metabolites or metabolic enzymes Epigenetic modiﬁcations Functions in cancer References Metabolites Acetyl-CoA DNA, RNA and Protein acetylation Plays a regulatory role in tumorigenesis and development Gopal and Pizzo (2017) NAD+ Protein dacetylation, desuccinylase, demalonylase Enables tumor progression, development and survival Hanahan and Weinberg (2011) Succinyl CoA Protein succinylation Promotes tumor growth and progression Li et al. (2020b) 2-hydroxyglutarate Protein hypermethylation Contributes to poor prognosis Cavalli and Heard (2019) Lactate Protein lactylation Contributes to tumorigenesis and indicates poor prognosis Yu et al. (2021) Palmitic acids Protein palmitoylation Participates in tumorigenesis and cell survival Yao et al. (2019) Farnesyl group Protein farnesylation Contributes to cancer cell growth Tamanoi et al. (2001) Geranylgeranyl group Protein geranylgeranylation Essential for cell proliferation and migration Dou et al. (2015), Mi et al. (2015); Lin and Yang (2016) β-hydroxybutyrate Protein β-hydroxybutyrylation Promotes tumor growth Liu et al. (2019) Glutamine Produce a variety of metabolites Replenishes the TCA cycle for biosynthesis to meet the needs of proliferation DeBerardinis et al. (2008), Kuhn et al. (2010) SAM DNA, RNA and Protein methylation Enhances subcutaneous tumor growth Dann et al. (2015) UDP-GlcNAc O-GlcNAcylation Promotes tumorigenesis Peng et al. (2017) Metabolic enzymes ACLY and ACSS1 Acetylation Contributes to cancer cell proliferation, migration and invasion Gopal and Pizzo (2017) α-KGDC Protein succinylation Promotes tumor growth and progression Wang et al. (2017) SDH Hypersuccinylation; Hypermethylation SDH loss causes drug resistance and promotes angiogenesis Isaacs et al. (2005); Selak et al. (2005); MacKenzie et al. (2007); Smestad et al. (2018) FH Hypersuccinylation; Hypermethylation FH deﬁciency results in angiogenesis and EMT Isaacs et al. (2005); Selak et al. (2005); MacKenzie et al. (2007); Cancer Genome Atlas Research et al. (2016); Sciacovelli et al. (2016) IDH1 and IDH2 Protein hypermethylation Contributes to poor prognosis Figueroa et al. (2010); Lu et al. References Contributes to poor prognosis Promotes tumorigenesis Contributes to poor prognosis Promotes tumorigenesis have been identiﬁed as driver mutations in cancer and mediators of epigenetic reprogramming (Pollard et al., 2003). In an SDH- loss cell model, the accumulation of succinate and succinyl-CoA results in global lysine hypersuccinylation, which modulates genome-wide transcription and hinders DNA repair ability and drug resistance (Smestad et al., 2018). In paraganglioma, SDH deﬁciency causes succinate accumulation and establishes a hypermethylation phenotype resulting from epigenetic silencing through the inhibition of 2-OG-dependent histone and DNA demethylases. In addition to SDH, inactivating FH mutations can also lead to this phenomenon (Letouze et al., 2013). The hypermethylation phenotype caused by SDH-inactivating mutations also exists in gastrointestinal stromal tumors (Killian et al., 2013). In renal cancer, the inactivating mutations of SDH or FH deﬁciency results in the subsequent accumulation of succinate or fumarate, respectively, thus, the inhibition of hypoxia-inducible factor (HIF) prolyl hydroxylases (HPH), which protect the stabilization of HIF and promote angiogenesis by increasing α-KG intake can alleviate this situation (Isaacs et al., 2005; Selak et al., 2005; MacKenzie et al., 2007). Moreover, germline mutations of FH cause an aggressive and metastatic form of type 2 papillary renal-cell carcinoma, which is linked to a widespread DNA the structure and function of proteins (Smestad et al., 2018). The increase of succinylation has different functions in the progression of a variety of tumors. α-KGDC binds to the promoter regions of lysine acetyltransferase 2A (KAT2A, also known as GCN5), and KAT2A also acts as a succinyltransferase and succinylates histone H3 on lysine 79, which facilitates histone succinylation and tumor cell proliferation (Wang et al., 2017). Lysine-222 succinylation is increased in gastric cancer, and LDHA lysine-222 succinylation, catalyzed by CPT1A via the stabilization of LDHA, promotes GC invasion and proliferation (Li X. et al., 2020). In pancreatic ductal adenocarcinoma, succinyl-CoA synthetase ADP-forming subunit β (SUCLA2) phosphorylation at S79 leads to dissociation from kidney-type glutaminase (GLS). Thus, the concentration of succinyl coenzyme A is increased and upregulates the succinylation of GLS at site K311. GLS K311 succinylation enhances the oligomerization, activity, and glutaminolysis to improve the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione, thereby, counteracting oxidative stress and promoting tumor growth (Tong et al., 2021). Nicotinamide Adenine Dinucleotide (NAD+) (2012) OGT O-GlcNAcylation Promotes tumorigenesis Slawson and Hart (2011); de Queiroz et al. (2014); Yang and Qian (2017) References Loss-of-function mutations in the TCA cycle enzymes fumarate hydratase (FH) and succinate dehydrogenase (SDH) December 2021 \| Volume 9 \| Article 793428 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 8 Metabolism Crosstalk with Epigenetic Modiﬁcations Huo et al. et al., 2014). In response to hypoxia, L-2HG is selectively produced to increase histone methylation levels, especially histone 3 lysine 9 (H3K9me3) (Intlekofer et al., 2015). hypermethylation pattern (Cancer Genome Atlas Research et al., 2016). Loss of FH triggers epigenetic suppression of miR-200 and epithelial-to-mesenchymal transition by inhibiting Tet-mediated demethylation (Sciacovelli et al., 2016). 2-Hydroxyglutarate Lactate, a metabolic by-product of pyruvate metabolism in cancer cells, is a substrate for histone lactylation. The lactylation of histone lysine residues (Kla) driven by lactate directly stimulates gene transcription from chromatin, linking metabolism and gene regulation. The production of intracellular lactic acid is the main determinant of lactylation modiﬁcation. Therefore, the balance between glycolysis and mitochondrial metabolism can regulate lactylation modiﬁcation (Izzo and Wellen, 2019; Zhang et al., 2019; Liberti and Locasale, 2020). Histone lactylation is elevated in ocular melanoma and is indicative of poor prognosis. Histone lactylation also contributes to tumorigenesis by facilitating YTHDF2 expression, which acts as an m6A reader to recognize and mediate the degradation of m6A-modiﬁed Period Circadian Regulator 1 PER1 and P53 mRNAs to accelerate tumorigenesis (Yu et al., 2021). Gain-of-function isocitrate dehydrogenase (IDH) mutations produce an oncometabolite, 2-hydroxyglutarate (2-HG), speciﬁcally the D enantiomer (D-2HG), and affect clinical therapy and prognosis (Dang et al., 2010; Cancer Genome Atlas Research et al., 2015). IDH1 and IDH2 mutations result in global hypermethylation and speciﬁc hypermethylation in AML by inhibiting demethylases such as KDM4C (Figueroa et al., 2010; Lu et al., 2012). 2-HG-mediated inhibition of the H3K9 demethylase KDM4C is induced during adipocyte differentiation (Lu et al., 2012). In addition to affecting the function of methyltransferase, 2-HG might also perturb the overall architecture of the genome and contribute to cancer and prognosis by affecting methyltransferase activity (Cavalli and Heard, 2019). Interestingly, kidney tumors present an accumulation of the L enantiomer of 2HG (L-2HG), because of the low expression of L-2HG dehydrogenase (L2HGDH). Similar to the function of D-2HG, L-2HG inhibits ten-eleven translocation (TET) as well, which converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). L-2HG is signiﬁcantly increased in tumors compared with their expression in normal kidney, and L2HGDH expression inhibits proliferation and colony formation in RCC cells (Shim Palmitic Acid Acetyl CoA produced by lipolysis can not only enter the TCA cycle for oxidation but also result in the synthesis of ketone bodies in the liver. β-Hydroxybutyrate (β-HB) is a ketone body produced by fatty acid hydrolysis. Elevated β-HB levels lead to a new type of histone mar, lysine β-hydroxybutyrylation (Kbhb) (Xie et al., 2016). β-HB is an endogenous and speciﬁc inhibitor of class I HDACs (Shimazu et al., 2013). In HCT116 cells, β-HB-mediated p53 Kbhb at K120, K319, and K370 sites results in lower levels of p53 acetylation and consequently, decreased activity of p53, leading to weakened tumor-suppressive function (Liu et al., 2019). MTA2 can transcriptionally regulate BDH1-mediated histone β-HB modiﬁcation through the R-loop structure in synergy with HDAC2/CHD4 and promote the proliferation of hepatoma stem cells (Zhang et al., 2021). Palmitoylation is a type of fatty acylation in which long-chain fatty acids (usually 16-carbon-long palmitic acid) are covalently modiﬁed to protein cysteine residues by thioester bonds. Palmitoylation plays a vital role in many biological processes (Zhang et al., 2018). In several human cancers, signal transducer and activator of transcription 3 (STAT3) is palmitoylated by zinc ﬁnger DHHC- Type palmitoyltransferase 19 (ZDHHC19), a palmitoyl acyltransferase, at the SRC homology 2 (SH2) domain, which promotes its dimerization and transcriptional activation. Under cytokine stimulation, the association between ZDHHC19 and STAT3 is promoted, which directly activates STAT3 by enhancing its palmitoylation with fatty acids (Niu et al., 2019). Palmitoylation mediated by the palmitoyltransferase zinc ﬁnger DHHC-Type palmitoyltransferase 3 (ZDHHC3) in the cytoplasmic domain of PD-L1 blocks its ubiquitination to stabilize PD-L1, thereby, suppressing PD-L1 degradation by lysosomes and PD-L1-mediated immune evasion in cancer (Yao et al., 2019). In addition to ZDHHC3, ZDHHC9 has been identiﬁed as a palmitoyltransferase for PD-L1 in breast cancer (Yang et al., 2019). In p53-mutant glioma, zinc ﬁnger DHHC-Type palmitoyltransferase 5 (ZDHHC5) is transcriptionally upregulated and mediates EZH2 palmitoylation, which affects methyltransferase activity of EZH2 and causing trimethylation of histone 3 at lysine 27 (H3K27me3 level) decreased, and then contributes to malignancy and tumor progression (Chen X. et al., 2017). MC1R palmitoylation mediated by the protein-acyl transferase zinc ﬁnger DHHC-Type palmitoyltransferase (ZDHHC13) activates MC1R signaling, which triggers increased pigmentation, ultraviolet-B-induced G1-like cell cycle arrest, and the control of senescence and melanomagenesis. Thus, MC1R palmitoylation protects against melanomagenesis (Chen S. et al., 2017). Lipid Metabolism Lipid signal transduction, especially the phosphatidylinositol 3 kinase (PI3K) pathway, is one of the most common signal transduction systems in cancer cells. Lipophilic groups are widespread modiﬁers and the following ﬁve types of lipids can be covalently attached to proteins: fatty acids, isoprenoids, sterols, phospholipids, and glycosylphosphatidylinositol anchors (Resh, 2013). In addition to FIGURE 3 \| Chromatin modiﬁcation by metabolites. Metabolites produced from cellular metabolic pathways are used as substrates for DNA, RNA, and chromatin modiﬁcation. Moreover, the intracellular pools of metabolites can modulate the activity of chromatin-modifying enzymes. TCA, tricarboxylic acid; αKG, α-ketoglutarate; SucCoA, succinyl-CoA; PAR, poly (ADP–ribose); GlcNAc, β-N-acetylglucosamine; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine. FIGURE 3 \| Chromatin modiﬁcation by metabolites. Metabolites produced from cellular metabolic pathways are used as substrates for DNA, RNA, and chromatin modiﬁcation. Moreover, the intracellular pools of metabolites can modulate the activity of chromatin-modifying enzymes. TCA, tricarboxylic acid; αKG, α-ketoglutarate; SucCoA, succinyl-CoA; PAR, poly (ADP–ribose); GlcNAc, β-N-acetylglucosamine; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine. December 2021 \| Volume 9 \| Article 793428 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 9 Huo et al. Metabolism Crosstalk with Epigenetic Modiﬁcations providing a substrate for rare acylation, fatty acid metabolism also produces acetyl-COA, as a substrate for energy metabolism and epigenetic modiﬁcations (Edmunds et al., 2014). atorvastatin and the geranylgeranyltransferase I inhibitor GGTI- 298 can impair cell proliferation and migration (Wei et al., 2020). Farnesyl Group SAM, a major methyl donor, is synthesized from the methionine cycle and serves as a substrate for methylation to maintain the epigenetic status, including histone methylation catalyzed by histone methyltransferases, DNA methylation mediated by DNA methyltransferase (DNMT), RNA methylation (Teperino et al., 2010; Varier and Timmers, 2011; Anderson et al., 2012; Locasale, 2013), and PTMs such as the methylation of lysine and arginine residues of nonhistone proteins (Chen et al., 2011; Yang and Bedford, 2013). Amino-acid transporters are often required for tumor cell import of essential amino acids. The amino acid transporters LAT1 and LAT4 are signiﬁcantly upregulated in tumors to facilitate the uptake of methionine for cell proliferation and differentiation in cancer cells (Haase et al., 2007). In lung cancer, cells that highly express LAT1 show increased abundance Protein farnesylation is a lipid PTM essential for the cancer- causing activity of proteins, such as gtpase Ras (Sebti, 2005). Farnesyltransferase inhibitors inhibit cancer cell growth and regulate cell cycle changes (Tamanoi et al., 2001). Glutamine Glutamine is the most abundant amino acid in the intracellular metabolite pool. After entering a cell, in the mitochondria, glutamine is transformed into glutamate, the precursor of the TCA cycle intermediate α-KG. Cells rely on glutamine to replenish the TCA cycle. Anaplerosis allows cancer cells to use the TCA cycle for biosynthesis to meet the needs of proliferation, and glutamine as an indispensable nutrient therein (DeBerardinis et al., 2008; Kuhn et al., 2010). Amino Acid and One-Carbon Metabolism Amino Acid and One-Carbon Metabolism One-carbon metabolism plays a crucial role in integrating metabolites from amino acids, glucose, and vitamins and participating in a variety of biosynthetic processes, including the biosynthesis of lipids, nucleotides, and proteins. Hyperactivation of the one-carbon metabolism pathway drives oncogenesis and forges links between cellular epigenetic status and metabolism (Locasale, 2013). Serine metabolism is also an important determinant of S-adenosylmethionine (SAM) levels. Serine is a single carbon donor of the folate cycle and is used for methionine regeneration and SAM synthesis (Locasale, 2013). Palmitic Acid The c-Met β-chain is palmitoylated at the cysteine site, which enhances its stability and release from the Golgi for transport to the plasma membrane, making it a novel therapeutic target for c-Met-driven cancers (Coleman et al., 2016). Palmitoyltransferases are upregulated in GBM and induce marked palmitoylation of proteins that participate in cell survival control and cell cycle regulation in GBM (Chen et al., 2020). Butyrate y Butyrate is a microbiota-derived SCFA that has been conﬁrmed to be an inhibitor of HDAC in vitro and in vivo. Exposure to butyrate causes the accumulation of acetylated histones (Candido et al., 1978; Wu S.-e. et al., 2020). In intestinal epithelia, histone H3K18 results in abundant crotonylation modiﬁcation, especially in small intestine crypts and the colon; the class I histone deacetylases HDAC1, HDAC2, and HDAC3 can catalyze histone decrotonylation. As an HDAC inhibitor, gut microbiota-derived butyrate affects histone decrotonylation and is linked to gene regulation (Fellows et al., 2018). Clostridial clusters, Anaerostipes, and Eubacterium cause histone acylation modiﬁcation through butyrate, thus, improving intestinal development and the immune balance (Wu J. et al., 2020). Butyrate increases histone methylation in the promoter region of NF-κB1, downregulating its expression (Liu et al., 2016; Yang and Yu, 2018). Geranylgeranyl Group GGylation is an essential modiﬁcation affecting cell survival in many types of cancer. In breast cancer, the Hippo pathway mediates GGylation-dependent cell proliferation and migration (Dou et al., 2015; Mi et al., 2015; Lin and Yang, 2016). In gastric cancer, GGylation promotes proliferation, migration, and invasion of gastric cancer cells through the YAP signaling pathway. Inhibition of GGylation by the mevalonate pathway inhibitor December 2021 \| Volume 9 \| Article 793428 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 10 Metabolism Crosstalk with Epigenetic Modiﬁcations Huo et al. of SAM, improving the activity of the histone methyltransferase EZH2 and consequently, increasing H3K27me3 levels to enhance subcutaneous tumor growth (Dann et al., 2015). The levels of SAM are controlled by multiple metabolic pathways and intracellular metabolism, as well as environmental inputs, such as nutrient availability, which also elevate SAM concentrations. to the risk, development, and progression of cancer. Metabolites produced by gut microbiota inﬂuence host metabolism via the modulation of metabolites, including the lipopolysaccharide endotoxin, bile acids, trimethylamine N-oxide, and short-chain fatty acids (SCFAs) (Schroeder and Backhed, 2016). Microbiota- derived metabolites represent stimuli that regulate epigenetic modifying enzymes and are involved in intestinal inﬂammation and carcinogenesis (Figure 4). Bile Acids l d Bile acids are produced in the liver and metabolized by enzymes produced by intestinal bacteria. Intestinal bacteria are very important for maintaining a healthy intestinal microbiota, balancing lipid and glucose metabolism, insulin sensitivity, and natural immunity. Bile acids have a wide range of gene- mediating effects, including bile acid metabolism, glucose and lipid metabolism, energy expenditure, intestinal motility and bacterial growth, inﬂammation, liver regeneration, and hepatocarcinogenesis (de Aguiar Vallim et al., 2013; Wahlstrom et al., 2016). The epigenetic regulation of cofactors senses changes in bile acid metabolism, regulates PTMs of histones, and causes chromatin remodeling to regulate gene transcription and maintain the balance of bile acids. Bile acids activate Farnesoid X receptor (FXR) and change the interaction between FXR and transcriptional cofactors, resulting in altered PTMs of FXR and histones to effectively modulate the expression of target genes (Kemper, 2011). An abnormal concentration of bile acids is believed to promote colorectal cancer (Fearon and Vogelstein, 1990). Uridine Diphosphate N-Acetylglucoseamine (UDP-GlcNAc) ( ) The hexosamine biosynthetic pathway produces nucleotide sugars that can be used as substrates for glycosylation. UDP-GlcNAc is a metabolite of the hexosamine biosynthetic pathway, which integrates glucose, amino acid, fatty acid, and nucleotide metabolism. O-GlcNAc transferase (OGT) adds O-GlcNAc groups from the donor substrate UDP- GlcNAc to proteins for O-GlcNAcylation (Yang and Qian, 2017; Li et al., 2018). The methylcytosine dioxygenases TET1, TET2, and TET3 can also become O-GlcNAcylated by OGT through protein–protein interactions (Hardiville and Hart, 2016; Yang and Qian, 2017). Previous studies have shown that O-GlcNAc glycosylation is abnormally upregulated in many tumors and plays an essential role in the proliferation of malignant cancer cells (Slawson and Hart, 2011; de Queiroz et al., 2014; Yang and Qian, 2017). Phosphoglycerate kinase 1 (PGK1) O-GlcNAcylation levels are signiﬁcantly upregulated in colorectal cancer; O-GlcNAcylation activates PGK1 activity to upregulate the TCA cycle and results in lactate production. The O-GlcNAcylation of PGK1 at T255 increases colon cancer cell proliferation. Furthermore, PGK1 T255 O-GlcNAcylation contributes to tumorigenesis by promoting glycolysis and upregulating the TCA cycle (Nie et al., 2020). Yes- associated protein (YAP) is O-GlcNAcylated at serine 109 by OGT, and the O-GlcNAcylated YAP disrupts the interaction with the upstream kinase large tumor suppressor kinases (LATS) 1. Consequently, O-GlcNAcylation activates the transcriptional activity of YAP by preventing the phosphorylation mediated by LATS1. Thus, glucose-induced YAP O-GlcNAcylation and activation promote tumorigenesis (Peng et al., 2017). OGT O-GlcNAcylates SRPK2 at a nuclear localization signal and triggers SRPK2 binding to importin α, which results in the import of SRPK2 into the nucleus and the phosphorylation of serine/arginine-rich proteins, promoting the splicing of lipogenic pre-mRNAs. O-GlcNAc promotes tumor growth by enhancing the intracellular localization of SRPK2 and regulating de novo lipid synthesis in tumor cells at the post-transcriptional level (Tan W. et al., 2021). Phytate h d Phytate digestion and inositol phosphates produced by phytate metabolism induce HDAC activity in intestinal epithelial cells. Dietary phytate metabolism mediated by microbiota-dependent mechanisms improves recovery from intestinal damage. Moreover, microbiota-derived phytate digestion is signiﬁcantly decreased in the intestinal contents of ulcerative colitis patients relative to that in inﬂammatory bowel disease patients. In addition, phytate-treated mice exhibit improved survival The biochemical reaction networks of metabolism can be manipulated by several factors including diet, nutrition, gut microbiota, and chemical exposure. These environmental factors regulate chromatin methylation and acetylation by modifying the intracellular pool of metabolites. There is increasing evidence that the gut microbiome is closely related December 2021 \| Volume 9 \| Article 793428 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 11 Huo et al. Huo et al. Metabolism Crosstalk with Epigenetic Modiﬁcations FIGURE 4 \| Metabolites produced by the intestinal microbiota inﬂuence host metabolism by modulating the metabolite pool. The gut microbiome regulates histone methylation and acetylation by modulating the intracellular pools of metabolites, such as SAM and acetyl-CoA. Metabolites from the gut microbiome, like phytate and butyrate, also cause changes in histone acetylation by affecting the enzyme activity of HDAC. Pyruvate can either be catabolized into succinate, lactate, or acetyl-CoA. SCFAs can provide acyl-CoA as a donor for histone acylation, while also directly inhibiting the activity of HDACs. FIGURE 4 \| Metabolites produced by the intestinal microbiota inﬂuence host metabolism by modulating the metabolite pool. The gut microbiome regulates histone methylation and acetylation by modulating the intracellular pools of metabolites, such as SAM and acetyl-CoA. Metabolites from the gut microbiome, like phytate and butyrate, also cause changes in histone acetylation by affecting the enzyme activity of HDAC. Pyruvate can either be catabolized into succinate, lactate, or acetyl-CoA. SCFAs can provide acyl-CoA as a donor for histone acylation, while also directly inhibiting the activity of HDACs. compared to vehicle-treated mice with dextran sodium sulfate- induced colitis (Wu S.-e. et al., 2020). According to a recent report, enterotoxigenic Bacteroides fragilis stimulation promotes PHF5A-mediated RNA alternative splicing of KAT2A via the downregulation of miR- 149–3p, promoting intestinal inﬂammation and malignancy. This process depends on METTL14-mediated m6A methylation (Cao et al., 2021). The human gut microbiota also catabolizes the conversion of pyruvate into succinate, lactate, or acetyl-CoA to further generate energy (Oliphant and Allen-Vercoe, 2019); this process is accompanied by changes in the metabolite pool. Phytate h d Dietary ﬁber, including non-starch polysaccharides, resistant starch, and oligosaccharides, are very rich and diverse in our diet and facilitate gut microbial and consequently beneﬁcial metabolite diversity (Riva et al., 2019). The gut microbiota break cellulose into SCFAs as a metabolic intermediate; SCFAs inhibit HDACs and serve as energy substrates. SCFAs also affect histone crotonylation and acetylation levels (Kasubuchi et al., 2015; Koh et al., 2016; Zhao et al., 2016; Fellows et al., 2018; Sabari et al., 2018). SCFAs induce epithelial anti-inﬂammatory IL-10 receptor alpha subunit (IL-10RA) mRNA and antimicrobial peptides by inhibiting HDACs (Campbell et al., 2012; Zheng et al., 2017). Lactobacillus and Biﬁdobacterium synthesize folate to increase DNA methylation and m6A mRNA in the intestine to ensure normal development of the intestine (Wu J. et al., 2020). In addition to folate, vitamins B2, B6, and B1 from intestinal microbiota can also lead to the synthesis of SAM as a methyl group donor to methylate DNA, RNA, and histones (Wu J. et al., 2020). Microbial metabolism also provides TCA cycle intermediates, such as α-KG, succinate, and fumarate, which serve as substitutes for epigenetic modiﬁcations (Wu J. et al., 2020). The gut microbiota also indirectly regulates the activity of epigenetically modiﬁed genes by regulating gene expression. CLINICAL TRIALS Recent studies have shown that tumor metabolism and epigenetic regulation are closely linked and important for maintaining cell growth and regulating cancer metastasis. Therefore, inhibitors of rate-limiting enzymes in key metabolic pathways used by cancer cells are actively being investigated (Erez and DeBerardinis, 2015). Inhibiting such key enzymes has potential for cancer treatment, but they would also have an effect on normal cells; therefore, inhibiting rate-limiting enzymes of metabolic pathways is often toxic (Rodríguez-Enríquez et al., 2014). g q In 2017, the FDA approved the ﬁrst cancer metabolism drug enasidenib, an IDH2 inhibitor, for which the main indication is relapsed and refractory AML (Mullard, 2017). IDH2 is the key rate-limiting enzyme in the TCA cycle and catalyzes the conversion of isocitrate to α-KG. Cancer cells show a functional mutation in IDH2 (Yao et al., 2021). Enasidenib, an IDH2 inhibitor, is undergoing phase 1b/2 clinical trials in multiple countries. The current research results show that compared to that with azacitidine treatment alone, the December 2021 \| Volume 9 \| Article 793428 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 12 Metabolism Crosstalk with Epigenetic Modiﬁcations Huo et al. Huo et al. TABLE 2 \| Clinical trials of IDH, HDAC, SAM cycle and others. CLINICAL TRIALS Category Drugs NCT number Title Condition Status Phase IDH inhibitors Enasidenib NCT02677922 A Safety and Efﬁcacy Study of Oral AG-120 Plus Subcutaneous Azacitidine and Oral AG-221 Plus Subcutaneous Azacitidine in Subjects with Newly Diagnosed Acute Myeloid Leukemia (AML) Active, not recruiting Leukemia, Myeloid, Acute Phase 1/ Phase 2 Enasidenib NCT01915498 Phase 1/2 Study of Enasidenib (AG-221) in Adults with Advanced Hematologic Malignancies with an Isocitrate Dehydrogenase Isoform 2 (IDH2) Mutation Active, not recruiting Hematologic Neoplasms Phase 1/ Phase 2 AG881 NCT02481154 Study of Orally Administered AG-881 in Patients with Advanced Solid Tumors, Including Gliomas, with an IDH1 and/or IDH2 Mutation Active, not recruiting Glioma Phase 1 HDAC inhibitors Romidepsin NCT00426764 A Trial of Romidepsin for Progressive or Relapsed Peripheral T-cell Lymphoma Completed Peripheral T-cell Lymphoma Phase 2 Romidepsin NCT00106431 A Single Agent Phase II Study of Romidepsin (Depsipeptide, FK228) in the Treatment of Cutaneous T-cell Lymphoma (CTCL) Completed Cutaneous T-cell Lymphoma Phase 2 Vorinostat NCT01266031 Phase I/II Adaptive Randomized Trial of Bevacizumab Versus Bevacizumab Plus Vorinostat in Adults with Recurrent Glioblastoma Completed Recurrent Glioblastoma Phase 1/ Phase 2 SAM cycle inhibitors Ethylornithine NCT01483144 Trial of Eﬂornithine Plus Sulindac in Patients with Familial Adenomatous Polyposis (FAP) Completed Familial Adenomatous Polyposis Phase 3 Ethylornithine NCT00033371 Celecoxib With or Without Eﬂornithine in Preventing Colorectal Cancer in Patients with Familial Adenomatous Polyposis Completed Colorectal Cancer, Familial Adenomatous Polyposis Phase 2 Ethylornithine NCT01059071 Safety Study for Refractory or Relapsed Neuroblastoma with DFMO Alone and in Combination with Etoposide Completed Neuroblastoma Phase 1 Others Physical activity and dietary change NCT00811824 Effects of Physical Activity and Dietary Change in Minority Breast Cancer Survivors Completed Breast Cancer Phase 2 Vitamin C NCT02877277 Epigenetics, Vitamin C and Abnormal Hematopoiesis - Pilot Study Completed Myelodysplastic Syndrome, Acute Myeloid Leukemia Not Applicable TABLE 2 \| Clinical trials of IDH, HDAC, SAM cycle and others. Histone deacetylase is a key target for tumor therapy. Therefore, many studies have explored compound inhibitors of histone deacetylase to ﬁnd new anti-tumor strategies. HDAC inhibitor romidepsin (also called FK228) affects iron metabolism, mediates increased intracellular iron accumulation, decreases the expression of export-type ferroprotein, and increases reactive oxygen species (ROS) production, thereby, mediating iron death. This result has a guiding role for the combined treatment strategy using HDAC inhibitors and iron-targeted chemotherapy (Oliveira et al., 2021). CLINICAL TRIALS The results of the second-phase clinical study show that FK228 can effectively control T-cell lymphoma, with safety and reliability (NCT00426764, NCT00106431) (Table 2) (Foss et al., 2014; Foss et al., 2016). The HDAC inhibitor BAS-2 inhibits HDAC6, affects the ENO1 gene and LDHA related to glucose metabolism, and ultimately affects glycolysis in breast cancer (Dowling et al., 2021). Studies have shown that 1A12 can inhibit the acetylation level of histone H3, and it can also affect the glucose metabolism level in preclinical test subjects (Chan et al., 2014). HDAC inhibitors (panobinostat, vorinostat, and romidepsin) reduce glycolysis in a c-MYC-dependent manner, triggering the metabolic reprogramming of glioblastoma (Nguyen et al., 2020). Currently, the HDAC inhibitors vorinostat, romidepsin, and panobinostat have been approved by the FDA for the treatment of clinical lymphoma and multiple myeloma (Cappellacci et al., 2020). However, current research overall drug response rate of enasidenib combined with azacitidine is improved, and the combination has good drug tolerance, thereby, improving the prognosis of patients with IDH2 AML (NCT02677922) (Table 2) (DiNardo et al., 2021). In addition, clinical studies have also shown that the drug responsiveness and tolerability are good in patients with IDH2-mutant myelodysplastic syndrome (NCT01915498) (Table 2) (Stein et al., 2020). overall drug response rate of enasidenib combined with azacitidine is improved, and the combination has good drug tolerance, thereby, improving the prognosis of patients with IDH2 AML (NCT02677922) (Table 2) (DiNardo et al., 2021). In addition, clinical studies have also shown that the drug responsiveness and tolerability are good in patients with IDH2-mutant myelodysplastic syndrome (NCT01915498) (Table 2) (Stein et al., 2020). IDH mutations result in the conversion of α-KG to 2- hydroxyglutarate, which competitively inhibits α-KG- dependent enzymes as the two small molecules, 2-HG and α-KG, are similar in structure (Pirozzi and Yan, 2021). These enzymes include histone demethylases (Jumonji domain- containing protein/lysine demethylase family, JMJD/KDM family) and DNA/RNA methylation-modiﬁcation-related enzymes (Tet family) (White et al., 2020; Waitkus and Yan, 2021). This causes epigenetic dysregulation and cell differentiation arrest, a key factor in tumor cell proliferation after mutations (White et al., 2020). In addition, in 2018, the mutant IDH1 inhibitor ivosidenib was used to treat relapsed or refractory AML. The IDH1 and IDH2 dual inhibitor AG-881 has also been shown to cross the blood-brain barrier. CLINICAL TRIALS Recently, the results of a phase I clinical trial (NCT02481154) (Table 2) of AG- 881 for low-grade gliomas demonstrated that it is safe and can shrink tumors in many non-enhanced glioma patients (Mellinghoff et al., 2021). December 2021 \| Volume 9 \| Article 793428 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 13 Metabolism Crosstalk with Epigenetic Modiﬁcations Huo et al. shows that vorinostat does not work well in glioma (NCT01266031) (Table 2) (Puduvalli et al., 2020). shows that vorinostat does not work well in glioma (NCT01266031) (Table 2) (Puduvalli et al., 2020). modiﬁcations, and some metabolic enzymes can also affect chromatin modiﬁcation. Therefore, we suggest that the metabolism-epigenome axis must be considered while approaching cancer biomarker studies. SAM is a methyl donor for DNA and histone methylation (Haws et al., 2020). Metformin has been found to affect the epigenetic metabolic regulation of tumors in recent years. It reduces SAH (a potent inhibitor of all SAM-dependent methylation reactions) and promotes the accumulation of SAM. This leads to an increase in the overall DNA methylation level of metastatic cancer cells (Cuyàs et al., 2018). One study found that ethylornithine has chemopreventative and therapeutic effects on colon cancer, reducing folate-dependent metabolites such as SAM, the thymidine pool, and other intermediate products of related pathways (Witherspoon et al., 2013). On one hand, ethylornithine has an important effect in delaying the progression of familial polyposis (NCT01483144, NCT00033371) (Table 2) (Burke et al., 2020), while on the other, studies have shown that it has the potential to prevent the recurrence of high-risk neuroblastoma (NCT01059071) (Table 2) (Saulnier Sholler et al., 2015). Additionally, the role of metabolism synergy and epigenetic modiﬁcations in the physiology and pathology of the gut microbiota and corresponding effect on the host is proposed to be valuable and have prospective applications in cancer research. On the one hand, the metabolism of the gut microbiota and the products provide substrates for epigenetic modiﬁcations, whereas on the other hand, they directly affect the activity of epigenetic modiﬁcation enzymes, resulting in epigenetic modiﬁcation changes. Lifestyle-related factors, such as exercise and nutrition, are very important factors that affect human health through the gut microbiota, and the emerging role of gut microbial diversity in epigenetics emphasizes the link between lifestyle choices and cancer. CLINICAL TRIALS Although great progress has been made in understanding the epigenetic–metabolism axis in the past few decades, the internal mechanism of this heritable change in tumorigenesis and development and its application to tumor prevention and treatment remain elusive. Therefore, the mechanisms of metabolically-regulated epigenomic landscape responses in tumorigenesis and development need to be investigated, and further potential therapeutic applications in this regard must be studied. In addition to drugs, lifestyle changes can affect epigenetic changes. A healthy lifestyle is associated with high levels of DNA methylation, whereas overall tissue and blood DNA hypomethylation are associated with an increased risk of cancer. The results of clinical trials (NCT00811824) (Table 2) show that lifestyle changes (diet and weight loss) can effectively change DNA methylation (Delgado-Cruzata et al., 2015). A high-fat diet can induce DNA methylation changes in the whole genome, which was found to involve metabolic disease-related genes and cancer- related genes (Jacobsen et al., 2012). Previous studies have shown that patients with malignant hematological tumors often lack vitamin C. Vitamin C can enhance the activity of TET protein in tumor cells, increase the level of 5hmC, decrease the level of 5mC in cells, and increase sensitivity to anticancer drugs. A clinical study in Denmark (NCT02877277) (Table 2) showed that vitamin C supplementation in patients with myeloma increases the 5hmC/ 5mC ratio of mononuclear myeloid cells, enhancing the clinical efﬁcacy of DNMT inhibitors (Gillberg et al., 2019). CONCLUSION AND PERSPECTIVES This work was supported by the National Natural Science Foundation of China (41,931,291, 42,125,707, 81,902,960), the Non-proﬁt Central Research Institute Fund of Chinese Academy of Medical Sciences (2019PT310027), the State Key Laboratory of Molecular Oncology (SKLMO-2021–21), Cooperative Project of Beijing-Tianjin-Hebei Regional Basic Research (19JCZDJC65700(Z)). This work was supported by the National Natural Science Foundation of China (41,931,291, 42,125,707, 81,902,960), the Non-proﬁt Central Research Institute Fund of Chinese Academy of Medical Sciences (2019PT310027), the State Key Laboratory of Molecular Oncology (SKLMO-2021–21), Cooperative Project of Beijing-Tianjin-Hebei Regional Basic Research (19JCZDJC65700(Z)). In this review, we elucidated how the crosstalk between epigenetic regulation and metabolic reprogramming changes during the process of tumorigenesis and development. Metabolic reprogramming in tumors changes the epigenetic landscape of tumor cells. Metabolites can serve as substrates for epigenetic AUTHOR CONTRIBUTIONS YW and WH designed the content of the review with input from all the co-authors. MH and JZ wrote the original review draft. YW and WH revised the article with feedback from all the co-authors. All authors approved this work for publication. Figures in this work were created with BioRender.com. 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Zheng, L., Kelly, C. J., Battista, K. D., Schaefer, R., Lanis, J. M., Alexeev, E. E., et al. (2017). Microbial-Derived Butyrate Promotes Epithelial Barrier Function through IL-10 Receptor-dependent Repression of Claudin-2. J. Immunol. 199 (8), 2976–2984. doi:10.4049/jimmunol.1700105 Zheng, Y., Wu, C., Yang, J., Zhao, Y., Jia, H., Xue, M., et al. (2020). Insulin-like Growth Factor 1-induced Enolase 2 Deacetylation by HDAC3 Promotes December 2021 \| Volume 9 \| Article 793428 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 20
https://openalex.org/W2319741988	http://journals.bg.agh.edu.pl/GEOMATICS/2012.6.4/geom.2012.6.4.83.pdf	English	null	Selected aspects of reclamation of soda waste landfill sites.	Geomatics and Environmental Engineering	2,012	cc-by	3,424	1. Introduction The nineteenth and twentieth centuries witnessed a rapid growth of industry, which resulted in changes in the natural environment. The development of the soda industry was a good example of that. Factories producing soda in the past century left their traces in the form of industrial waste landę ll sites, which are diĜ cult to handle to this day. In the case of soda waste, there are at least a few adverse eě ects which hin- der conducting the reclamation process, such as the content of readily soluble com- pounds [17, 26] which, when penetrating into the environment, may be included in various interactions with it, or the fact that they are produced in large quantities and are stored in large sedimentation tanks, which are signię cantly noticeable in the landscape. p For these reasons, their reclamation becomes an important, socially desirable and sometimes very costly objective. GEOMATICS AND ENVIRONMENTAL ENGINEERING • Volume 6 • Number 4 • 2012 http://dx.doi.org/10.7494/geom.2012.6.4.83 GEOMATICS AND ENVIRONMENTAL ENGINEERING • Volume 6 • Number 4 • 2012 http://dx.doi.org/10.7494/geom.2012.6.4.83 Joanna UrbaÚska, Krzysztof UrbaÚski Joanna UrbaÚska, Krzysztof UrbaÚski * AGH University of Science and Technology, Faculty of Mining Surveying and Environmental Engi- neering, Department of Development and Environmental Protection, Krakow, Poland The paper has been prepared within the scope of the AGH UST statutory research no. 11.11.150.008 3) management of sediments. g p p The paper has been prepared within the scope of the AGH UST statutory research no. 11.11. 3. Spontaneous Succession Spontaneous succession exists on a variety of industrial waste landę ll sites. Spontaneous overgrowth of such wasteland, as numerous researchers have proved [16, 21, 42] is still very slow, mainly due to: lack of appropriate plant species on-site or in the surrounding, water shortages or excesses, high concentrations of salts or heavy metals, nutrient deę ciencies, too alkaline or too acidic reaction, or strong erosion. The process of natural succession can be divided into several stages. The ę rst one is the colonization of the surface by plants which are the most resistant to ex- tremely adverse conditions of a habitat. The second one refers to the creation of the turf, or the total coverage of the area with herbaceous vegetation. The third one is the possibility of introducing shrubs and trees. The individual steps depend in par- ticular on the origin of the wasteland, and therefore on the nature of the formations which are deposited in dumps or sedimentation tanks. According to HewleĴ , in the case of soda waste landę ll sites, the stages of spon- taneous succession could be as follows [14]: Stage I – associated with highly alkaline and saline sedimentary material, intro- ducing pioneering alkaliphilic and halophilic vegetation. Stage I – associated with highly alkaline and saline sedimentary material, intro- ducing pioneering alkaliphilic and halophilic vegetation. Stage II – associated with transformed sedimentary material, with a marginal salinity and alkaline pH; emergence of herbaceous vegetation and tree seedlings. Stage II – associated with transformed sedimentary material, with a marginal salinity and alkaline pH; emergence of herbaceous vegetation and tree seedlings. Stage III – associated with sedimentary material subject to far-reaching trans- formation as a result of the constant interaction of atmospheric variables, and no- ticeably marked processes resembling in its characteristics the phenomenon of rock weathering; areas with well-developed woody vegetation are mainly dominating. Stage III – associated with sedimentary material subject to far-reaching trans- formation as a result of the constant interaction of atmospheric variables, and no- ticeably marked processes resembling in its characteristics the phenomenon of rock weathering; areas with well-developed woody vegetation are mainly dominating. 2. Trends in Land Management Following the literature on the issue of soda waste sedimentation tanks, three basic ways of managing the area of tailing ponds can be distinguished: 1) leaving the sedimentation tanks to the spontaneous succession, 2) conducting selected technical and biological reclamation treatm 2) conducting selected technical and biological reclamation treatments, 3) management of sediments. 83 J. UrbaÚska, K. UrbaÚski 84 3. Spontaneous Succession Stage IV – the entire area is dominated by a well-developed ecosystem The tailing ponds of soda plants are classię ed, according to Krzaklewski [21], as the objects with a diĜ cult level of biological reclamation, where the lack of sponta- neous overgrowth of the wasteland is maintained for a minimum of 10 years. How- ever, leaving the sedimentation tanks to the spontaneous succession as “the direc- tion of reclamation” has some reasons in the case of soda waste tanks. One of the arguments may be the time of creating and ę lling the tank, during which climatic factors may systematically lead to the reduction of harmful substances in the surface layers of the sediment. It may be accompanied by the ę rst aĴ empts to colonize the surface by the plants and the emergence of the ę rst stage of plant succession, which could later be destroyed or inhibited by reclamation treatments. Examples of spontaneous plant succession on soda waste sedimentation tanks can be observed in Germany near Bernburg [10, 11] and in the Czech Republic – near Petrovice [13, 32]. There are sedimentation tanks where the development of vegetation derived from spontaneous succession, which has lasted for over 70 years, can be seen. The research conducted in Germany allows us to observe and com- pare the individual stages of plants conglomerations formed in this way, and the Selected Aspects of Reclamation of Soda Waste Landę ll Sites 85 transformation of the stored material and the formation of the substrate under their inĚ uence. They visualized the impact of natural succession on the accumulation of organic carbon (i.e. the phenomenon conę rming soil-forming processes [9]) and the stabilization of organic maĴ er. And the presence of organic maĴ er was associated with the age of the sedimentation tank and the fact how long it was covered with the vegetation layer. The soil formed in these specię c conditions has been classię ed as lime regosols mixed with a great amount of industrial waste (Calcareous spolic regosols) [41]. The research carried out in the Czech Republic focused mainly on observations of growing vegetation within the sedimentation tanks, emerging by spontaneous succession and its inĚ uence on the abundance and diversity of microfauna, includ- ing mainly representatives of the Nematoda type (nematodes). 3. Spontaneous Succession The observations have shown that the greening of landę ll sites continues to tens of years, and with the growth of vegetation and the emergence of new species of plants, the number of micro-organisms increases as well, but even after almost 80 years of plant suc- cession, their specię c composition shows numerous diě erences, compared with the semi-natural forest nearby [13]. 4. Assisted Reclamation Vast areas of wasteland are often subjected to a standard three-stage technical and biological reclamation [8, 15, 28] or only selected treatments aimed at support- ing the spontaneous processes of environment restoration. The examples of a standard (three-stage) reclamation are, among others, the pro- cesses on the selected complexes of the sedimentation tanks of the former Soda Plant “Solvay” in Krakow, where the reclamation works were carried out in 1989–1995. Preliminary design works (stage I) preceded the implementation of the technical as- sumptions of the reclamation project (stage II). The treatments included: leveling of the dikes separating the individual sedimentation tanks, shaping and securing the slopes, application of topsoil layer as well as execution of water drainage system [20, 31]. Soil material used to secure the crown of the sedimentation tanks was mostly coming from the building site excavations, which meant that it was characterized by a great diversity of mechanical composition [1, 22]. A total of about 185,000 m3 of soil material was applied on the tailing ponds, of which about 35,000 m3 was the material for reinforcing the slopes, while 150,000 m3 was the material used to construct the topsoil [1]. Upon the completion of the technical works, the biological stage of the reclamation followed (stage III), which consisted mainly of seeding the topsoil with a mix of grasses and legumes [1]. Due to the high labour and cost consumption of such a reclamation method, various modię cations are often applied, mainly consisting of the replacement of soil material building topsoil layer with an alternative material. This function is often J. UrbaÚska, K. UrbaÚski 86 taken over by all kinds of sewage sludge [33]. In such cases, the sediments play a dual role, and thus function as an insulating layer, and due to the high nutrient content are also able to intensify the growth of grass and herbaceous vegetation. In addition, the sediments in the form of sludge can be placed on the landę lls with grass seeds through the so-called hydro-seeding, which dramatically reduces the workload. It should be remembered, though, that the sediments in contrast to the natural organic fertilizers in their composition may have signię cant amounts of heavy metals, es- pecially when derived from the highly urbanized and industrialized areas [2, 33]. These metals can penetrate and accumulate in the plants and become included in the cycle of maĴ er and energy [12, 27]. 4. Assisted Reclamation An example of such a sediment reclamation con- cept are landę ll sites in Soda Plant in Janikowo. Technical reclamation was brought here to the liquidation of the dikes separating individual sedimentation tanks, and to leveling of their surface. Biological reclamation was carried out based on sewage sludge and sowing seeds of meadow grass mixtures. In addition, until intensive growth of grasses, plants protecting and increasing the process of vegetation were planted [33, 34]. These reclamation treatments are often supplemented with additional qualities aimed at protecting the environment from the harmful eě ects of soda waste landę lls. One such quality is the use of fast-growing vegetation having the ability to bioaccu- mulate, which is used to ę lter the leachate from the landę lls. This becomes a partic- ularly important issue when the stored waste contains a lot of easily soluble salts, which, together with the inę ltrating rainwater, can penetrate the groundwater or wa- tercourses, highly polluting them [38, 40]. The test solution based on the exploitation of osier willow as a ę lter medium was introduced in the United States in Syracuse [5, 39], where huge deposits of sediments collected in the close vicinity of the lake are contributing to the changes in its physical and chemical parameters [7, 29]. 5. Management of Sediments The management of the sediments would be the best solution to the problem of soda waste, but due to their physical and chemical properties, at least for now, this type of waste treatment is very limited. The high content of calcium chloride (CaCl2) and sodium chloride (NaCl) signię cantly reduces the possibility of soda waste man- agement. The methods of removing these compounds or of signię cantly reducing their amount in waste were already worked on since 1938 [35]. Poorly developed waste disposal techniques were due to the fact that the issue of waste management in those days was marginalized [18, 36]. As the interest in the problem of accumu- lated sediments and negative impacts that may occur on the environment was in- creased, works on potential soda waste management methods were being devel- oped. As a result, it turned out that this waste can be used in various manufacturing industries, to a limited extent. 87 Selected Aspects of Reclamation of Soda Waste Landę ll Sites One of the proposed solutions to the problem of soda waste was using it in agriculture as a substitute for agricultural lime [3, 6], however, as a detailed study revealed [30], raw waste did not qualify as a fertilizer. This was mainly due to the high chloride content and excessive humidity which was resulted from high hy- groscopicity [30]. Only after partial processing, yielding fertilizer chalk or fertilizer calcium carbonate [23], the waste could be used in agriculture. The “Solvay” Soda Plant launched this type of production line in1960 [4, 19]. The usability of soda waste was also examined in terms of its use as raw mate- rial for the preparation of mortar or cement mixtures [3, 6, 35]. Partial management of soda waste, especially that in the solid form, is also carried out in the process of using it to produce various kinds of earth structures (construction of tank and road embankments) [24, 25], while the semi-liquid waste is trying to be used as the ę lling material for excavation voids and depressions caused by mining damage, or in the liquid form – directly injected into the deep layers of soil [23] 6. Summary The provided examples of various manners of soda waste management prove that the development of an eě ective method for reclamation which would give tan- gible results poses numerous problems. They should be seen mainly in the specię c nature of the waste, among others, in their semi-liquid form, heterogeneity with- in a sedimentation tank, a large content of easily soluble salts or strongly alkaline reaction. Taking into account the standard methods of reclamation for large area sedi- mentation tanks, very high costs should be expected, associated with transporting the soil masses needed to protect the tanks against the eě ects of weathering. Ad- ditionally, there are hydraulic engineering works to capture any rainwater leach- ate and inę ltrating from the tanks, as well as the works associated with seeding or planting of plants shaping the biological architecture of the reclaimed facility. How- ever, such works are carried out, but their intensity is usually adapted to the local requirements and current needs. As a result, despite the continued development of various technologies for soda waste management, still a very large mass remains within sedimentation tanks [3]. [1] BoroÚ K., Zajc E., Klatka S.: Rekultywacja terenu skÙadowania odpadów KZS „Solvay” w Krakowie. InČynieria Ekologiczna, nr 1, 2000, pp. 58–64. 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https://openalex.org/W2071288560	https://www2.ifrn.edu.br/ojs/index.php/HOLOS/article/download/1713/754	Portuguese	null	ANÁLISE ESPAÇO TEMPORAL DA EVAPOTRANSPIRAÇÃO SOBRE A MICRORREGIÃO DO SERIDÓ NO ESTADO DO RIO GRANDE DO NORTE	Holos	2,013	cc-by	5,863	RESUMO correspondeu a valores baixos na porção central e a altos no extremo norte e sul da microrregião do Seridó tal como a ETP. Relacionando com a disponibilidade de energia concluiu-se que durante o ano essa variável fosse mais importante no processo de evapotranspiração do que a precipitação sobre o Seridó, tornando o Modelo adequado para avaliar quando houver a baixa na precipitação. Este estudo teve como objetivo estimar as evapotranspirações potencial e real de área dos municípios pertencentes a microrregião do Seridó no Estado do Rio Grande do Norte, utilizando do modelo complementar de Morton. Após isso procurou- seespacializar os resultados para verificar como se distribui a ETR e ETP sobre a região. Pela análise feita ficou evidente que nas regiões onde a primeira é alta a outra tende a ser baixa. Foi visto que a ETR PALAVRAS-CHAVE: evapotranspiração; precipitação; disponibilidade hídrica. JESUS & MATTOS (2013) ANÁLISE ESPAÇO TEMPORAL DA EVAPOTRANSPIRAÇÃO SOBRE A MICRORREGIÃO DO SERIDÓ NO ESTADO DO RIO GRANDE DO NORTE Edmir dos Santos Jesus1 e Arthur Mattos2 Universidade Federal do Rio Grande do Norte E-mail: edmir.jesus@gmail.com1; armattos@ct.ufrn.br2 Edmir dos Santos Jesus1 e Arthur Mattos2 Universidade Federal do Rio Grande do Norte E-mail: edmir.jesus@gmail.com1; armattos@ct.ufrn.br2 Artigo submetido em outubro/2013 e aceito em dezembro/2013 KEYWORDS: evapotranspiration; precipitation; hidric availability. 1 INTRODUÇÃO Como uma das mais importantes variáveis no ciclo hidrológico, a evapotranspiração refere-se a perda conjunta d’água para a atmosfera que sob condições de restrições hídricas, constitui um processo muito complexo e de difícil dedução por métodos climatológicos. Sob estas condições, o estado da água disponível ao ambiente radicular das plantas cultivadas e na superfície do solo, afeta significativamente a razão de evapotranspiração (MOTA,1977). No Estado do Rio Grande do Norte, o Seridó é uma das microrregiões que abrange dezessete municípios, o qual possui área de aproximadamente 9.372 km², correspondendo a quase 18% do Estado, com uma população estimada de 260.000 habitantes (IBGE, 2000). Esta região está inserida na área semiárida da Região Nordeste do Brasil (NEB), com regime pluviométrico bastante irregular tanto espacialmente como temporalmente. A atividade econômica principal do Seridó é a agropecuária. Entretanto, a falta de água no solo torna-se a principal limitação para o pequeno agricultor, não apenas pelo seu volume total anual de precipitação pluvial, mas pela distribuição irregular. Este fato reforça a necessidade de um monitoramento contínuo hidrológico da microrregião do Seridó. Esse tipo de informação terminada é de extrema necessidade para diversos fins na agricultura, prevenção de incêndios florestais e o planejamento de consumo de água nas grandes comunidades (ISAIAS et al. 1992). Segundo Bezerra Júnior & Souza (2007), o clima semi-árido com suas altas temperaturas, baixas médias pluviométricas, dentre outras características influenciam o sistema hidrográfico. Devido ao déficit hídrico intenso, a região seridoense abriga inúmeras barragens que trazem oportunidades para prática de irrigação, uso humano e turístico no intuito de diminuir as dificuldades socioeconômicas que se apresentam na época da seca. Em regiões semiáridas o déficit observado no balanço hídrico anual constitui um grave problema as atividades agropecuárias, pois a deficiência hídrica limita à produção agrícola, diminui a disponibilidade de água para dessedentação animal e consumo humano, sendo assim fonte de risco agrícola nestas áreas. Desta forma a quantificação da evapotranspiração assume particular acepção em virtude destes eventos de déficit hídrico, ajudando no planejamento agrícola, indicando o período de escassez de água e assim buscando-se formas de as culturas para que não haja perda da agrícola (MENDONÇA & DANTAS, 2010). Devido à dificuldade de se estimar a evapotranspiração diversos modelos empíricos são encontrados na literatura, os quais respondem e que levam em consideração as variáveis climáticas reinantes da região de estudo. JESUS & MATTOS (2013) ABSTRACT that the ETR corresponded to low values in the central and high in the extreme north and south of micro Seridó as the ETP. Relating to the availability of energy concluded that during the year this variable was more important in the process of evaporation than precipitation on Seridó, making the model suitable for assessing when there low in precipitation. that the ETR corresponded to low values in the central and high in the extreme north and south of micro Seridó as the ETP. Relating to the availability of energy concluded that during the year this variable was more important in the process of evaporation than precipitation on Seridó, making the model suitable for assessing when there low in precipitation. This study objective to estimate the potential evapotranspiration and actual area of the municipalities belonging to the micro Seridó in the state of Rio Grande of the North, using the complementary model of Morton. After that we tried to spatialize the results to see how it distributes the ETR and ETP on the region. For the analysis it became clear that in regions where the first one is high the other tends to be low. It was seen KEYWORDS: evapotranspiration; precipitation; hidric availability. HOLOS, Ano 29, Vol. 6 22 2.1 Área de estudo A região de estudo abrange a microrregião do Seridó no Estado do Rio Grande do Norte, situada entre os paralelos de 6°06’ e 6°55’ Sul e meridianos de 37°25’ e 36°22’ Oeste com altitudes entre 134 a 572 metros (Figura 1). Segundo a classificação climática de Thornthwaite, o clima desta região é DdA’a’, ou seja, semi-árido, megatérmico, com excedente hídrico pequeno ou nulo e concentração da evapotranspiração nos meses de verão igual a 26%, atingindo uma média anual de 1.464mm. FIGURA 1. Localização geográfica dos municípios na microrregião do Seridó. (Fonte: do Autor) FIGURA 1. Localização geográfica dos municípios na microrregião do Seridó. (Fonte: do Autor) JESUS & MATTOS (2013) área, isto é, existe uma resposta complementar, igual e de sinal contrário entre ambas, devido as mudanças de disponibilidade de água para a evapotranspiração real de área. área, isto é, existe uma resposta complementar, igual e de sinal contrário entre ambas, devido as mudanças de disponibilidade de água para a evapotranspiração real de área. Alguns autores como (JESUS & ASSIS, 2004), GOSSON (2005), JESUS et al. (2012) mostram que a utilização do modelo de relação complementar de Morton para a obtenção da evapotranspiração potencial e evapotranspiração real de área obteve bons resultados comparados aos dados observados. Neste sentido, este trabalho teve como objetivo analisar a estimativa das evapotranspirações potencial e real de área sobre os municípios localizados na microrregião do Seridó. 1 INTRODUÇÃO Dessa forma, BOUCHET (1963) formulou a hipótese de que variações nas evapotranspirações potencial e real de área, devido à variações no fornecimento de umidade regional são complementares. MORTON (1965) desenvolveu uma equação para a hipótese de Bouchet, a qual foi sendo aperfeiçoada pelo mesmo até chegar no modelo de relação complementar. Este modelo está fundamentado no conceito de que existe uma relação complementar entre a evapotranspiração potencial e a evapotranspiração real de HOLOS, Ano 29, Vol. 6 23 2.2 Dados meteorológicos utilizados Para a realização deste trabalho foram selecionados os dados meteorológicos mensais dos dezessete municípios como a temperatura do ar (°C), umidade relativa do ar (%), insolação (horas), temperatura do ponto de orvalho (°C), pressão real (mb) e de saturação do vapor (mb) e total anual de precipitação (mm). A série de dados foi obtida de estações meteorológicas de superfície monitoradas pelo 3º DISME (Distrito de Meteorologia) e pela Empresa de Pesquisa Agropecuária do Rio Grande do Norte (EMPARN)que correspondem ao período de 1963 a 2008. HOLOS, Ano 29, Vol. 6 24 JESUS & MATTOS (2013) Os dados climatológicos de temperatura média do ar foram estimados em cada município da microrregião por meio do softwareEstima_T (Cavalcanti et al. 2006), do Departamento de Ciências Atmosféricas (DCA), da Universidade Federal de Campina Grande (UFCG). Estes dados estimados de temperatura do ar levam em consideração uma série temporal de 30 anos (1961 a 1990) através das estações meteorológicas convencionais instaladas no Rio Grande do Norte pelo Instituto Nacional de Meteorologia (INMET). Para estimar as evapotranspirações potencial e real de área, foi utilizado o programa computacional WREVAP, desenvolvido em linguagem Fortran, desenvolvido por MORTON et al. 1985. Este programa possui três opções: modelo CRAE (relação complementar de evapotranspiração de área), modelo CRWE (relação complementar de evaporação de superfície saturada) e modelo CRLE (relação complementar de evaporação de lago). A opção de programa WREVAP utilizada neste trabalho foi o modelo CRAE. O programa WREVAP foi desenvolvido para ter como valores de entrada osdados climatológicos medidos para um período que varia de um dia a um mês. Os dados meteorológicos utilizados para inicializaro modelo CRAE, foram: médias mensais de temperatura do ponto de orvalho (°C), da temperatura do ar (°C), da insolação real (horas) e normal anual de precipitação (mm); além da altitude (m) e latitude (graus) da estação meteorológica. Obtendo como valores de saída a ETP (evapotranspiração potencial), ETR (evapotranspiração real), e Rn (saldo de radiação solar). JESUS & MATTOS (2013) A ordenada representa a evapotranspiração e a abscissa representa o fornecimento de água às superfícies solo-planta da área, uma quantidade que é normalmente desconhecida. Quando não existe água disponível para a evapotranspiração real de área (extremo esquerdo da Figura 2) a R ET é igual a zero, e o ar é muito quente e seco. P ET tem valor máximo que é igual a W ET 2 . Quando o fornecimento de água para as superfícies solo-planta aumenta, o aumento equivalente resultante em R ET modifica as condições do ar que está circulando sobre a região, tornando-o mais frio e mais úmido, o qual, em retorno (feedback), produz um decréscimo equivalente em P ET . Finalmente, quando o fornecimento de água para as superfícies solo-planta da área aumenta suficientemente, os valores de R ET e P ET convergem para W ET . No modelo de Morton, a P ET é estimada de uma solução de equações do balanço de energia (Equação 2) e saldo de radiação à temperatura de equilíbrio Np R (Equação 3)proposta por Priestley-Taylor (1972), ajustada para levar em conta os efeitos de advecção (divergência horizontal) de Larga Escala.    T T + T f εσ + γp f R = ET p T T N P       3 273 4 (2)   T T pf ET = R p T P Np   (3) (2) (3) (3) em que p T é a temperatura de equilíbrio (°C), T é a temperatura do ar (°C), Tf é o coeficiente de transferência de vapor d’água, é o coeficiente de transferência de calor (2,45MJ.kg-1), sendo a constante psicrométrica (kPa.°C-1), p é a pressão atmosférica (kPa),  é a constante de Stefan-Boltzman (4,8989x10-9MJ.m-2.K-4.dia-1), e a emissividade da superfície. 2.2 Dados meteorológicos utilizados A base conceitual do modelo CRAE leva em consideração os efeitos de fenômenos de Larga Escala que contribuem para variar a taxa de evapotranspiração e a equação que descreve a relação complementar, segundo MORTON (1983), é expressa da seguinte forma: R W P ET ET ET  2 (1) R W P ET ET ET  2 (1) onde : P ET é a evapotranspiração potencial que é estimada de uma solução de equações do balanço de energia e do transporte de vapor d’água, representando a evapotranspiração que ocorreria em uma superfície úmida hipotética, com características de absorção de radiação e de transporte de vapor semelhantes àquelas de uma extensa área; R ET é a evapotranspiração real de área que ocorre em uma área bastante extensa e W ET representa a evapotranspiração de um ambiente úmido. A Figura 2 mostra a relação esquemática entre as três variáveis da Equação (1), considerando o fornecimento de energia radiante constante. onde : P ET é a evapotranspiração potencial que é estimada de uma solução de equações do balanço de energia e do transporte de vapor d’água, representando a evapotranspiração que ocorreria em uma superfície úmida hipotética, com características de absorção de radiação e de transporte de vapor semelhantes àquelas de uma extensa área; R ET é a evapotranspiração real de área que ocorre em uma área bastante extensa e W ET representa a evapotranspiração de um ambiente úmido. A Figura 2 mostra a relação esquemática entre as três variáveis da Equação (1), considerando o fornecimento de energia radiante constante. FIGURA 2. Representação esquemática da relação complementar entre evapotranspiração potencial e evapotranspiração real de área, com fornecimento de energia radiante constante (MORTON, 1983). Fonte: Adaptação PEREIRA (1997). Evapotranspiração ETW = Evapotranspiração Potencial em superfície úmida ← ETP = Evapotranspiração Potencial ← ETR = Evapotranspiração Real de área ← 2ETW = Evapotranspiração Potencial em regiões secas Disponibilidade de água Solo seco FIGURA 2. Representação esquemática da relação complementar entre evapotranspiração potencial e evapotranspiração real de área, com fornecimento de energia radiante constante (MORTON, 1983). Fonte: Adaptação PEREIRA (1997). HOLOS, Ano 29, Vol. 6 25 0,007 0,71 1 273 4 (7) (7)              τ a + + τ R = R a o g . 1 1 1 . 0 0   Z ω η = R cos 180 1354 2 0                        τ a + + τ R = R a o g . 1 1 1 . 0 0   (8) Z ω η = R cos 180 1354 2 0           (9) (8) (9) onde : 0 g R é a radiação global (MJ.m-2.d-1) a céu claro; 0 R é a irradiância solar (MJ.m-2d-1) no topo da atmosfera; é a excentricidade da órbita da Terra em torno do Sol; N n é a razão de insolação; se é a pressão de saturação de vapor(mb) à temperatura do ponto de orvalho dT em (°C); sp p é a onde : 0 g R é a radiação global (MJ.m-2.d-1) a céu claro; 0 R é a irradiância solar (MJ.m-2d-1) no topo da atmosfera; é a excentricidade da órbita da Terra em torno do Sol; N n é a razão de insolação; se é a pressão de saturação de vapor(mb) à temperatura do ponto de orvalho dT em (°C); sp p é a razão da pressão atmosférica na estação pela pressão ao nível do mar com a correção da pressão para atmosfera padrão; a  é a razão de transmitância a céu claro pela fração absorvida; é o ângulo horário, Z é o ângulo zenital ao meio dia. A estimativa do saldo de radiação ( n R ) para superfície solo-planta à temperatura do ar (T ), envolve as variáveis: albedo médio ();albedo médio a céu claro ( 0 );radiação solar global ( g R ) e perda de radiação de onda longa para o conjunto solo-planta ( ol R ), conforme descrito as Equações:   ol g n R R = R   1 (4)   ol g n R R = R   1 (4) com as contribuições : com as contribuições :             330 1 1 0 Z N n + N n =   (5) o g R N n N n + + N n = R         1 . 0,30 0,08 . Rg0 (6) ç             330 1 1 0 Z N n + N n =   (5) o g R N n N n + + N n = R         1 . 0,30 0,08 . Rg0 (6) HOLOS, Ano 29, Vol. 6 26 JESUS & MATTOS (2013)                 ρ + p p e + + T εσ = R s s ol 1 . 0,007 0,71 1 273 4                 ρ + p p e + + T εσ = R s s ol 1 . 0,007 0,71 1 273 4 (7)              τ a + + τ R = R a o g . 1 1 1 . 0 0   (8) Z ω η = R cos 180 1354 2 0           (9)                 ρ + p p e + + T εσ = R s s ol 1 . HOLOS, Ano 29, Vol. 6 3 RESULTADOS E DISCUSSÃO 3.1 Análise anual da evapotranspiração real de área e potencial em função da precipitação, saldo de radiação, temperatura, umidade relativa e insolação. 3.1 Análise anual da evapotranspiração real de área e potencial em função da precipitação, saldo de radiação, temperatura, umidade relativa e insolação. 3.1 Análise anual da evapotranspiração real de área e potencial em função da precipitação, saldo de radiação, temperatura, umidade relativa e insolação. Na discussão dos resultados destacamos as Figuras 3a e 3b, onde tem-se a distribuição espacial da evapotranspiração real e potencial anual para a microrregião do Seridó, e neste, observa-se que os menores valores de ETP foram registrados na região nordeste e leste do Seridó. Segundo Ross (2003) essa região é formada pela Depressão Sertaneja e pelo Planalto da Borborema favorecendo a menor demanda hídrica pelas plantas através da evapotranspiração, devido a alguns condicionantes climáticos, que segundo Pereira et al.(2002) o efeito combinado de temperatura, umidade relativa e velocidade do vento define a demanda atmosférica por vapor d’águaem cada região. Os maiores valores registrados da evapotranspiração foram observados nas regiões oeste e centro-oeste da microrregião do Estado. Analisando a distribuição espacial da ETR (Figura 3a), verifica-se o aumento gradativo do centro-oeste do Seridó ocidental ao norte e sudeste da microrregião. Em parte das localidades de Timbaúba dos Batistas, Caicó, São João do Sabugi e Jardim do Seridó seu valor foi superior a 1.120mm. Ainda pode ser visto que os menores valores de ETR estiveram concentrados na porção da região centro-oeste, como em Caicó (1.111,4mm) e São João do Sabugi (1.123,6mm) e na região extremo nordeste, como em Currais Novos (1.291,1mm) os quais demonstram superestimados comparados aos valores encontrados por Gosson (2005). A Figura 3b mostra a distribuição da ETP, onde se observa o aumento da variável na direção oeste do Seridó, com valores de 2.300 a 2.500mm, principalmente nas localidades de HOLOS, Ano 29, Vol. 6 27 JESUS & MATTOS (2013) Jardim de Piranhas, São Fernando, Timbaúba dos Batistas, Serra Negra do Norte, Caicó e Ipueira (2.520,4mm). Osmenores valores concentraram-se na região nordeste, como em Currais Novos e numa pequena faixa ao sul com valores abaixo de 2.200mm. Dado aos fatores do saldo de radiação, superior a 4.300MJ.m-2.dia-1 (Figura 4c), da insolação, entre 2.800 e 2.900 horas (Figura 4d) da alta temperatura em torno de 27°C (Figura 4a) e a baixa na umidade relativa do ar (em torno de 60%, Figura 4b) que somadas ocasionam a alta taxa de evapotranspiração em toda a microrregião. Como a relação complementar de Morton entre a evapotranspiração potencial e real de área é baseada na interação entre a superfície evaporante e o ar ambiente, quando ocorre uma redução na disponibilidade de água para a evapotranspiração real de área, o ar torna-se mais quente e mais seco, o que causa o aumento da evapotranspiração potencial. Por isso, é que nas regiões onde a ETR é baixa, a ETP é alta, e há restrições na disponibilidade de água que é representada pela precipitação (Figura 3c). Ressalta-se que em Caicó, na porção central, a ETR foi bem abaixo da ETP, com uma diferença em torno de 1.439,9mm. A precipitação (Figura 3c) observada ficou acima de 700mm, mas a alta demanda evaporativa para a atmosfera foi influenciada por temperaturas médias elevadas associada com a disponibilidade de energia (Figura 4c). Ainda segundo Santos et al. (2010) que calcularam o balanço hídrico climatológico para a microrregião do Seridó pelo método de Thornthwaite e Mather (1955) verificaram as variabilidades sazonais conforme a evapotranspiração potencial e real e viram que a deficiência hídrica é bastante elevada na região, concentrando-se no período de junho a dezembro, não ocorrendo excedente em nenhum outro mês. Comparando os valores encontrados pelos métodos na região, vimos os estimados pelo método de Morton superestimaram os valores encontrados por Thornthwaite e Matter. Ao verificar os valores de ETR, ETP e PRP percebe-se o quanto há de perda de água em toda a microrregião tornando a região em completa escassez comprometendo a toda a população do Seridó, confirmando o levantamento feito por Bezerra Junior e Silva (2007). HOLOS, Ano 29, Vol. 6 28 JESUS & MATTOS (2013) (a) (b) (c) uição espacial do total anual da Evapotranspiração Real de Área(a), Evapotranspiração Pote (b) e Precipitação (c). 1000 1200 1400 1600 1800 2000 2200 2400 2600 Paraíba 6°06' 6°12' 6°18' 6°24' 6°30' 6°36' 6°42' 6°48' Latitude Sul Paraíba 6°54' 37°24' 37°12' 37°00' 36°48' 36°36' 36°24' Longitude Oeste Acari Caicó Carnaúba dos Dantas Cruzeta Currais Novos Equador Ipueira Jardim de Piranhas Jardim do Seridó Ouro Branco Parelhas Santana do Seridó São Fernando São João do Sabugi São José do Seridó Serra Negra do Norte Timbaúba dos Batistas mm 1000 1200 1400 1600 1800 2000 2200 2400 2600 Paraíba 6°06' 6°12' 6°18' 6°24' 6°30' 6°36' 6°42' 6°48' Latitude Sul Paraíba 6°54' 37°24' 37°12' 37°00' 36°48' 36°36' 36°24' Longitude Oeste Acari Caicó Carnaúba dos Dantas Cruzeta Currais Novos Equador Ipueira Jardim de Piranhas Jardim do Seridó Ouro Branco Parelhas Santana do Seridó São Fernando São João do Sabugi São José do Seridó Serra Negra do Norte Timbaúba dos Batistas mm 400 450 500 550 600 650 700 750 800 Paraíba 6°06' 6°12' 6°18' 6°24' 6°30' 6°36' 6°42' 6°48' Latitude Sul Paraíba 6°54' 37°24' 37°12' 37°00' 36°48' 36°36' 36°24' Longitude Oeste Acari Caicó Carnaúba dos Dantas Cruzeta Currais Novos Equador Ipueira Jardim de Piranhas Jardim do Seridó Ouro Branco Parelhas Santana do Seridó São Fernando São João do Sabugi São José do Seridó Serra Negra do Norte Timbaúba dos Batistas mm (a) 1000 1200 1400 1600 1800 2000 2200 2400 2600 Paraíba 6°06' 6°12' 6°18' 6°24' 6°30' 6°36' 6°42' 6°48' Latitude Sul Paraíba 6°54' 37°24' 37°12' 37°00' 36°48' 36°36' 36°24' Longitude Oeste Acari Caicó Carnaúba dos Dantas Cruzeta Currais Novos Equador Ipueira Jardim de Piranhas Jardim do Seridó Ouro Branco Parelhas Santana do Seridó São Fernando São João do Sabugi São José do Seridó Serra Negra do Norte Timbaúba dos Batistas mm (a) (b) 1000 1200 1400 1600 1800 2000 2200 2400 2600 Paraíba 6°06' 6°12' 6°18' 6°24' 6°30' 6°36' 6°42' 6°48' Latitude Sul Paraíba 6°54' 37°24' 37°12' 37°00' 36°48' 36°36' 36°24' Longitude Oeste Acari Caicó Carnaúba dos Dantas Cruzeta Currais Novos Equador Ipueira Jardim de Piranhas Jardim do Seridó Ouro Branco Parelhas Santana do Seridó São Fernando São João do Sabugi São José do Seridó Serra Negra do Norte Timbaúba dos Batistas mm (b) (c) 400 450 500 550 600 650 700 750 800 Paraíba 6°06' 6°12' 6°18' 6°24' 6°30' 6°36' 6°42' 6°48' Latitude Sul Paraíba 6°54' 37°24' 37°12' 37°00' 36°48' 36°36' 36°24' Longitude Oeste Acari Caicó Carnaúba dos Dantas Cruzeta Currais Novos Equador Ipueira Jardim de Piranhas Jardim do Seridó Ouro Branco Parelhas Santana do Seridó São Fernando São João do Sabugi São José do Seridó Serra Negra do Norte Timbaúba dos Batistas mm (c) FIGURA 3. Distribuição espacial do total anual da Evapotranspiração Real de Área(a), Evapotranspiração Potencial (b) e Precipitação (c). HOLOS, Ano 29, Vol. 6 29 JESUS & MATTOS (2013) (a) (b) (c) (d) o espacial da média anual de Temperatura do ar (a) e Umidade Relativa do ar (b), e o total anual do Saldo de Radiação (c) e Insolação (d). Paraíba 6°36' 6°42' 6°48' Latitud Paraíba 6°54' 37°24' 37°12' 37°00' 36°48' 36°36' 36°24' Longitude Oeste Equador Ipueira Jardim do Seridó Ouro Branco Parelhas Santana do Seridó São João do Sabugi Serra Negra do Norte 23°C 23.5°C 24°C 24.5°C 25°C Paraíba 6°06' 6°12' 6°18' 6°24' 6°30' 6°36' 6°42' 6°48' Latitude Sul Paraíba 6°54' 37°24' 37°12' 37°00' 36°48' 36°36' 36°24' Longitude Oeste Acari Caicó Carnaúba dos Dantas Cruzeta Currais Novos Equador Ipueira Jardim de Piranhas Jardim do Seridó Ouro Branco Parelhas Santana do Seridó São Fernando São João do Sabugi São José do Seridó Serra Negra do Norte Timbaúba dos Batistas 60% 62% 64% 66% 68% 70% 3550 3650 3750 3850 3950 4050 4150 4250 4350 4450 Paraíba 6°06' 6°12' 6°18' 6°24' 6°30' 6°36' 6°42' 6°48' Latitude Sul Paraíba 6°54' 37°24' 37°12' 37°00' 36°48' 36°36' 36°24' Longitude Oeste Acari Caicó Carnaúba dos Dantas Cruzeta Currais Novos Equador Ipueira Jardim de Piranhas Jardim do Seridó Ouro Branco Parelhas Santana do Seridó São Fernando São João do Sabugi São José do Seridó Serra Negra do Norte Timbaúba dos Batistas MJ/m².d Paraíba 6°06' 6°12' 6°18' 6°24' 6°30' 6°36' 6°42' 6°48' Latitude Sul Paraíba 6°54' 37°24' 37°12' 37°00' 36°48' 36°36' 36°24' Longitude Oeste Acari Caicó Carnaúba dos Dantas Cruzeta Currais Novos Equador Ipueira Jardim de Piranhas Jardim do Seridó Ouro Branco Parelhas Santana do Seridó São Fernando São João do Sabugi São José do Seridó Serra Negra do Norte Timbaúba dos Batistas Horas 2730 2770 2810 2850 2890 2930 2970 (a) (a) (b) Longitude Oeste Paraíba 6°06' 6°12' 6°18' 6°24' 6°30' 6°36' 6°42' 6°48' Latitude Sul Paraíba 6°54' 37°24' 37°12' 37°00' 36°48' 36°36' 36°24' Longitude Oeste Acari Caicó Carnaúba dos Dantas Cruzeta Currais Novos Equador Ipueira Jardim de Piranhas Jardim do Seridó Ouro Branco Parelhas Santana do Seridó São Fernando São João do Sabugi São José do Seridó Serra Negra do Norte Timbaúba dos Batistas 60% 62% 64% 66% 68% 70% (b) (b) (c) 37 24 37 12 37 00 36 48 36 36 36 24 Longitude Oeste 3550 3650 3750 3850 3950 4050 4150 4250 4350 4450 Paraíba 6°06' 6°12' 6°18' 6°24' 6°30' 6°36' 6°42' 6°48' Latitude Sul Paraíba 6°54' 37°24' 37°12' 37°00' 36°48' 36°36' 36°24' Longitude Oeste Acari Caicó Carnaúba dos Dantas Cruzeta Currais Novos Equador Ipueira Jardim de Piranhas Jardim do Seridó Ouro Branco Parelhas Santana do Seridó São Fernando São João do Sabugi São José do Seridó Serra Negra do Norte Timbaúba dos Batistas MJ/m².d Longitude Oeste (c) (c) (d) Longitude Oeste Paraíba 6°06' 6°12' 6°18' 6°24' 6°30' 6°36' 6°42' 6°48' Latitude Sul Paraíba 6°54' 37°24' 37°12' 37°00' 36°48' 36°36' 36°24' Longitude Oeste Acari Caicó Carnaúba dos Dantas Cruzeta Currais Novos Equador Ipueira Jardim de Piranhas Jardim do Seridó Ouro Branco Parelhas Santana do Seridó São Fernando São João do Sabugi São José do Seridó Serra Negra do Norte Timbaúba dos Batistas Horas 2730 2770 2810 2850 2890 2930 2970 (d) 00 36 48 Longitude Oeste FIGURA 4. mostra a média anual da temperatura do ar umidade relativa e déficit d Paraíba 6°06' 6°12' 6°18' 6°24' 6°30' 6°36' 6°42' 6°48' Latitude Sul Paraíba 6°54' 37°24' 37°12' 37°00' 36°48' 36°36' 36°24' Longitude Oeste Acari Caicó Carnaúba dos Dantas Cruzeta Currais Novos Equador Ipueira Jardim de Piranhas Jardim do Seridó Ouro Branco Parelhas Santana do Seridó São Fernando São João do Sabugi São José do Seridó Serra Negra do Norte Timbaúba dos Batistas 23°C 23.5°C 24°C 24.5°C 25°C 25.5°C 26°C 26.5°C 27°C Paraíba 6°06' 6°12' 6°18' 6°24' 6°30' 6°36' 6°42' 6°48' Latitude Sul Paraíba 6°54' 37°24' 37°12' 37°00' 36°48' 36°36' 36°24' Longitude Oeste Acari Caicó Carnaúba dos Dantas Cruzeta Currais Novos Equador Ipueira Jardim de Piranhas Jardim do Seridó Ouro Branco Parelhas Santana do Seridó São Fernando São João do Sabugi São José do Seridó Serra Negra do Norte Timbaúba dos Batistas 60% 62% 64% 66% 68% 70% 3550 3650 3750 3850 3950 4050 4150 4250 4350 4450 Paraíba 6°06' 6°12' 6°18' 6°24' 6°30' 6°36' 6°42' 6°48' Latitude Sul Paraíba 6°54' 37°24' 37°12' 37°00' 36°48' 36°36' 36°24' Longitude Oeste Acari Caicó Carnaúba dos Dantas Cruzeta Currais Novos Equador Ipueira Jardim de Piranhas Jardim do Seridó Ouro Branco Parelhas Santana do Seridó São Fernando São João do Sabugi São José do Seridó Serra Negra do Norte Timbaúba dos Batistas MJ/m².d Paraíba 6°06' 6°12' 6°18' 6°24' 6°30' 6°36' 6°42' 6°48' Latitude Sul Paraíba 6°54' 37°24' 37°12' 37°00' 36°48' 36°36' 36°24' Longitude Oeste Acari Caicó Carnaúba dos Dantas Cruzeta Currais Novos Equador Ipueira Jardim de Piranhas Jardim do Seridó Ouro Branco Parelhas Santana do Seridó São Fernando São João do Sabugi São José do Seridó Serra Negra do Norte Timbaúba dos Batistas Horas 2730 2770 2810 2850 2890 2930 2970 (a) Paraíba 6°06' 6°12' 6°18' 6°24' 6°30' 6°36' 6°42' 6°48' Latitude Sul Paraíba 6°54' 37°24' 37°12' 37°00' 36°48' 36°36' 36°24' Longitude Oeste Acari Caicó Carnaúba dos Dantas Cruzeta Currais Novos Equador Ipueira Jardim de Piranhas Jardim do Seridó Ouro Branco Parelhas Santana do Seridó São Fernando São João do Sabugi São José do Seridó Serra Negra do Norte Timbaúba dos Batistas 23°C 23.5°C 24°C 24.5°C 25°C 25.5°C 26°C 26.5°C 27°C (a) (b) (c) (d) FIGURA 4. Distribuição espacial da média anual de Temperatura do ar (a) e Umidade Relativa do ar (b), e o total anual do Saldo de Radiação (c) e Insolação (d). 4 CONCLUSÕES O presente estudo procurou mostrar a distribuição dos potenciais evapotranspirométricos sobre a microrregião do Seridó através do Modelo de relação complementar de Morton,o qual foi usado para gerar as evapotranspirações real de área e potencial. Como a precipitação em toda a região é baixaemrelação aos valores de evapotranspiração, a ETR responde a essa baixa, ficando bem abaixo da ETP. Dessa forma, tem-se que a ETR foisuperior ao extremo norte e sul e baixa na porção central do Seridó. Pode-se concluir que durante o ano a disponibilidade de energia seja a mais importante no processo de evapotranspiração do que a precipitação sobre a microrregião, sendo assim, o Modelo se torna adequado para avaliar a baixa na precipitação. JESUS & MATTOS (2013) estiveram nas localidades do Seridó Ocidental, tal como, Caicó (com 2.551,3mm), cujos osvalores combinam com a temperatura, umidade, déficit de saturação, saldo de radiação e insolação. estiveram nas localidades do Seridó Ocidental, tal como, Caicó (com 2.551,3mm), cujos osvalores combinam com a temperatura, umidade, déficit de saturação, saldo de radiação e insolação. TABELA 1. Média Anual da de temperatura do ar (T), Umidade relativa do ar (UR), déficit de saturação de vapor (e) e o total anual de evapotranspiração real de área (ETR), evapotranspiração potencial (ETP), precipitação (PRP), saldo de radiação solar (RN) e insolação (INS) dos municípios da microrregião do Seridó. (PRP), saldo de radiação solar (RN) e insolação (INS) dos municípios da microrregião do Seridó. Média Anual Total Anual Município T (°C) UR (%) e (mb) ETR (mm) ETP (mm) PRP (mm) RN (MJ.m-2.d-1) INS (hrs) Acari 25,2 68,8 10,1 1.270,0 2.325,9 492,0 4.206,4 2.860,0 Caicó 26,5 64,2 12,5 1.111,4 2.551,3 666,0 4.135,1 2.925,0 Carnaúba dos Dantas 25,0 69,2 9,8 1.277,0 2.296,5 621,0 4.192,3 2.850,0 Cruzeta 25,7 68,3 10,5 1.271,9 2.378,8 583,0 4.243,6 2.890,0 Currais Novos 24,6 70,4 9,1 1.291,1 2.207,4 444,0 4.134,6 2.740,0 Equador 23,1 68,3 9,0 1.175,6 2.184,6 400,0 3.984,4 2.795,0 Ipueira 26,2 65,0 12,0 1.166,0 2.520,4 805,0 4.204,9 2.936,0 Jardim de Piranhas 26,9 65,8 12,2 1.223,6 2.549,7 744,0 4.292,9 2.945,0 Jardim do Seridó 25,7 65,0 11,6 1.124,4 2.463,4 570,0 4.104,3 2.907,5 Ouro Branco 25,9 65,0 11,8 1.131,7 2.486,6 568,0 4.128,9 2.925,0 Parelhas 25,4 69,2 10,0 1.312,6 2.338,3 563,0 4.276,9 2.875,0 Santana do Seridó 24,9 65,8 10,8 1.131,8 2.385,0 723,0 4.064,3 2.875,0 São Fernando 26,7 65,8 12,0 1.210,6 2.532,3 701,0 4.264,3 2.925,0 São João do Sabugi 26,4 65,0 12,1 1.123,6 2.495,8 632,0 4.093,9 2.830,0 São José do Seridó 25,9 65,8 11,5 1.176,2 2.470,5 578,0 4.180,2 2.910,0 Serra Negra do Norte 26,7 65,0 12,4 1.185,5 2.559,4 742,0 4.253,2 2.985,0 Timbaúba dos Batistas 26,5 64,2 12,5 1.117,5 2.561,3 665,0 4.154,3 2.955,0 Distribuição espacial da média anual de Temperatura do ar (a) e Umidade Relativa do ar (b), e o total anual do Saldo de Radiação (c) e Insolação (d). A Tabela 1 mostra a média anual da temperatura do ar, umidade relativa e déficit de saturação, além do total anual da precipitação e insolação dos municípios da microrregião do Seridó. Além disso, os valores obtidos pelo modelo complementar de Morton, como o total anual de ETR, ETP e RN para cada munícipio citado. Verificou-se que os valores mais elevados de ETP HOLOS, Ano 29, Vol. 6 30 6 REFERÊNCIAS BIBLIOGRÁFICAS 1. BEZERRA JUNIOR, J. G.O.; SILVA, N. M. Caracterização Geoambiental Da Microrregião Do Seridó Oriental Do Rio Grande Do Norte. RevistaHolos, Ano 23, Vol. 2. 2007. 2. BOUCHET, R.J. Evapotranspiration Réelleetpotentielle, signification climatique. In: International Association of Scientific Hydrology Symposium, 62., 1963, Berkeley, Anais...p.134-142. 3. CAVALCANTI, E.P.; SILVA, V.P.R.; SOUSA, F.A.S. Programa computacional para a estimativa da temperatura do ar para a Região Nordeste do Brasil. Revista Brasileira de Engenharia Agrícola e Ambiental, Campina Grande, PB, v.10, n.1, p.140-147, 2006. 4. GOSSON, P. C. R. Avaliação de métodos de balanço de energia para determinação da evaporação em lago na região semi-árida do RN. Dissertação de mestrado. Universidade Federal do Rio Grande do Norte. Natal (RN), 2005. 5. ISAIAS, E. M. B. I.; ISAIA, T.; VERSLYPE, C.; GARIGLIO, M. A. Avaliação do estoque lenheiro do Estado do Rio Grande do Norte – 1ª etapa: Estratificação e mapeamento da vegetação nativa lenhosa através de composições coloridas do TM Landsat. PNUD/FAO-IBAMA. Nota técnica. N. 4, 29p, 1992. 6. JESUS, E. S.; SCHMIDT, D. M.; MATTOS, A.; GOMES JESUS, N. V. Estimativa da Evapotranspiração de alguns Municípios da Região do Seridó–RN. Congresso Brasileiro de Meteorologia, Gramado-RS. 2012. Anais do XVII CBMET. 7. JESUS, E.S. Avaliação Mensal da Evapotranspiração no Estado do Rio Grande do Sul utilizando o Modelo Complementar de Morton. Dissertação de mestrado. Universidade Federal de Pelotas. 2004. 89p. 8. JESUS, E.S., ASSIS, S.V. Evapotranspiração Mensal da Região da Campanha do Estado do Rio Grande do Sul. Congresso Brasileiro de Meteorologia, Fortaleza-CE. 2004. Anais do XIII CBMET. 9. MENDONÇA, E. A.; DANTAS, R. T. Estimativa da Evapotranspiração de Referência no município de Capim, PB. Revista Brasileira de Engenharia Agrícola e Ambiental, v. 14, n. 2, p. 196–202, 2010. 10. MORTON, F.I. Operation estimatives of areal evapotranspiration and their significance to the science and practice of hydrology. JournalofHydrology, v.66,p.1-76.1983. 11. MORTON, F.I., RICARD, F., FOGARASI, S. Operational estimates of areal evapotranspiration and lake evaporation – Program WREVAP. National Hydrology Research Institute. Inlan Waters Directorate. Ottawa, Canada, NHRI PAPER, n.24, 1985. 12. MOTA, F. S. Meteorologia Agrícola, São Paulo, NOBEL, 1977. 13. PEREIRA, A. R.; VILLA NOVA, N. A.; SEDIYAMA, G. C. Evapotranspiração. Piracicaba: FEALQ, 183p. 1997. 14. PRIESTLEY CHB; TAYLOR RJ. On the assessment of surface heat flux and evapotranspiration using large scale parameters. Mon Weather Rev 100:81–92.1972. 15. ROSS, Jurandir L. Sanches. Geografia do Brasil. São Paulo: Edusp, p.57, p.63,p.103 16. SANTOS, A. JESUS & MATTOS (2013) 5 AGRADECIMENTOS Os autores agradecem ao Programa de Pós-Graduação em Ciências Climáticas ao Laboratório de Recursos Hídricos e Saneamento Ambiental e a CAPES pelo apoio a esta pesquisa. HOLOS, Ano 29, Vol. 6 31 6 REFERÊNCIAS BIBLIOGRÁFICAS S.; MATTOS, A.; LOPO, A. B.; MEDEIROS, F. C.; BRISTOT, G.; PINHEIRO, J. U. Estimativa do Balanço Hídrico Climático para a Microrregião do Seridó - Estado do Rio Grande do Norte. Congresso Brasileiro de Meteorologia, Belém-PA. 2010. Anais do XVI CBMET. HOLOS, Ano 29, Vol. 6 32
https://openalex.org/W4282588699	http://bura.brunel.ac.uk/bitstream/2438/24687/1/FullText.pdf	English	null	Frataxin Deficit Leads to Reduced Dynamics of Growth Cones in Dorsal Root Ganglia Neurons of Friedreich’s Ataxia YG8sR Model: A Multilinear Algebra Approach	Frontiers in molecular neuroscience	2,022	cc-by	14,768	Edited by: Benoit Laurent, Université de Sherbrooke, Canada 1 Chemical Biology Group, Department of Biomedical Engineering, Eindhoven University of Technology (TU/e), Eindhoven, Netherlands, 2 Department of Genetics, Faculty of Biological Sciences, University of Valencia, Valencia, Spain, 3 CIBER de Enfermedades Raras (CIBERER), Valencia, Spain, 4 Brain Connectivity Laboratory, Joint Unit FISABIO & Prince Felipe Research Centre (CIPF), Valencia, Spain, 5 Regional Ministry of Health in Valencia, Hospital Sagunto (CEIB-CSUSP), Valencia, Spain, 6 CIBER de Salud Mental (CIBERSAM), Valencia, Spain, 7 The Mathworks B.V., Eindhoven, Netherlands, 8 Biosciences, Brunel University London, Uxbridge, United Kingdom, 9 Department of Physiology, Faculty of Medicine and Dentistry, University of Valencia, Valencia, Spain, 10 Biomedical Research Institute INCLIVA, Valencia, Spain, 11 Department of Genetic and Molecular Medicine IPER, Institut de Recerca Sant Joan de Déu, Hospital Sant Joan de Déu, Barcelona, Spain, 12 Division of Pediatrics, University of Barcelona School of Medicine and Health Sciences, Barcelona, Spain Reviewed by: Elisabetta Indelicato, Medizinische Universität Innsbruck, Austria David Lynch, University of Pennsylvania, United States Correspondence: Pilar Gonzalez-Cabo pilargc@uv.es †These authors have contributed equally to this work ‡These authors share senior authorship Reviewed by: Elisabetta Indelicato, Medizinische Universität Innsbruck, Austria Reviewed by: Elisabetta Indelicato, Medizinische Universität Innsbruck, Austria David Lynch, University of Pennsylvania, United States David Lynch, University of Pennsylvania, United States Correspondence: Pilar Gonzalez-Cabo pilargc@uv.es †These authors have contributed equally to this work ‡These authors share senior authorship Correspondence: Pilar Gonzalez-Cabo pilargc@uv.es †These authors have contributed equally to this work ‡These authors share senior authorship Computational techniques for analyzing biological images offer a great potential to enhance our knowledge of the biological processes underlying disorders of the nervous system. Friedreich’s Ataxia (FRDA) is a rare progressive neurodegenerative inherited disorder caused by the low expression of frataxin, which is a small mitochondrial protein. In FRDA cells, the lack of frataxin promotes primarily mitochondrial dysfunction, an alteration of calcium (Ca2+) homeostasis and the destabilization of the actin cytoskeleton in the neurites and growth cones of sensory neurons. In this paper, a computational multilinear algebra approach was used to analyze the dynamics of the growth cone and its function in control and FRDA neurons. Computational approach, which includes principal component analysis and a multilinear algebra method, is used to quantify the dynamics of the growth cone (GC) morphology of sensory neurons from the dorsal root ganglia (DRG) of the YG8sR humanized murine model for FRDA. Keywords: growth cone, DRG neurons, neurobiology of the disease, Friedreich’s ataxia, tensor decompositions, multilinear algebra ORIGINAL RESEARCH published: 13 June 2022 doi: 10.3389/fnmol.2022.912780 ORIGINAL RESEARCH published: 13 June 2022 doi: 10.3389/fnmol.2022.912780 Frataxin Deﬁcit Leads to Reduced Dynamics of Growth Cones in Dorsal Root Ganglia Neurons of Friedreich’s Ataxia YG8sR Model: A Multilinear Algebra Approach Diana C. Muñoz-Lasso1, Belén Mollá2,3†, Jhon J. Sáenz-Gamboa4,5,6†, Edwin Insuasty7, Maria de la Iglesia-Vaya4,5,6, Mark A. Pook8, Federico V. Pallardó3,9,10, Francesc Palau3,11,12‡ and Pilar Gonzalez-Cabo3,9,10‡ Diana C. Muñoz-Lasso1, Belén Mollá2,3†, Jhon J. Sáenz-Gamboa4,5,6†, Edwin Insuasty7, Maria de la Iglesia-Vaya4,5,6, Mark A. Pook8, Federico V. Pallardó3,9,10, Francesc Palau3,11,12‡ and Pilar Gonzalez-Cabo3,9,10‡ Edited by: Benoit Laurent, Université de Sherbrooke, Canada It was conﬁrmed that the dynamics and patterns of turning were aberrant in the FRDA growth cones. In addition, our data suggest that other cellular processes dependent on functional GCs such as axonal regeneration might also be affected. Semiautomated computational approaches are presented to quantify differences in GC behaviors in neurodegenerative disease. In summary, the deﬁciency of frataxin has an adverse effect on the formation and, most importantly, the growth cones’ function in adult DRG neurons. As a result, frataxin deﬁcient DRG neurons might lose the intrinsic capability to grow and regenerate axons properly due to the dysfunctional GCs they build. Specialty section: This article was submitted to Neuroplasticity and Development, a section of the journal Frontiers in Molecular Neuroscience Specialty section: This article was submitted to Neuroplasticity and Development, a section of the journal Frontiers in Molecular Neuroscience Received: 04 April 2022 Accepted: 17 May 2022 Published: 13 June 2022 Edited by: Benoit Laurent, Université de Sherbrooke, Canada INTRODUCTION spatial patterns of the dynamics of growth cone morphology in control and Friedreich’s Ataxia neurons (FRDA, OMIM 229300, ORPHA 95). Computational methods for analyzing biological time-lapse experiments are accelerating the pace of research of neurological diseases every day. In this context, the application of linear algebra methods is well established. Recently, Goodhill et al. (2015) have used principal component analysis (PCA) to analyze the dynamics of growth cone (GC) morphology. In this paper, ﬁrstly, we validate the use of PCA to determine principal motion patterns of the GC. Secondly, a generalization of the methods described in Goodhill et al. (2015), is presented by implementing multilinear algebra approaches using tensor decompositions to quantify the dynamics of the growth cone morphology in the neurological disease: Friedreich’s ataxia. The FRDA is a rare progressive neurodegenerative inherited disorder aﬀecting the Caucasian population with two to four individuals per 100,000 (Polo et al., 1991; López-Arlandis et al., 1995). The most common mutation in FRDA patients is a homozygous guanine-adenine-adenine (GAA) trinucleotide repeat expansion in the ﬁrst intron of FXN gene encoding frataxin, which leads to a reduced expression of this protein in all the cells. FRDA is characterized by the dorsal root ganglia (DRG) neurodegeneration through a wholly unknown mechanism. Nevertheless, previous ﬁndings, including our research, suggest a link between the DRG neurons’ growth cone (GC) and the pathological hallmarks of FRDA. FRDA cells have impaired mitochondrial ATP production (González-Cabo and Palau, 2013; Bolinches-Amorós et al., 2014), increased oxidative stress (Rodríguez et al., 2020b), calcium dysregulation (Mollá et al., 2017; Rodríguez et al., 2020a) and cytoskeletal destabilization (Pastore et al., 2003; Piermarini et al., 2016; Mollá et al., 2017; Muñoz-Lasso et al., 2020b), which turn on to be essential to regulate the motility of GC and the axonal pathﬁnding (McCormick and Gupton, 2020; Muñoz-Lasso et al., 2020a). The growth cone (GC) is a sensitive and motile structure accountable for directing and guiding axons and neurites through the tissues and ensuring an accurate connection with their target cells. It appears at the tip of growing axons and neurites during neurite development or axonal regeneration. The GC morphology changes quickly in response to attractive and repulsive clues. It is characterized by presenting two parts, a broad, ﬂattened structure called lamellipodia and thin peaks called ﬁlopodia. Citation: Muñoz-Lasso DC, Mollá B, Sáenz-Gamboa JJ, Insuasty E, de la Iglesia-Vaya M, Pook MA, Pallardó FV, Palau F and Gonzalez-Cabo P (2022) Frataxin Deﬁcit Leads to Reduced Dynamics of Growth Cones in Dorsal Root Ganglia Neurons of Friedreich’s Ataxia YG8sR Model: A Multilinear Algebra Approach. Front. Mol. Neurosci. 15:912780. doi: 10.3389/fnmol.2022.912780 June 2022 \| Volume 15 \| Article 912780 1 Frontiers in Molecular Neuroscience \| www.frontiersin.org Growth Cone Dynamic in FRDA Muñoz-Lasso et al. MATERIALS AND METHODS Computational multilinear algebra approaches preserve the spatial-temporal data structures for the analysis, avoiding the vectorization process required by the classical PCA. For this reason, multilinear algebra methods and tensor decompositions for signal and image processing have been applied successfully in medical technology. Yahyanejad et al. (2019) have provided a review of recent applications of tensor analysis methods for biological systems. Also, in bioinformatics, Omberg et al. (2007) used tensor higher-order singular value decomposition for integrative analysis of DNA microarray data. Additionally, in medical imaging, Mathew and Kumar applied Multilinear Principal Component Analysis (MPCA) in order to reduce the dimensionality of the raw data for the classiﬁcation of diseases using Support Vector Machines (Mathew and Vijaya Kumar, 2019). Furthermore, in neuroscience, Miwakeichi et al. (2004) have applied tensor decompositions to electroencephalographic (EEG) data to identify spatial and temporal patterns. INTRODUCTION Moreover, three regions are distinguished based on cytoskeletal content: a central domain (C) rich in microtubules; a peripheral domain (P) enriched with actin ﬁlaments; and a transition zone consisting of an area at the interface of the P and C domains. Consequently, when a GC extends, retracts or turns, the cytoskeletal components within the GC are reorganized. This paper focuses on studying the dynamics of GC morphology in FRDA. Given the spatial-temporal nature of the GC dynamics, this fundamental question is addressed by applying a well-established computational linear algebra approach (principal component analysis), and extending it by using tensor decompositions to quantify the dynamics of the GC cone morphology. These promise to open a new research direction in imaging and tool development and it is hoped that they will contribute to an enhanced understanding of the role of GC in neurodegenerative diseases. The growth cone moves toward its target in three phases: protrusion, obstruction, and consolidation. First, the lamellipodia and ﬁlopodia extend (protrusion). Secondly, the lamellipodia swells while the growth cone swallows it (obstruction). Third, the growth cone narrows and becomes a new axon or neurite (consolidation). Frontiers in Molecular Neuroscience \| www.frontiersin.org Animals Dr. Mark Pook donated YG8sR and Y47R mice used in this study. The YG8sR and control mice are available in The Jackson Laboratory Repository (YG8sR, Stock No: 024113 and Y47R, Stock No: 024097). The YG8sR mouse model has been derived from natural breeding of YG8R mouse. YG8sR mice are homozygous for a mutation in the Fxn gene (knock-out for the Fxn allele; Fxn−) and hemizygous for the YG8s transgene (a copy of the F.X.N. gene with ∼250–300 repeats of the GAA triplet). Compared to other human FXN YAC transgenic rescue mouse models such as the YG8R mouse, the YG8sR model expresses contracted human frataxin, resulting in much more signiﬁcant FXN deﬁciency (Anjomani Virmouni et al., 2015). The mouse model was ampliﬁed by crossing YG8sR mice with a single copy of the YG8s transgene (Fxn−/−; FXN±) with the control Y47R mice containing a single copy of the YG8 transgene containing 9 regular repetitions of the GAA triplet on an Fxn−/−background. Animals were maintained and genotyped as previously described (Anjomani Virmouni et al., 2015). For this study, only males were included in the tests. To study the dynamics of neuronal morphology, researchers typically design time-lapse experiments in order to record axonal growth. Time-lapse experiments record images with two spatial coordinates and time as the third coordinate. In this paper, the use of tensor decompositions is extended to identify the main June 2022 \| Volume 15 \| Article 912780 Frontiers in Molecular Neuroscience \| www.frontiersin.org 2 Growth Cone Dynamic in FRDA Muñoz-Lasso et al. All procedures were carried out following the United Kingdom Home Oﬃce’ Animals (Scientiﬁc Procedures) Act 1986’ and with approval from the Brunel University Animals Welfare and Ethical Review Board. the glass slides to air-dry overnight in a bio-safety cabinet. We assembled the coated cover glasses for the Silicone Device the same day. For this, we ﬁrst removed any debris remaining on the Silicone Device by using 3MTM Scotch Brand 471 Vinyl tape. Then, we proceeded to sterilize the Silicone Devices by rinsing them with 70% ethanol and allowing them to air dry the feature side up for 1 h in the bio-safety cabinet, ensuring that the Devices were thoroughly dry before proceeding. To assemble the devices, we put each PDL coated glass into a new sterile 60 mm dish and placed each device evenly on top of each coated glass using two tweezers (previously sterilized by autoclave). Animals After that, we added 150 µl of laminin to the top left well, allowed the laminin to enter the main channel for 1 min, and then ﬁlled the top right well with 150 µl of laminin. Returned to the incubator and incubated overnight. The next day, the day of primary culture, we removed the laminin. We added 150 µl to the top right well, allowed the media to enter the main channel for 1 min, then ﬁlled the bottom right well with 150 µl of media and returned to the incubator 2–3 h (the time expended to isolate the DRG neurons). Preparation of the Non-plasma Bonded Devices We prepared the microﬂuidic devices following the instructions recommended by Xona Microﬂuidics, Inc. and choosing the best conditions for primary culture of dorsal root ganglia neurons. The ﬁrst step is sterilizing the Corning (24 mm × 40 mm) cover glasses. For this, we placed them in a stainless-steel staining rack and immersed them in a glass staining dish full of 95% ethanol (sealed with paraﬁlm). Later, we sonicated them in a water bath sonicator for 30 min. We left the slides to dry in a biosafety cabinet overnight. The next day, we coated the cover glasses with 0.5 mg/mL Poli-DL-Ornitin (PDL, Sigma-Aldrich). To do this, we put the sterile cover glasses in sterile 100 mm dishes (2 per each 10 cm petri dish) and added 1 mL of the sterile 0.5 mg/ml PDL solution on each cover glass and incubated overnight. The next day, we proceeded to rinse the glass slides three times with sterile dH2O (taking care they do not overlap), placed each cover glass in sterile 60 mm dishes, and ﬁlled the dish with 3 mL sterile dH2O (cover slices are covered) and incubated for 3 h. Immediately after, we rinsed the cover glass with sterile dH2O three more times, then aspirated oﬀthe water and allowed In vitro Axotomy Primary culture of DRGs isolated from mice with 2 months of age in microﬂuidic chambers was performed and maintained at 37◦C in a humidiﬁed 5% CO2 incubator as described above. The medium was supplemented with murine NGF and GDNF as follows; Days 0–1: 100 ng/ml of each neurotrophin; Days 1–3: somal compartment, 50 ng/ml; axonal compartment, 100 ng/ml; Day 3 onwards: somal compartment, 25 ng/ml; axonal compartment, 100 ng/ml and were grown for 48 h. Axotomy was performed at 3 DIV (days in vitro) by transecting the axons away from the cell bodies with suction for 5 s with a ﬁne tip glass pipette. The axonal compartment was immediately washed and replaced with a fresh medium. Then, cultures were incubated for 2 days. Time-Lapse Experiment Primary cultures in glass-bottom dishes were incubated during 12–14 h at 37◦C, 5% CO2. Phase-contrast images of neurons and their GCs were acquired with a LD Achroplan 40×/0.60 dry objective (Zeiss) for 1 h at 30-s intervals with an incubator- inverted microscope (Axiovert 200 M, Zeiss). Images of SOMA and AXON chambers were acquired at diﬀerent steps of the in vitro axotomy. Five DIV (before axotomy), immediately after, and 48 h after axotomy. Phase- contrast images of cell bodies (SOMA chamber) and axonal network (AXON chamber) were acquired at 40× with an incubator-inverted microscope (Axiovert 200 M, Zeiss). Primary Culture of Adult Dorsal Root Ganglia Neurons g Y47R and YG8sR mice were euthanized by cervical dislocation following the laboratory animals’ care and use guidelines. Whole DRGs were dissected from the entire length of the vertebral column and maintained in ice-cold L-15 (Leibovitz) medium. DRGs were incubated with collagenase and 2.5% Trypsin, then washed in Nutrient Mixture F-12 Ham (Sigma), followed by gentle mechanical trituration in 1 ml of medium. Isolated neurons were pelleted by centrifugation (59 g for 8 min) through a 15% bovine serum albumin (BSA) cushion (BSA 30%: HBSS−/−in a ratio 1:1). Neurons were suspended in media and then seeded either in glass-bottom dishes (MatTek) at 10 neurons/mm2, or in microﬂuidic chambers (Xona Microﬂuidics, SND150) at a density of 3,5 × 104 neurons per device (see Supplementary Methods for a detailed protocol). Dishes and microﬂuidic devices were previously coated with 0.5 mg/mL of poly-DL-Ornitin and 0.01 mg/mL of laminin (Sigma). Glass bottom dishes were ﬁlled with 2 mL of growth medium and supplemented with mouse nerve growth factor (mNGF, 10 ng/mL, Peprotech bioNova), and mouse glial cell line-derived neurotrophic factor (mGDNF, 10 ng/mL). Frontiers in Molecular Neuroscience \| www.frontiersin.org Morphometric Analysis of the Growth Cone We established and followed customized parameters to quantify the growth cone’s morphology (GC) (Goodhill et al., 2015) based on our expertise and others. We chose to analyze the morphology of the growth cone with phase-contrast imaging with in vivo conditions to diminish the eﬀect of external factors other than the reduced expression of frataxin in DRG neurons. Images were captured of entire neurons to choose the GC at the tip of the thickest neurite, which probably corresponds to the axon. We selected GCs that were not overlapping with other neurites/axons or debris. Furthermore, we chose to analyze GC exhibiting a hand-like shape. Images were processed and analyzed with the Fiji/Image- J software (N.I.H). A polygon selection was manually traced Frontiers in Molecular Neuroscience \| www.frontiersin.org Frontiers in Molecular Neuroscience \| www.frontiersin.org June 2022 \| Volume 15 \| Article 912780 3 Growth Cone Dynamic in FRDA Muñoz-Lasso et al. network. The parameters settled here were slightly adjusted from those proposed by Sasaki et al. (2009), who used the primary culture of DRGs isolated from embryonic mice as a model. following the peripheral region of each GC, and the measurement tool was executed. We selected what we considered the 4 most biological relevant parameters: the area, Feret’s diameter, circularity and solidity. On the one hand, the area and Feret’s diameter (the longest distance between any two points along the selection boundary) reﬂect the GC’s size. The circularity and solidity are shape descriptors that reﬂect the geometry of the GC. Circularity (4π∗area/perimeter2) indicate how circular is the GC, a value of 1.0 indicates a perfect circular GC. As values approach 0, it means a more elongated GC. Besides, solidity is the ratio of GC area over their convex area; then, it shows how much a GC ﬁlls the area of its ﬁlopodial spread (Chitsaz et al., 2015). Motion Tracking Analysis With MtrackJ The GCs were imaged every 30 s for 1 h using Zeiss Axiovision software. Active GCs (spread growth cones, not retracted) and growing in all directions were chosen for these measurements. Therefore, we handle all sequence images to get a common reference point. Sequence images of GCs were extracted from the original ﬁles. Then, they were rotated to get the base of the growth cone centered on the x-axis and the GC (ﬁlopodia and lamellipodia) pointing to the right. Analysis of Axonal Growth and Regeneration in Microﬂuidic Chambers g Phase-contrast images were acquired with in vivo conditions and processed following the method proposed by Sasaki et al. (2009) with some modiﬁcations. Images were acquired from random ﬁelds in the axon chamber from three independent experiments. Phase-Contrast images were processed with the Analyze particles – plugin of Fiji/ImageJ (N.I.H) software (Schindelin et al., 2012) to obtain the measurements of the total and the fragmented area of the axonal network. Later, we calculated the degeneration index as the ratio of the fragmented area and total area as proposed by Sasaki et al. (2009). In order to use the Analyze particle plugin, we had to establish some conditions and parameters based on those settled by Sasaki et al. (2009), (Supplementary Figure 4). First, we extracted the minimum 5 and maximum of 10 quadrangular regions (700 pixels × 700 pixels) from images of separated ﬁelds of the axonal chamber. Therefore, per each genotype, a minimum of 15 and a maximum of 30 quadrangular regions were analyzed (N = 3 mice per genotype, Supplementary Table 1). Those areas where the axons were stacking were excluded for this analysis. In order to optimize the image processing and avoid biases, images were ordered in stacks based on similarities of the grayscale and then processed with the Process module of Fiji/ImageJ (NIH) to increase contrast and resolution with the steps described next: (a) Filters: Median (radius: 0.2 pixels), (b) Subtract background (Rolling ball radius: 5 pixels, Light background, Sliding paraboloid), (c) Enhance contrast (Saturated pixels: 0.3%, Normalize, Equalize histogram). Later we obtained binary images following the next steps and parameters: (a) Threshold (Method: Otsu), Black and White (B&W), Light background (dark background inactive). Morphometric Analysis of the Growth Cone The base of the GCs was tracked manually using the MtrackJ plugin (Meijering et al., 2012). A 5 min time interval was chosen to avoid variability (Goodhill et al., 2015). The average value obtained for length, velocity and α [◦] in each track (one-time lapse per GC) was used for statistical analysis. α [deg] is the angle of the in-plane component of the most recent displacement vector (pointing from the previous point to the current point of the track) with respect to the x–y- coordinate system of the image (with the origin taken in the previous point). The values range from −180 to +180 degrees, where 0 degrees means the vector component runs parallel to the positive x-axis (pointing to the right), +90 degrees (or −90 degrees) means it runs parallel to the positive (or negative) y-axis (pointing downward, or upward, respectively), and +180 degrees (which is the same as −180 degrees) means it runs parallel to the negative x-axis (pointing to the left). Study of the Growth Cone Dynamics by Principal Component Analysis The dynamics of the growth cone morphology is complex and diﬃcult to quantify due to the size, the shape and/or the position of the growth cone change within minutes (Goldberg and Burmeister, 1986; Goodhill et al., 2015). Here, we have implemented the method based on Principal Component Analysis (PCA) proposed by Goodhill et al. (2015) with customized modiﬁcations. The methodology included several steps (Figure 3A). First, we obtained segmented images from the time-lapse experiments. To do this, we developed a customized platform in MATLAB R⃝(The MathWorks, Inc.) that allowed us to process the experiments semi-automatically (∼1 h per time-lapse) (Supplementary Figure 3). We captured the growth cone outline from the segmented time-lapse experiments and obtained a list of coordinates (x-, y-). At this point, we got coordinates that represented each growth cone outline as a single point in a 500-dimensional space. Last, we performed a PCA algorithm in MATLAB R⃝to obtain the directions of the space’s main patterns. The two main components explaining most of the data’s variance were visualized using the bi-plot algorithm in MATLAB R⃝. Once we got the binary images, we proceeded to obtain the measurements for the axonal network’s fragmented and total area. To calculate the fragmented area, we used the analyze particle plugin of Fiji/ImageJ (N.I.H) following the next steps and parameters: (a) particle size: 20–10000 Pixel Units, (b) circularity: 0.5–1.0, (c) exclude analysis at edges: yes. To quantify the total area, we obtained the total sum of pixels (RawIntDen) for each binary-region with the R.O.I. manager tools of Fiji/ImageJ (NIH). We calculated the degeneration index with these data, which consisted of dividing the area occupied by the axonal network’s fragments by the value of the total area occupied by the axonal Frontiers in Molecular Neuroscience \| www.frontiersin.org Tensor Decomposition Analysis The method proposed by Goodhill et al. (2015) is based on the application of principal component analysis (PCA), which is fundamentally suitable for 2-dimensional data. The image June 2022 \| Volume 15 \| Article 912780 4 Growth Cone Dynamic in FRDA Muñoz-Lasso et al. sequence of living cells can be stacked as spatial temporal 3- dimensional data structures, and 4–dimensional data structures if we consider an additional coordinate for the type of cells. In linear algebra, tensors are the generalizations of vectors and matrices for multiple coordinates. For this reason, tensors are the most convenient mathematical form to handle the 3- dimensional or 4-dimensional data structures generated by time- lapse biology experiments. Traditionally in PCA (a technique for 2-dimensional data), the images (which are spatial data structures in two coordinates) are converted into vectors (1- dimensional data structures) and then stacked in a data matrix (2-dimensional data structure). This process destroys the spatial structure and the spatial correlations relevant to the subsequent analysis. This section has applied methods based on multilinear algebra and tensor analysis to overcome this limitation. Tensors are multidimensional generalizations of the concept of vectors and matrices. Since the 2-dimensional spatial structure in the images is preserved, it is also possible to maintain the correlation space-time in a sequence of images. Figure 1). This approach allowed us to analyze the morphology of the GCs from DRG neurons at 2 and 6 months of age in a natural state without aﬀecting neurons’ viability (Figure 1A). As described in methods, we manually selected the GCs following the GC-outline in Fiji/ImageJ (NIH) of the images acquired and got measurements for several shape parameters: circularity, Feret’s diameter, solidity, and area. Figure 1). This approach allowed us to analyze the morphology of the GCs from DRG neurons at 2 and 6 months of age in a natural state without aﬀecting neurons’ viability (Figure 1A). As described in methods, we manually selected the GCs following the GC-outline in Fiji/ImageJ (NIH) of the images acquired and got measurements for several shape parameters: circularity, Feret’s diameter, solidity, and area. At 2 months of age we found that 73.3% of the mice YG8sR-GCs had an area between 0 and 100 µm2, contrasting with the 78.9% obtained for the control GCs (Figure 1B). Growth Cones From Frataxin Deﬁcient Dorsal Root Ganglia Neurons Show Abnormal Morphology Growth Cones From Frataxin Deﬁcient Dorsal Root Ganglia Neurons Show Abnormal Morphology Frataxin Deﬁciency Reduces the Neurite Extension Rate and Alters the Growth Cone Turning in Adult Dorsal Root Ganglia Neurons The murine models that emulate the mutation responsible for the FRDA present a series of complications, mainly because the genomic environment is diﬀerent. Overall, they show signs of the start of the neurodegeneration cascade; however, the dorsal root ganglia (DRG), dentate nucleus (DN), or cerebellum do not degenerate as observed in the pathology of the human disease, probably because of the short lifespan of murine models. On the other hand, these models, particularly the YG8R/YG8sR mice (Mollá et al., 2017; Muñoz-Lasso et al., 2020a), have been broadly characterized by several researchers who have determined that they are suitable for investigating the ﬁrst neuropathological changes occurring in the FRDA and establishing the molecular and cellular mechanisms that underlie the neurodegenerative process. We previously showed that GCs of frataxin-deﬁcient neurons from a 24 months old mice model for FRDA, the YG8R mouse, were smaller and showed qualitative morphological changes compared to their controls (Muñoz-Lasso et al., 2020a). Consequently, we aimed to conﬁrm quantitatively whether these morphological observations were present in the living GCs of adult neurons isolated from YG8sR mice and determine their evolution. To quantify changes in the GCs of DRG neurons, we performed phase-contrast imaging with in vivo conditions of the dorsal root ganglia’s primary culture (DRGs) (Supplementary The GCs of DRG neurons are highly motile structures that change their morphology quickly during growth (Supplementary Video 1). Because the morphology and the function of the growth cone are directly connected, we were aimed to determine whether the aberrant morphology observed for the adult YG8sR-GCs was also aﬀecting the GC-dynamics by measuring the speed, displacement and GC-turning events. To reach this goal, here we used Time-Lapse phase-contrast microscopy to record living GCs (Figure 2A) and a particle tracking plugin: MTrackJ (Meijering et al., 2012) for tracking the GC base (Figure 2B and see Section “Materials and Methods” for detailed explanation). We choose to track the base of the GC (Figure 2B) because, (i) it is easy to detect and, therefore, (ii) the position of the base changes as soon as the GC advances (Supplementary Videos 1–6). Before performing the manual tracking, all time-lapse experiments were orientated so that the GC’s base was centered to the x-axis and pointing to the right, so there is a common point of reference for all the time-lapse experiments (see Section “Materials and Methods”). Tensor Decomposition Analysis Then, mice YG8sR-GCs showed a reduced area compared with control neurons GCs (YG8sR: 105.74 ± 15.83 µm2; control: 110.16 ± 9.66 µm2; ∗∗P-value = 0.001) and reduced Feret’s diameter (YG8sR: 17.56 ± 0.71 µm2; control: 19.27 ± 0.51 µm; P-value = 0.001) (Figure 1C). Furthermore, the mice YG8sR-GCs were more circular (YG8sR: 0.36 ± 0.02; control: 0.31 ± 0.02; P-value = 0.014) (Figure 1D) and were characterized for showing a solid geometry (YG8sR: 0.69 ± 0.02; control: 0.65 ± 0.02; P-value = 0.024) (Figure 1E) compared to the controls. Signiﬁcant diﬀerences were not detected at 6 months for any shape parameters, including the area and Feret’s diameter (Table 1 and Figures 1B–D). These results are explained by the high variability of the data observed at 6 months of age in both genotypes (Table 1), particularly in the control mice. Furthermore, the control and YG8sR data distributions are similar (Figures 1B–D) for any shape parameters tested. Similarly to the matrix’s case, there is a multilinear principal component analysis (MPCA) used to ﬁnd principal patterns of movement in image sequences. Particularly in cell biology, there are no references to applying these numerical techniques to analyze growth cones. The algorithms (see Supplementary Methods) have been programed in MATLAB R⃝based on (de Lathauwer et al., 2000). These results conﬁrm that frataxin deﬁciency produces morphological alterations in the GCs of DRG neurons of early young adult mice, at 2 months of age, and generates aberrant morphology. Frontiers in Molecular Neuroscience \| www.frontiersin.org Frataxin Deﬁciency Reduces the Neurite Extension Rate and Alters the Growth Cone Turning in Adult Dorsal Root Ganglia Neurons By analyzing the average angle of displacement of each GC during the time- lapse, we observed that the GC turning angles of the YG8sR-GCs diﬀer signiﬁcantly from neurons from the control mice at 2 (P-value = 0.0006) and 6 (P-value = 0.046) months of age. At 2 months, while 24% of the YG8sR-GCs turned horizontally (0◦, −180◦, +180◦) and 57% diagonally (−45◦, +45◦), 57% of the controls turned vertically (−90◦, +90◦) and 31% diagonally (Figure 2E, left and Table 2). At 6 months, while 67% of the YG8sR-GCs turned vertically and 29% diagonally, 32% of the controls turned vertically and 47% diagonally (Figure 2E, right and Table 2). Then, at 2 months, YG8sR-GCs turning is limited to horizontal and diagonal turnings compared to the control. A broader distribution of turning angles is observed (Supplementary Videos 1–6). We investigated the behavior of GCs at 2 months of age by applying the methods to the same time-lapse experiments used for the manual approach described above. The ﬁrst method is a Principal Components Analysis (PCA) (Goodhill et al., 2015), which uses matrix analysis fundamentally suitable for 2-dimensional data, with customized modiﬁcations (see Section “Materials and Methods” and Supplementary Data 1 for details of the algorithms). To perform PCA analysis of the images, sequences of living cells, which are spatial-temporal data structures in 2 coordinates, were converted into vectors (1- dimensional data structures) and then stacked in a data matrix (2-dimensional data structure). Figure 3A shows the steps that were followed in the process. First, we obtained binary images from the phase-contrast time- lapse experiments (segmentation). In doing so, we created a semi-automated platform to get binary images for each time- lapse with MATLAB R⃝(The MathWorks) to optimize the image’s processing and reduce the variability introduced by the observer (see Section “Materials and Methods” for more details). Secondly, we obtained an outline of the GC that was later processed to get each GC’s vectorization in the time-lapse (Figure 3A). Finally, the PCA is performed with these vectorized data. PCA- analysis results showed that components 1 and 2 explain 91 and 73% of the variance for the control and YG8sR GCs, respectively (Figure 3B). The 2D scatter plots of these two components (Figure 3C) show that control GCs have long trajectories (concentration of dots giving shape to lines in the graph) of movement in several directions (from −180◦to 180◦). Frataxin Deﬁciency Reduces the Neurite Extension Rate and Alters the Growth Cone Turning in Adult Dorsal Root Ganglia Neurons These trajectories are characterized as a well-deﬁned path from the beginning (Figure 3C, left graph), possibly indicating the high exploratory and growth activity of the control GCs. On the contrary, the YG8sR-GCs show mostly short trajectories with random and curved movement patterns (Figure 3C, right graph), with absent well-deﬁned paths indicating weak exploratory activity and low growth capacity. Interestingly, the YG8sR-GCs turning patterns are lost at 6 months. However, it remains distinct from the turning patterns observed for the controls. Overall, the changes in the spatial behavior of YG8sR-GCs suggest that frataxin deﬁcient YG8sR neurons can build GCs, but these would be dysfunctional. Frataxin Deﬁciency Reduces the Neurite Extension Rate and Alters the Growth Cone Turning in Adult Dorsal Root Ganglia Neurons This difference is lost at 6 months of age (Y47R: 147 growth cones from n = 5, YG8sR: 154 growth cones from n = 4, P = 0.001, Mann–Whitney test, P = 0.587). (C–E) Shape parameters of the GCs. (C) The GCs from the YG8sR mice at 2 months of age were smaller (C) (Y47R: 150 growth cones from n = 3, YG8sR: 133 growth cones from n = 3, ∗∗P = 0.001), more circular (D) (Y47R: 150 growth cones from n = 3, YG8sR: 133 growth cones from n = 3, ∗P = 0.014) and more convex (E) (Y47R: 150 growth cones from n = 3, YG8sR: 133 growth cones from n = 3, ∗P = 0.024). These differences are lost at 6 months of age (Y47R: 147 growth cones from n = 5, YG8sR: 154 growth cones from n = 4, P = 0.001, Mann–Whitney test, P = 0.587). The data distribution is presented with violin plots. The median (red horizontal bar) and interquartile (25 and 75%, colored horizontal bars) are shown. Figures beside the y-axis of the graphs visualize examples of the shape descriptors calculated. Statistical signiﬁcance: ∗∗∗∗P-value < 0.0001. P-value = 0.014) and 6 months (control: 27.45 ± 4.31 µm; YG8sR: 22.75 ± 2.89 µm; P-value = 0.044) of age compared to the controls (Table 2 and Figure 2C). Furthermore, we observed a signiﬁcant decrease in the velocity of displacement at 2 months of age (control: 0.010 ± 0.001 µm/sec; YG8sR: 0.008 ± 0.001 µm/sec; P-value = 0.012) that was lost at 6 months of age (control: 0.009 ± 0.002; YG8sR: 0.007 ± 0.001; P-value = 0.108) (Figure 2D). Mathworks). The signiﬁcant advantages of using these methods are the capacity to analyze extensive datasets and the obtention of important information of the GC morphology lost with the previous manual tracking of the GC’s base. Therefore, it is essential to mention that with these computer-based methods, we can get valuable information on changes in the whole GCs, including the ﬁlopodia. The reason is that here we are detecting changes in the time of the entire structure of the GC. Since GC turning is also a critical behavior that allows the GCs to seek the correct synaptic target, we were aimed to analyze GC turning angles in our time-lapse experiments. Frataxin Deﬁciency Reduces the Neurite Extension Rate and Alters the Growth Cone Turning in Adult Dorsal Root Ganglia Neurons We found that YG8sR-GCs traveled reduced distances at 2 (control: 32.39 ± 4.13 µm; YG8sR: 24.81 ± 3.14 µm; 2 June 2022 \| Volume 15 \| Article 912780 Frontiers in Molecular Neuroscience \| www.frontiersin.org 5 Growth Cone Dynamic in FRDA Muñoz-Lasso et al. RE 1 \| Growth cones of frataxin deﬁcient neurons of the YG8sR mouse are smaller and abnormal. (A) Representative phase-contrast images of three SPREAD Cs observed in the primary culture of DRG neurons from the control (Y47R) and the YG8sR model at 2 and 6 months of age. We observed disorganization in tribution of ﬁlopodia and lamellipodia of YG8sR-GCs. Notice the hand-like shape in the controls and the absence of this shape in the YG8sR-GCs. Also, we ved small structures, probably rich in actin, growing from the axon or neurite close to the YG8sR-GCs (black arrows). These structures are not observed in the (Continued) FIGURE 1 \| Growth cones of frataxin deﬁcient neurons of the YG8sR mouse are smaller and abnormal. (A) Representative phase-contrast images of three SPREAD like GCs observed in the primary culture of DRG neurons from the control (Y47R) and the YG8sR model at 2 and 6 months of age. We observed disorganization in the distribution of ﬁlopodia and lamellipodia of YG8sR-GCs. Notice the hand-like shape in the controls and the absence of this shape in the YG8sR-GCs. Also, we observed small structures, probably rich in actin, growing from the axon or neurite close to the YG8sR-GCs (black arrows). These structures are not observed in the (Continued) Frontiers in Molecular Neuroscience \| www.frontiersin.org June 2022 \| Volume 15 \| Article 912780 6 Growth Cone Dynamic in FRDA Muñoz-Lasso et al. FIGURE 1 \| controls. Images were acquired with in vivo conditions after 14–16 h of culture. Each image is representative of independent experiments (three mice per each genotype and age). Scale bar, 10 µm. (B) Cumulative distribution of the growth cone area was plotted for both genotypes at 2 and 6 months of age. Most of the growth cones of DRG neurons from the YG8sR mice were smaller (0–100 µm2) than those from the controls at 2 months of age (Y47R: 150 growth cones from n = 3, YG8sR: 133 growth cones from n = 3, ∗∗P = 0.001, Mann–Whitney test). Frataxin Deﬁciency Reduces the Dynamics of the Growth Cone Morphology of Adult Dorsal Root Ganglia Neurons Previously, we have shown a morphological alteration of the GCs of frataxin deﬁcient neurons from the YG8sR mouse model. These observations are signiﬁcant at 2 months of age but it is lost at 6 months of age. Interestingly, we have also shown that YG8sR- GC has reduced motility, which is more prominent at 2 months by tracking the GC’base (Figure 2B), they did not include the GC’s macrostructural features (e.g., ﬁlopodia and lamellipodia). Therefore, we chose to quantify the neuronal GC morphology dynamics with two computer-based methods in MATLAB R⃝(The From a mathematical perspective, classical PCA methods are based on matrix analysis and are not best suited to June 2022 \| Volume 15 \| Article 912780 Frontiers in Molecular Neuroscience \| www.frontiersin.org 7 Growth Cone Dynamic in FRDA Muñoz-Lasso et al. TABLE 1 \| Overview of the data obtained for the morphometric analysis of the growth cone. 2 months Control (Y47R) YG8sR Mean SEM N Mean SEM N 126.44 11.30 50 108.16 17.47 48 Area (µm2) 99.30 7.55 50 116.78 19.09 33 104.75 10.15 50 92.29 10.92 52 Total mean 110.16 9.66 N = 150 105.74 15.83 N = 133 20.10 0.94 17.03 1.01 Feret’s diameter (µm) 18.13 0.80 19.54 1.82 19.58 0.92 16.79 1.04 Total mean 19.27 0.89 17.78 1.29 0.32 0.01 0.38 0.02 Circularity (a.u.) 0.32 0.02 0.34 0.02 0.30 0.02 0.36 0.02 Total mean 0.31 0.02 0.36 0.02 0.67 0.02 0.71 0.02 Solidity (a.u) 0.66 0.02 0.67 0.02 0.63 0.02 0.68 0.02 Total mean 0.65 0.02 0.69 0.02 6 months Control (Y47R) YG8sR Mean SEM N Mean SEM N 127.43 115.59 12 84.64 48.39 33 121.65 72.11 21 134.53 112.55 51 Area (µm2) 110.43 59.57 35 130.11 74.12 46 157.84 124.70 22 99.63 61.37 24 145.93 278.25 57 Total mean 132.66 130.04 N = 147 112.23 74.11 N = 154 20.07 1.77 15.04 0.76 17.67 1.15 19.11 1.00 Feret’s diameter (µm) 18.46 0.91 20.01 1.07 20.81 1.79 17.92 1.34 18.69 1.93 Total mean 19.14 1.51 18.02 1.04 0.29 0.03 0.42 0.03 Circularity (a.u.) 0.39 0.03 0.39 0.02 0.34 0.02 0.41 0.02 0.38 0.03 0.40 0.02 0.45 0.02 Total mean 0.37 0.03 0.40 0.02 TABLE 1 \| Overview of the data obtained for the morphometric analysis of the growth cone. identify patterns in spatial-temporal phenomena such as time- lapse microscopy. To overcome this limitation, we have used unconventional numerical methods based on Tensor analysis. Frontiers in Molecular Neuroscience \| www.frontiersin.org Frataxin Deﬁciency Reduces the Dynamics of the Growth Cone Morphology of Adult Dorsal Root Ganglia Neurons Tensors are multidimensional generalizations of the concept of vectors and matrices. Since the 2-dimensional spatial structure in the images is preserved, it is also possible to safeguard the correlations space-time in a sequence of images (Figure 4A). we have programmed in MATLAB R⃝is based on the Tensor Toolbox work (de Lathauwer et al., 2000; Brett and Tamara, 2021) (Supplementary Data 2, 3). The results show that the pattern of movement of the control-GCs was mostly vertical (Figure 4B, left). In contrast, the YG8sR-GCs, showed combined vertical and horizontal lines, indicating a diﬀerential and insuﬃcient exploratory activity of the GCs (Figure 4B, right). Similarly to the matrix’s case, a multilinear principal component analysis (MPCA) is used to ﬁnd principal movement patterns in image sequences. There are no references to applying these numerical techniques to analyze growth cones, particularly cell biology. The code to compute the tensor decompositions The results obtained with the PCA and tensor analysis coincide in showing alterations of the dynamics of the GCs of frataxin deﬁcient neurons at 2 months of age. These observations may explain the reduced dynamics observed when we manually tracked the GC base. June 2022 \| Volume 15 \| Article 912780 Frontiers in Molecular Neuroscience \| www.frontiersin.org 8 Growth Cone Dynamic in FRDA Muñoz-Lasso et al. GURE 2 \| Neurites of frataxin-deﬁcient neurons of the YG8sR mouse grow slower and with not deﬁned trajectories. (A) Representative sequences of frames 5 min part from a time-lapse movie of control and YG8sR- growth cone (GC) at 2 and 6 months of age. Scale bar, 10 µm. (B) Workﬂow to obtain the motion tracking with e MTrackJ plugin. First, rotation of the movie to a ﬁxed position to start. Secondly, selection of the points of the track with the points located at the center of the ase of the growth cone (red points) and in frames 5 min apart; and Third, obtention of the ﬁnal measurement results. (C–E) Violin plots show the average Length (C) nd velocity (D) data’s general distribution. (E) Graphical representation of the frequency distributions for the GC’s average turning angles (α[◦]) at 2 (left, Y47R: 42 rowth cones from n = 3, YG8sR: 21 growth cones from n = 3) and 6 months of age (right, Y47R: 19 growth cones from n = 3, YG8sR: 24 growth cones from = 3). Frataxin Deﬁciency Reduces the Dynamics of the Growth Cone Morphology of Adult Dorsal Root Ganglia Neurons The GCs of YG8sR neurons travel fewer distances with lower speed and distinctive turning angles than the controls at 2 (∗∗∗: exact P-value = 0.0006) and 6 : exact P-value = 0.046). The median (red horizontal bar) and interquartile (25 and 75%, colored horizontal bars) are shown. Statistical signiﬁcance was determined sing the Holm–Sidak method for length and velocity and using the Mann–Whitney test for the turning angle. FIGURE 2 \| Neurites of frataxin-deﬁcient neurons of the YG8sR mouse grow slower and with not deﬁned trajectories. (A) Representative sequences of frames 5 min apart from a time-lapse movie of control and YG8sR- growth cone (GC) at 2 and 6 months of age. Scale bar, 10 µm. (B) Workﬂow to obtain the motion tracking with the MTrackJ plugin. First, rotation of the movie to a ﬁxed position to start. Secondly, selection of the points of the track with the points located at the center of the base of the growth cone (red points) and in frames 5 min apart; and Third, obtention of the ﬁnal measurement results. (C–E) Violin plots show the average Length (C) and velocity (D) data’s general distribution. (E) Graphical representation of the frequency distributions for the GC’s average turning angles (α[◦]) at 2 (left, Y47R: 42 growth cones from n = 3, YG8sR: 21 growth cones from n = 3) and 6 months of age (right, Y47R: 19 growth cones from n = 3, YG8sR: 24 growth cones from n = 3). The GCs of YG8sR neurons travel fewer distances with lower speed and distinctive turning angles than the controls at 2 (∗∗∗: exact P-value = 0.0006) and 6 (∗: exact P-value = 0.046). The median (red horizontal bar) and interquartile (25 and 75%, colored horizontal bars) are shown. Statistical signiﬁcance was determined using the Holm–Sidak method for length and velocity and using the Mann–Whitney test for the turning angle. June 2022 \| Volume 15 \| Article 912780 Frontiers in Molecular Neuroscience \| www.frontiersin.org 9 Growth Cone Dynamic in FRDA Muñoz-Lasso et al. TABLE 2 \| Overview of the data obtained from the quantiﬁcation of the growth cone dynamics. Frataxin Deﬁciency Reduces the Dynamics of the Growth Cone Morphology of Adult Dorsal Root Ganglia Neurons 2 months Control (Y47R) YG8sR Mean SEM N Mean SEM N Velocity (µm/sec) 0.010 0.002 9 0.009 0.001 4 0.009 0.001 7 0.008 0.001 7 0.011 0.002 10 0.006 0.001 10 0.010 0.001 5 0.009 0.001 11 Total mean 0.010 0.001 N = 42 0.008 0.001 N = 21 Length (µm) 29.26 4.76 23.61 3.57 31.04 3.17 26.05 2.93 38.31 5.61 24.76 2.92 33.17 4.20 30.18 2.94 Total mean 32.39 4.13 N = 42 24.81 3.14 N = 21 Turning angle (◦) −63.15 10.95 −38.34 21.46 −66.09 9.73 −44.53 12.95 −82.89 7.24 −43.73 5.81 −63.20 25.55 −62.43 6.65 Total mean −67.55 12.02 N = 42 −42.20 13.41 N = 21 6 months Control (Y47R) YG8sR Mean SEM N Mean SEM N Velocity (µm/sec) 0.011 0.003 3 0.010 0.002 7 0.010 0.003 3 0.006 0.001 5 0.008 0.002 4 0.008 0.002 6 0.008 0.001 9 0.006 0.001 3 0.007 0.001 3 Total mean 0.009 0.002 N = 19 0.007 0.001 N = 24 Length (µm) 32.82 7.57 23.39 1.06 22.92 1.48 21.81 3.18 26.66 5.95 24.72 5.40 27.39 2.24 22.50 1.73 21.31 3.06 Total mean 27.45 4.31 N = 19 22.75 2.89 N = 24 Turning angle (◦) −45.22 23.55 −66.30 6.76 −71.43 24.74 −81.08 2.96 −56.58 6.96 −78.36 6.86 −59.48 11.02 −73.81 19.10 −41.19 28.17 Total mean −58.18 16.56 N = 19 −68.15 12.77 N = 24 Adult Frataxin Deﬁcient Dorsal Root Ganglia Neurons Lose Their Intrinsic Ability to Regenerate in vitro As a consequence of the morphological and functional changes observed in the growth cones (GCs) of YG8sR-DRG neurons, it was possible to postulate that critical cellular processes, such as axonal regeneration, were also being aﬀected. In axonal regeneration peripheral neurons can reactivate axonal growth transport to clear the debris or recycle damaged proteins and ensure the new axon’s materials. Therefore, the growth cone (GC) plays a vital role in the unique capability of peripheral axons to regenerate and reinnervate their target tissues and organs after an injury during adulthood (Blanquie and Bradke, 2018; Muñoz-Lasso et al., 2020b). To investigate whether the DRG-neurons from YG8sR mouse model can extend healthy axons and regenerate appropriately after an injury we performed an axotomy in vitro as a model TABLE 2 \| Overview of the data obtained from the quantiﬁcation of the growth cone dynamics. Frataxin Deﬁciency Reduces the Dynamics of the Growth Cone Morphology of Adult Dorsal Root Ganglia Neurons When compared to the controls, the YG8sR-growth cones exhibited fewer trajectories and traveled short distances. Besides, the trajectories of the YG8sR-growth cones are less deﬁned and more dispersed. FIGURE 3 \| The PCA analysis shows that YG8sR growth cones have a reduced dynamic morphology at 2 months of age. (A) Workﬂow used for the principal component analysis. (B) The principal component analysis showed that components 1 and 2 explain 91 and 73% of the variance for the control and YG8sR GCs, respectively. (C) Bi-plots of components 1 and 2 visualizing the movement of the growth cone for 1 h. The principal components indicate the main patterns of movement of all points that form the growth cone, including the axonal segment that the growth cone builds over the time-lapse. The lines start in the lower central part of the images (initial position of the cones at t = 0) and are directed to the right, left or above in the images with different angles (ﬁnal position of the cones at t = 60 min). Therefore, the more dispersed the points are in the graphs, there are fewer trajectories of movement. The length of each trajectory indicates the distance traveled by the growth cones in the time-lapse. When compared to the controls, the YG8sR-growth cones exhibited fewer trajectories and traveled short distances. Besides, the trajectories of the YG8sR-growth cones are less deﬁned and more dispersed. FIGURE 3 \| The PCA analysis shows that YG8sR growth cones have a reduced dynamic morphology at 2 months of age. (A) Workﬂow used for the principal component analysis. (B) The principal component analysis showed that components 1 and 2 explain 91 and 73% of the variance for the control and YG8sR GCs, respectively. (C) Bi-plots of components 1 and 2 visualizing the movement of the growth cone for 1 h. The principal components indicate the main patterns of movement of all points that form the growth cone, including the axonal segment that the growth cone builds over the time-lapse. The lines start in the lower central part of the images (initial position of the cones at t = 0) and are directed to the right, left or above in the images with different angles (ﬁnal position of the cones at t = 60 min). Therefore, the more dispersed the points are in the graphs, there are fewer trajectories of movement. Frataxin Deﬁciency Reduces the Dynamics of the Growth Cone Morphology of Adult Dorsal Root Ganglia Neurons Adult Frataxin Deﬁcient Dorsal Root Ganglia Neurons Lose Their Intrinsic Ability to Regenerate in vitro Adult Frataxin Deﬁcient Dorsal Root Ganglia Neurons Lose Their Intrinsic Ability to Regenerate in vitro transport to clear the debris or recycle damaged proteins and ensure the new axon’s materials. Therefore, the growth cone (GC) plays a vital role in the unique capability of peripheral axons to regenerate and reinnervate their target tissues and organs after an injury during adulthood (Blanquie and Bradke, 2018; Muñoz-Lasso et al., 2020b). As a consequence of the morphological and functional changes observed in the growth cones (GCs) of YG8sR-DRG neurons, it was possible to postulate that critical cellular processes, such as axonal regeneration, were also being aﬀected. In axonal regeneration, peripheral neurons can reactivate axonal growth programs, including axonal elongation and axonal pathﬁnding, active during the development. Besides, neurons rely on axonal To investigate whether the DRG-neurons from YG8sR mouse model can extend healthy axons and regenerate appropriately after an injury, we performed an axotomy in vitro as a model for axonal damage (Figure 5A). Here, we have chosen to evaluate DRG neurons’ regenerative response at 2 months of age because June 2022 \| Volume 15 \| Article 912780 Frontiers in Molecular Neuroscience \| www.frontiersin.org 10 Growth Cone Dynamic in FRDA Muñoz-Lasso et al. FIGURE 3 \| The PCA analysis shows that YG8sR growth cones have a reduced dynamic morphology at 2 months of age. (A) Workﬂow used for the principal component analysis. (B) The principal component analysis showed that components 1 and 2 explain 91 and 73% of the variance for the control and YG8sR GCs, respectively. (C) Bi-plots of components 1 and 2 visualizing the movement of the growth cone for 1 h. The principal components indicate the main patterns of movement of all points that form the growth cone, including the axonal segment that the growth cone builds over the time-lapse. The lines start in the lower central part of the images (initial position of the cones at t = 0) and are directed to the right, left or above in the images with different angles (ﬁnal position of the cones at t = 60 min). Therefore, the more dispersed the points are in the graphs, there are fewer trajectories of movement. The length of each trajectory indicates the distance traveled by the growth cones in the time-lapse. Frontiers in Molecular Neuroscience \| www.frontiersin.org Frataxin Deﬁciency Reduces the Dynamics of the Growth Cone Morphology of Adult Dorsal Root Ganglia Neurons June 2022 \| Volume 15 \| Article 912780 Frontiers in Molecular Neuroscience \| www.frontiersin.org 11 Growth Cone Dynamic in FRDA Muñoz-Lasso et al. FIGURE 4 \| A tensor analysis conﬁrms the reduced dynamics of DRG neurons’ growth cone morphology from the YG8sR mouse. (A) Visualization of the methodology applied for the tensor analysis. (B) Graph of the two principal components obtained for tensor analysis. Like the PCA analysis, the principal components indicate the main patterns of movement of all points that form the growth cone, including the axonal segment built during the time-lapse. These patterns are seen in the graphs as continuous lines that stand out from the background color. The lines start in the lower central part of the images (initial position of the cones at t = 0) and are directed to the right, left or above in the images with different angles (ﬁnal position of the cones at t = 60 min). When the main components of the YG8sR mouse (right) and the control are compared (left), we observed that movement patterns of the growth cones of the YG8sR mouse (yellow and blue) are different from those of the control, presenting shorter movement patterns directed to the right or left (horizontal). FIGURE 4 \| A tensor analysis conﬁrms the reduced dynamics of DRG neurons’ growth cone morphology from the YG8sR mouse. (A) Visualization of the methodology applied for the tensor analysis. (B) Graph of the two principal components obtained for tensor analysis. Like the PCA analysis, the principal components indicate the main patterns of movement of all points that form the growth cone, including the axonal segment built during the time-lapse. These patterns are seen in the graphs as continuous lines that stand out from the background color. The lines start in the lower central part of the images (initial position of the cones at t = 0) and are directed to the right, left or above in the images with different angles (ﬁnal position of the cones at t = 60 min). When the main components of the YG8sR mouse (right) and the control are compared (left), we observed that movement patterns of the growth cones of the YG8sR mouse (yellow and blue) are different from those of the control, presenting shorter movement patterns directed to the right or left (horizontal). Frataxin Deﬁciency Reduces the Dynamics of the Growth Cone Morphology of Adult Dorsal Root Ganglia Neurons The length of each trajectory indicates the distance traveled by the growth cones in the time-lapse. When compared to the controls, the YG8sR-growth cones exhibited fewer trajectories and traveled short distances. Besides, the trajectories of the YG8sR-growth cones are less deﬁned and more dispersed. The objective was to determine how the low density of axons could aﬀect the regenerative capacity of YG8sR-DRG neurons and ﬁnd out whether the axotomy stimulates other axons to cross the microchannels. After 2 days of performing the axotomy, we observed that YG8sR-DRG neurons could not regenerate their axons as the control axons did it. Also, the axotomy did not produce additional axons to cross the microchannels. Next, we evaluate signs of degeneration such as axonal swelling and axonal fragmentation in the axons that crossed the microchannels. To do this, we performed a quantiﬁcation method proposed by Sasaki et al. (2009) based on Phase-Contrast Microscopy and DRG culture from mouse embryos to measure an axonal degeneration index. This method uses the Particle analyzer algorithm of Fiji/ImageJ for detecting particles with shape and size characteristic of degenerated axons (see Section “Materials and Methods”). Our data conﬁrmed our previous observations, showing that YG8sR-DRG neurons respond poorly to the neurotrophic gradient and grow less axons than control neurons we have shown throughout this work that GCs of YG8sR-DRG are signiﬁcantly defective at this age. Also, a high regenerative capacity is expected for young neurons. An equivalent number of neuronal somas isolated from the YG8sR and control mice were plated in the soma chambers (3 × 104 neurons per device) for the experiment. Then, neurons were progressively exposed to a neurotrophic gradient for 3 days (see Section “Materials and Methods” for details). Later, we performed the axotomy and then allowed the axons to regenerate for 2 days. Phase-Contrast Microscopy with in vivo conditions was used to capture and examine the morphology of living neurons and their axons. After 3 days in culture and before performing the axotomy, we observed a considerably reduced number of axons in YG8sR- DRG neurons than in the control neurons (Figure 5B). In addition to this, we observed that axons of the YG8sR-DRG neurons that crossed the microchannels were stacked once they passed the microchannel contrasting with the axonal arrays observed in the control neurons (Figure 5B and Tables 3, 4). Frataxin Deﬁciency Reduces the Dynamics of the Growth Cone Morphology of Adult Dorsal Root Ganglia Neurons FIGURE 4 \| A tensor analysis conﬁrms the reduced dynamics of DRG neurons’ growth cone morphology from the YG8sR mouse. (A) Visualization of the methodology applied for the tensor analysis. (B) Graph of the two principal components obtained for tensor analysis. Like the PCA analysis, the principal components indicate the main patterns of movement of all points that form the growth cone, including the axonal segment built during the time-lapse. These patterns are seen in the graphs as continuous lines that stand out from the background color. The lines start in the lower central part of the images (initial position of the cones at t = 0) and are directed to the right, left or above in the images with different angles (ﬁnal position of the cones at t = 60 min). When the main components of the YG8sR mouse (right) and the control are compared (left), we observed that movement patterns of the growth cones of the YG8sR mouse (yellow and blue) are different from those of the control, presenting shorter movement patterns directed to the right or left (horizontal). regenerative potential compared to control neurons, and the molecular mechanism involved probably is axonal-speciﬁc. (Figure 5C). However, we did not detect signiﬁcant diﬀerences in the degeneration index before or after the axotomy (Figure 5D). Besides the axons, we were also interested in evaluating the neuronal somas after the axotomy. Interestingly, we did not observe noticeable changes in the number or the morphology of the cell bodies that evidenced a process of cell death (Supplementary Figure 2). Overall, our results show that while control DRG neurons can grow healthy axons with well-deﬁned tracks in response to a neurotrophic gradient, the YG8sR-DRG neurons cannot do it. This behavior is maintained even after the axotomy and without aﬀecting neuronal cell bodies, which do not show changes in the number or the morphology of the cell bodies that evidenced a process of cell death (Supplementary Figure 2). All of them suggest that adult YG8sR-DRG neurons have reduced Frontiers in Molecular Neuroscience \| www.frontiersin.org DISCUSSION In this work, a link between the growth cone, a critical neuronal structure, and the pathophysiology of FRDA is revealed by using in vivo phase-contrast imaging and computational methods in the YG8sR mouse. When an axon grows or regenerates, the mitochondrial network, the neuronal cytoskeleton and motor proteins are carefully coordinated to create a growth cone (GC). The GC plays a critical role in guiding axons to their targets. They navigate to their targets, driven by several molecular In this work, a link between the growth cone, a critical neuronal structure, and the pathophysiology of FRDA is revealed by using in vivo phase-contrast imaging and computational methods in the YG8sR mouse. When an axon grows or regenerates, the mitochondrial network, the neuronal cytoskeleton and motor proteins are carefully coordinated to create a growth cone (GC). The GC plays a critical role in guiding axons to their targets. They navigate to their targets, driven by several molecular June 2022 \| Volume 15 \| Article 912780 Frontiers in Molecular Neuroscience \| www.frontiersin.org Frontiers in Molecular Neuroscience \| www.frontiersin.org 12 Growth Cone Dynamic in FRDA Muñoz-Lasso et al. FIGURE 5 \| Adult DRG neurons from the YG8sR mouse reduce their natural regenerative response after an injury in vitro. (A) Scheme of a microﬂuidic device (upper panel) and steps of the experiment (bottom panel). Each device has two identical compartments connected with microﬂuidic channels (150 µm in length). Neuronal somas were seeded into one of the chambers (soma-chamber), and were allowed to grow under a neurotrophic gradient pushing them to grow their axons crossing the microchannels. Axotomy (or cutting of axons) was performed 3 days in culture, and neurons were cultured for 2 days more. Images show a typical view of the (Continued) FIGURE 5 \| Adult DRG neurons from the YG8sR mouse reduce their natural regenerative response after an injury in vitro. (A) Scheme of a microﬂuidic device (upper panel) and steps of the experiment (bottom panel). Each device has two identical compartments connected with microﬂuidic channels (150 µm in length). Neuronal somas were seeded into one of the chambers (soma-chamber), and were allowed to grow under a neurotrophic gradient pushing them to grow their axons crossing the microchannels. Axotomy (or cutting of axons) was performed 3 days in culture, and neurons were cultured for 2 days more. DISCUSSION Images show a typical view of the (Continued) June 2022 \| Volume 15 \| Article 912780 Frontiers in Molecular Neuroscience \| www.frontiersin.org Frontiers in Molecular Neuroscience \| www.frontiersin.org 13 Growth Cone Dynamic in FRDA Muñoz-Lasso et al. FIGURE 5 \| neuronal somas and axons in the device after 3 days of culture. (B) Representative images of control and YG8sR-living axons during the experiment. Before (3 days in culture), immediately after axotomy (3 days in culture), and after axotomy (2 days after axotomy). Compared to the controls (top-left), few DRG neurons of the YG8sR-mice were able to cross the microchannels and extend axonal networks (bottom-left). After the axotomy, DRG neurons of the YG8sR-mice tried to regenerate the axons with visible difﬁculty. (C) Violin plots show the distribution of the means obtained for the area covered by the axons before and after the axotomy. Data show a reduced area covered by the axons of DRG neurons of the YG8sR-mice before and after the axotomy. The area covered by the control axons is visibly higher before and after the axotomy. (D) Violin plots show the distribution of the means obtained for the fragmentation index (fragmented area versus total area) before and after the axotomy. Data show a non-signiﬁcant tendency to increase the fragmented area in the control and YG8sR axons. All the means corresponded to three independent experiments and were obtained by averaging the values collected from 10 random ﬁelds in each experiment’s axonal chamber. The median (red horizontal bar) and interquartile (25 and 75%, colored horizontal bars) are shown. Signiﬁcance was determined using an unpaired t-test with the Holm–Sidak method. Statistical signiﬁcance: P-value < 0.05. clues that trigger the reorganization of the GC-cytoskeleton producing changes in the morphology and determining which way the growth cone will turn and direct the new axon’s growth (Muñoz-Lasso et al., 2020b). the morphology of the GCs are available, most of them imply technical limitations. First, high-resolution microscopy allows quantifying individual ﬁlopodia present in the GCs; nevertheless, it is a diﬃcult task and time-consuming. It relies heavily on human intuition to determine the most signiﬁcant variance in the growth cone morphology (Goodhill et al., 2015). Secondly, the use of ﬂuorescent probes facilitates the tracking and increases the resolution of the ﬁlopodia; then, images from ﬁxed cells or in vivo experiments can be analyzed using computer-based tools to quantify the ﬁlopodia automatically. DISCUSSION However, ﬁxation can alter ﬁlopodia’s real amount and the architecture of the GCs. In addition, adult DRG neurons are not easy to transfect or transduce, even at young ages, such as 2 months of age. Several transfection methods of adult DRG neurons produce high cell death levels (glial cells and neurons) proportional to the mice’s age. Thus, the use of ﬂuorescent proteins or probes could induce changes in the morphology of the growth cone that would have no relation with frataxin producing misleading results. In contrast, Phase-Contrast microscopy with in vivo conditions reduces external factors to the media, neurotrophic elements, and frataxin deﬁcit. The model of degeneration described in the FRDA involves a distal degeneration of peripheral sensory neurons, with initial deterioration of synapses, slowly progressing to the spinal cord’s posterior columns and corticospinal and spinocerebellar tracts. This model of degeneration of the dying-back type is the most common form of axonal degeneration described in the PNS. Nevertheless, there is increasing evidence that the neurodegeneration process has to be very early in life (Indelicato et al., 2018) or even during neurodevelopment (Koeppen et al., 2017). Nerve ﬁber loss in patients is heterogeneous and correlates with the size of the short GAA allele. However, there is no association between the severity of the neuropathy and the duration of the disease. This suggests that the loss of neurons is early and stable over time. Indeed, a neuropathological study of tissue samples from FRDA patients supports the conclusion that it is during development that the dorsal root is altered, due to inappropriate growth and guidance of the axons of neural crest-derived neurons (Koeppen et al., 2017). We think our ﬁndings support this hypothesis. We observed that frataxin deﬁcient DRG neurons from the YG8sR mice built smaller and morphologically aberrant GCs than their controls at 2 months. These signiﬁcant morphological changes were not observed in mice at 6 months of age. Therefore, morphological alterations on the GCs might appear at embryonic stages when long axons of DRG neurons are actively being produced. Although it is speculative, our results suggest that developmental defects may induce the neurodegenerative process that determines the observations made by these authors. This hypothesis has to be conﬁrmed at an early postnatal age. YG8sR-GCs grow smaller neurites and exhibit altered dynamics compared to control-GCs by using two computer- based methods, PCA and Tensor Decomposition analysis. Frontiers in Molecular Neuroscience \| www.frontiersin.org DISCUSSION Despite the fact that additional eﬀorts are required to reveal the molecular changes associated with these ﬁndings, these two- practical computer-based approaches can quickly be adapted to further analyses of the GC motility in the FRDA and other disorders of the nervous system, which is an unexplored ﬁeld due to the GC spatial-temporal complexity. Among the potential implications of dysfunctional GCs in the frataxin-deﬁcient DRG neurons was the aﬀection of axonal growth programs such as axonal pathﬁnding and axonal regeneration, which ensure the proper development and maintenance of DRG neurons throughout their lifetime. These two critical processes were investigated using an in vitro axotomy of DRG axons as a model for damage. After dissection of DRGs, the intuitive ability to grow axons in vitro was not aﬀected in both the control and YG8sR-DRG neurons. However, after the axotomy, the YG8sR-neurons did not regenerate their axons to the extent the controls did. Additionally, damage or evident loss of neuronal cell bodies of YG8sR neurons was not apparent, before and after the axotomy; these results indicate that an axonal speciﬁc disturbing condition might prevent the proper formation of functional GCs. In conclusion, frataxin deﬁcient DRG neurons from the YG8sR mice may lose the intrinsic ability The morphology and function of the GCs are tightly connected. Consequently, the YG8sR-GCs function was observed to be reduced and aberrant. These results indicate that the GCs of frataxin deﬁcient DRG neurons indeed have reduced motility compared to the control GCs at 2 and 6 months of age. This reduced motility is characterized by a reduced speed of migration, broader turning angles and more short and complex trajectories, indicating that frataxin-deﬁcient DRG neurons can create GCs, but they are dysfunctional. The knowledge about the FRDA’s neurons’ GC dynamics is lacking, showing evidence of a functional aﬀection of the DRG-GCs in an FRDA mouse model. Despite the fact that several quantitative approaches to measure the dynamics of June 2022 \| Volume 15 \| Article 912780 14 Growth Cone Dynamic in FRDA Muñoz-Lasso et al. TABLE 3 \| Data obtained for the quantiﬁcation of the degeneration index of the control mice. DISCUSSION Control (Y47R) 2 months pre-axotomy (N = 3) # ﬁelds analyzed Fragmented area (µm2) Total axon area Degeneration index (DI) Mean SEM Mean SEM Mean SEM 10 1704,7 106,5 117819,6 12341,0 0,016 0,002 10 2540,0 170,5 79655 6168,3 0,033 0,003 10 1685,6 129,3 47331,8 5766,4 0,038 0,003 Control (Y47R) 2 months post-axotomy (N = 3) # ﬁelds analyzed Fragmented area (µm2) Total axon area Degeneration index (DI) Mean SEM Mean SEM Mean SEM 10 2766,7 108,7 78336,6 5215,4 0,016 0,002 10 2461,5 109,8 48792,2 3937,5 0,033 0,003 10 1976,8 134,8 63100,4 4315,5 0,038 0,002 TABLE 4 \| Data obtained for the quantiﬁcation of the degeneration index of the YG8sR mice. Control (YG8sR) 2 months pre-axotomy (N = 3) # ﬁelds analyzed Fragmented area (µm2) Total axon area Degeneration index (DI) Mean SEM Mean SEM Mean SEM 7 538.2 78.0 10676.4 1693.1 0.054 0.007 6 372.2 114.7 10185.2 1999.9 0.038 0.007 10 419.7 42.4 16390.0 2319.8 0.027 0.003 Control (YG8sR) 2 months post-axotomy (N = 3) # ﬁelds analyzed Fragmented area (µm2) Total axon area Degeneration index (DI) Mean SEM Mean SEM Mean SEM 7 1128.1 100.3 12660.1 1359.3 0.092 0.008 6 397.7 116.5 6384.3 1631.4 0.090 0.034 9 453.4 56.8 29539.1 4357.5 0.017 0.002 TABLE 3 \| Data obtained for the quantiﬁcation of the degeneration index of the control mice. to regenerate their axons after injury, a protective phenomenon observed mainly in DRG neurons (McQuarrie and Grafstein, 1973; Richardson and Issa, 1984). A possible explanation for this observation is the failure of axonal regeneration programs in the YG8sR-DRG neurons, which in normal conditions allows them to protect against cumulative axon damage. work may be masking a more pronounced alteration than that observed. GDNF and NGF are neurotrophic factors well known to stimulate axonal regeneration and pathﬁnding in peripheral axons in vitro and in vivo (Menesini Chen et al., 1978; Fine et al., 2002; Dudanova et al., 2010; Schuster et al., 2010; Mashanov et al., 2020). Furthermore, GDNF and NGF have been shown to modulate other pathﬁnding pathways such as sema3A pathway in adult DRG neurons (Wanigasekara and Keast, 2006). Therefore, we cannot exclude that the inability of the YG8sR-DRG to cross the microchannels may reﬂect a defect on both neurotrophin and axonal pathﬁnding pathways such as sema3A. The low capacity of YG8sR axons to cross the microﬂuidic channels and their inability to explore the axon area is striking. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnmol.2022. 912780/full#supplementary-material Supplementary Video 1 \| Phase-contrast imaging of living growth cone two months old isolated from the ﬁrst Y47R mouse. Supplementary Video 1 \| Phase-contrast imaging of living growth cone two months old isolated from the ﬁrst Y47R mouse. Supplementary Video 1 \| Phase-contrast imaging of living growth cone two months old isolated from the ﬁrst Y47R mouse. ETHICS STATEMENT observed in necropsies studies on FRDA patients proposed that FRDA neuropathology could be a developmental delay (Koeppen et al., 2017). How and why this problem occurs during development remains to be solved. Our results support the hypothesis that an incorrect innervation could occur due to axonal growth programs alteration, such as axonal pathﬁnding pathways and axonal regeneration. When an axon cannot innervate correctly, it can be eliminated through physiological neuronal death (Clarke and Cowan, 1976). Therefore, both a dysfunctional GC and the inability to regenerate axons against cumulative damage explain why the DRG is a subject of a hypoplastic process. The animal study was reviewed and approved by Brunel University Animals Welfare and Ethical Review Board. FUNDING This work was supported by grants from the Ministerio de Economía y Competitividad de España (SAF2015-66625-R) and the Agencia Estatal de Investigación (PID2020-115190RB-I00) within the framework of the National R + D + I Plan and co- funded by the Instituto de Salud Carlos III (ISCIII)-Subdirección General de Evaluación y Fomento de la Investigación and FEDER funds; the Generalitat Valenciana (ACOMP/2014/058 and PROMETEO/2018/135). CIBERER is an initiative developed by the Instituto de Salud Carlos III in cooperative and translational research on rare diseases. EMBO Short Fellowship (ASTF 562-2015). Further analysis of the GC dynamics and axonal regeneration response is needed to reveal the cascade of molecular events that leads to the dysfunction of the DRG-GC in the FRDA. In this context, the morphology and motility of GC rely on three critical central functions of the mitochondria that are known to be aﬀected in FRDA cells: (a) ATP production, (b) the maintenance of cellular redox status and (c) buﬀering of cytosolic calcium [Ca2+]i. Interestingly, evidence from patients and cellular and animal models show that FRDA cells present alterations in these three processes. Defects on mitochondrial dynamics (Puccio et al., 2001; Hick et al., 2013; Bolinches-Amorós et al., 2014) and neuronal cytoskeleton (Pastore et al., 2003; Sparaco et al., 2009; Piermarini et al., 2016; Muñoz-Lasso et al., 2020a) have been described. Moreover, recent studies have settled the Ca2+- mediated signaling and homeostasis in the sequence of pathologic events observed in FRDA neurons. Defects in the Ca2+ buﬀering capacity have been observed in neurons (Bolinches-Amorós et al., 2014; Mincheva-Tasheva et al., 2014; Mollá et al., 2017; Purroy et al., 2018) and cerebellar granule neurons from the YG8R mice (Abeti et al., 2018). ACKNOWLEDGMENTS We highly appreciate MP and Sahar Al-Mahdawi for providing the Y47R and YG8sR mice, facilities and resources at Brunel University London. In summary, we have evidenced an adverse eﬀect of the deﬁcit of frataxin in the formation and, most importantly, the growth cones’ function in adult DRG neurons by applying both classical and novel computer-assisted analysis. As a result, we propose that frataxin deﬁcient DRG neurons might lose the intrinsic capacity to grow and regenerate axons properly due to the dysfunctional GCs they build. DISCUSSION Within the microﬂuidic chamber, axons are incited to cross the microchannels by physical (volume of the media), and, more importantly, a neurotrophic gradient of GDNF and NGF. Previous works have shown the therapeutic potential of diﬀerent neurotrophic factors for Friedreich’s ataxia, including insulin- like growth factor-1 (IGF-1) (Franco et al., 2012; Sanz-Gallego et al., 2014), Brain-derived Neurotrophic Factor (BDNF) (Katsu- Jiménez et al., 2016) and the range of neuroprotective factors secreted by mesenchymal stem cells (MSCs) (Kemp K. C. et al., 2017; Kemp K. et al., 2017). Therefore, we have to consider that the presence of neurotrophins in the experiments of this In the FRDA pathophysiology context, the aﬀection of axonal pathﬁnding pathways and axonal regeneration programs in DRG neurons implies that several axons could not reach their target tissues eﬀectively. Consequently, the innervation of DRG neurons would be ineﬃcient. The evaluation of nerve biopsies from FRDA patients (McLeod, 1971; Santoro et al., 1999; Nolano et al., 2001; Indelicato et al., 2018) supports this hypothesis. In addition to this, the evidence of pathological axonal phenotypes June 2022 \| Volume 15 \| Article 912780 Frontiers in Molecular Neuroscience \| www.frontiersin.org 15 Growth Cone Dynamic in FRDA Muñoz-Lasso et al. AUTHOR CONTRIBUTIONS DM-L conducted and designed experiments, analyzed the results, and wrote the manuscript. BM performed the experiments. MP provided the mouse model and funding acquisition. EI performed the Tensor Analysis in MATLAB R⃝(Mathworks) and wrote the manuscript. MI-V and JS-G performed the PCA analysis. FVP interpreted the data and wrote the manuscript. FP funding acquisition, interpreted the data, and wrote the manuscript. PG-C designed the study, supervised the experiments, analyzed the data, funded acquisition, and wrote the manuscript. All authors read and approved the ﬁnal manuscript. 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Supplementary Video 5 \| Phase-contrast imaging of living growth cone two months old isolated from the second YG8sR mouse. Supplementary Video 5 \| Phase-contrast imaging of living growth cone two months old isolated from the second YG8sR mouse. DATA AVAILABILITY STATEMENT Supplementary Video 2 \| Phase-contrast imaging of living growth cone two months old isolated from the second Y47R mouse. 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. Supplementary Video 3 \| Phase-contrast imaging of living growth cone two months old isolated from the third Y47R mouse. Supplementary Video 4 \| Phase-contrast imaging of living growth cone two months old isolated from the ﬁrst YG8sR mouse. Supplementary Video 4 \| Phase-contrast imaging of living growth cone two months old isolated from the ﬁrst YG8sR mouse. 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https://openalex.org/W2069039798	https://research.tilburguniversity.edu/files/27070021/SP_d_Vries_values_clarification_BMC_MIaDM_open_access_2014.pdf	English	null	Values clarification in a decision aid about fertility preservation: does it add to information provision?	BMC medical informatics and decision making	2,014	cc-by	12,434	Values clarification in a decision aid about fertility preservation Garvelink, M.M.; ter Kuile, M.M.; Stiggelbout, A.M.; de Vries, M. Published in: BMC Medical Informatics and Decision Making DOI: 10.1186/1472-6947-14-68 Publication date: 2014 Document Version Publisher's PDF, also known as Version of record Link to publication in Tilburg University Research Portal Citation for published version (APA): Garvelink, M. M., ter Kuile, M. M., Stiggelbout, A. M., & de Vries, M. (2014). Values clarification in a decision aid about fertility preservation: Does it add to information provision? BMC Medical Informatics and Decision Making, 14, Article 68. https://doi.org/10.1186/1472-6947-14-68 Values clarification in a decision aid about fertility preservation Garvelink, M.M.; ter Kuile, M.M.; Stiggelbout, A.M.; de Vries, M. Citation for published version (APA): Garvelink, M. M., ter Kuile, M. M., Stiggelbout, A. M., & de Vries, M. (2014). Values clarification in a decision aid about fertility preservation: Does it add to information provision? BMC Medical Informatics and Decision Making, 14, Article 68. https://doi.org/10.1186/1472-6947-14-68 Citation for published version (APA): Garvelink, M. M., ter Kuile, M. M., Stiggelbout, A. M., & de Vries, M. (2014). Values clarification in a decision aid about fertility preservation: Does it add to information provision? BMC Medical Informatics and Decision Making, 14, Article 68. https://doi.org/10.1186/1472-6947-14-68 Tilburg University * Correspondence: m.m.garvelink@lumc.nl 1Department of Gynecology, Leiden University Medical Center (LUMC), Mail zone VRSP, P/O Box 9600, 2300 RC Leiden, the Netherlands Full list of author information is available at the end of the article General rights C i ht d General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private st • You may not further distribute the material or use it for any profit making activity or commercial gain y p py y p p p p p p y • You may not further distribute the material or use it for any profit-making activity or commercial gain Yo ma freel distrib te the URL identif ing the p blication in the p blic portal • You may not further distribute the material or use it for any profit-making activity o • You may freely distribute the URL identifying the publication in the public portal y y p g y • You may freely distribute the URL identifying the publication in the public portal Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim Download date: 24. Oct. 2024 Garvelink et al. BMC Medical Informatics and Decision Making 2014, 14:68 http://www.biomedcentral.com/1472-6947/14/68 Garvelink et al. BMC Medical Informatics and Decision Making 2014, 14:68 http://www.biomedcentral.com/1472-6947/14/68 Open Access Open Access Values clarification in a decision aid about fertility preservation: does it add to information provision? Mirjam M Garvelink1, Moniek M ter Kuile1, Anne M Stiggelbout2 and Marieke de Vries3 Mirjam M Garvelink1, Moniek M ter Kuile1, Anne M Stiggelbout2 and Marieke de Vries3 Abstract Background: We aimed to evaluate the effect of a decision aid (DA) with information only compared to a DA with values clarification exercise (VCE), and to study the role of personality and information seeking style in DA-use, decisional conflict (DC) and knowledge. Methods: Two scenario-based experiments were conducted with two different groups of healthy female participants. Dependent measures were: DC, knowledge, and DA-use (time spent, pages viewed, VCE used). Respondents were randomized between a DA with information only (VCE-) and a DA with information plus a VCE(VCE+) (experiment 1), or between information only (VCE-), information plus VCE without referral to VCE(VCE+), and information plus a VCE with specific referral to the VCE, requesting participants to use the VCE(VCE++) (experiment 2). In experiment 2 we additionally measured personality (neuroticism/conscientiousness) and information seeking style (monitoring/blunting). Results: Experiment 1. There were no differences in DC, knowledge or DA-use between VCE- (n=70) and VCE+ (n=70). Both DAs lead to a mean gain in knowledge from 39% at baseline to 73% after viewing the DA. Within VCE+, VCE-users (n=32, 46%) reported less DC compared to non-users. Since there was no difference in DC between VCE- and VCE+, this is likely an effect of VCE-use in a self-selected group, and not of the VCE per se. Experiment 2. There were no differences in DC or knowledge between VCE- (n=65), VCE+ (n=66), VCE++ (n=66). In all groups, knowledge increased on average from 42% at baseline to 72% after viewing the DA. Blunters viewed fewer DA-pages (R=0.38, p<.001). More neurotic women were less certain (R=0.18, p<.01) and felt less supported in decision making (R=0.15, p<.05); conscientious women felt more certain (R=−0.15, p<.05) and had more knowledge after viewing the DA (R=0.15, p<.05). Conclusions: Both DAs lead to increased knowledge in healthy populations making hypothetical decisions, and use of the VCE did not improve knowledge or DC. Personality characteristics were associated to some extent with DA-use, information seeking styles with aspects of DC. More research is needed to make clear recommendations regarding the need for tailoring of information provision to personality characteristics, and to assess the effect of VCE use in actual patients. Keywords: Values clarification method, Decision aid, Decisional conflict, Knowledge, Personality, Information seeking style, Experiment © 2014 Garvelink et al.; licensee BioMed Central Ltd. The decision d A good example of a preference sensitive decision with a difficult decision making process is the decision whether or not to undergo fertility preserving procedures (fertil- ity preservation, FP) before the start of the cancer treat- ment when diagnosed with breast cancer. The last decades, chemotherapy for breast cancer has increased survival chances, but with an increased possibility of los- ing fertility as a consequence [14]. Since many young cancer patients have a future child wish, interest has risen in possibilities to preserve fertility before undergo- ing cancer treatment. At this moment one can try to spare fertility by cryopreserving embryos, oocytes, or ovarian tissue [15]. However, since chances to become infertile are never 100%, not undergoing any fertility sparing treatment (wait and see) is also an option [14,16]. All these FP options come with risks and success rates [15,16]. For some years, FP has been offered to young women with breast cancer (18–40 years old). Not only are there many aspects to consider in deciding about FP, but the decision also has to be made in the short time frame (often a few days to a week) between diagnosis and start of the chemotherapy treatment, with competing demands from other breast cancer-related decisions and emotions [17]. In order to increase participation in decision making and improve decision making processes and outcomes for preference sensitive decisions, decision aids (DAs) are increasingly used. DAs are tools that provide at minimum some information about the (medical) prob- lem, possible solutions, including an option to wait and see, information about risks and uncertainties, and a bal- anced overview of advantages and disadvantages of each option [4]. Despite availability of quality criteria for the develop- ment and evaluation of DAs [5], which are used by most DA developers, DAs differ with regard to the type of medium (e.g. brochures, booklets, DVD’s, CD-ROMs, websites), their content, and the offered decision making support [6-8]. Some DAs provide patients with informa- tion only, summaries, or patient narratives, with which patients can implicitly clarify what is important for them. Others combine information with explicit values clarification methods (VCM), in which patients are sup- ported in active deliberation about what is important to them. In order to assist decision making about FP, we have developed a DA for women with breast cancer who have to decide about FP treatments [18]. Preference sensitive decision making and decision aids (DAs) Preference sensitive decision making and decision aids (DAs) An increasing number of medical decisions are prefer- ence sensitive, indicating that the “best” decision or treatment option does not only depend on what is best from a medical point of view, but depends on patient preferences with regard to the treatment options as well, and should therefore take into account the values a pa- tient attaches to the advantages and disadvantages of those option(s). In other words, with preference sensitive decisions, patients should be actively invited to partici- pate in decision making [1-3]. Abstract 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 credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 13 Garvelink et al. BMC Medical Informatics and Decision Making 2014, 14:68 http://www.biomedcentral.com/1472-6947/14/68 Background VCM) and analytical reasoning may not always be bene- ficial for decision making [11], since deliberation may overshadow important intuitive feelings that are more difficult to formulate but may be just as important in de- cision making [11]. The decision d BMC Medical Informatics and Decision Making 2014, 14:68 http://www.biomedcentral.com/1472-6947/14/68 Table 1 Content of the web-based decision aid “Breast cancer and wish for children” [18] Chapter # Titles and subtitles 1. Can I still achieve a pregnancy (after my treatment for breast cancer)? • Chemotherapy • Hormonal therapy • Other treatments 2. What can I do now to be able to have children later? • Wait and see • Cryopreservation of embryos • Cryopreservation of ovarian tissue • Cryopreservation of oocytes 3. What if I cannot achieve a pregnancy later? • No children • Oocyte donation • Adoption • Foster parenting 4. Background information • Fertility • Pregnancy and breast cancer • Genetics and breast cancer 5. Deciding about fertility preservation • What is important to me? - Wait and see - Cryopreservation of embryos - Cryopreservation of ovarian tissue • Question prompt list Table 1 Content of the web-based decision aid “Breast cancer and wish for children” [18] can indicate the extent to which they are in favor of the treatment options, and make a decision based on their own values. Patients are not provided with a clear-cut advice about which treatment to choose. This overview was chosen, rather than a summary bar indicating how much someone favors one of the FP treatments [9], be- cause we did not want to steer patients towards one of the treatments. In previous studies with this DA, acceptability, comprehensibility and user-friendliness were assessed in patients and clinicians and both the textual information and the VCE were considered relevant, coherent and understandable [18]. We hypothesized that the use of our DA with VCE in deciding about FP would decrease DC compared to information only [7,13]. Emotions, coping styles and personal characteristics may influence decision processes and the extent to which informational sources are used [19-22]. Since pa- tients may react with feelings of anxiety and depression to the news about a diagnosis with a life threatening dis- ease such as breast cancer and the prospect of a fertility threatening cancer treatment [23-25], it may be import- ant to acknowledge these emotions. Furthermore, emo- tions may affect values related to the decision, and risk perception [26]. Additionally, patients may have their own coping styles when it comes to getting informed about threatening medical situations, which is reflected in their preferred role in decision making and conse- quently their behavior with regard to seeking informa- tion. The decision d The DA has been designed for use by individual patients, as preparation for a consultation with a clinician in which the final de- cision is made. The DA consists of textual information (Table 1), and a fine-grained, explicit VCE. The few studies that have evaluated the impact of VCEs found in- dications for a beneficial effect of adding a VCE to a DA with regard to the percentage of patients who made an informed decision that was in agreement with their per- sonal values [3,12]. We have chosen for an explicit VCE since explicit VCEs showed indications of being more ef- fective than implicit VCEs with regard to satisfaction with preparation for decision making [13] and decisional conflict [7]. We have chosen a fine grained, additive VCE (comparing attributes of one treatment option at a time) [9], as we wanted patients to choose only between pursuing (or not) the options for which they are eligible. Each VCE consists of statements about the conse- quences of a FP option, for each of which patients are asked to indicate the extent to which they were consid- ered a benefit or disadvantage (Figures 1 and 2). Patients In general, DAs as a whole have been found to be ef- fective in reducing DC, to increase knowledge on the subject, to lead to more realistic expectations, and to lead to a higher percentage of patients who are able to decide on a course of action [3]. However, the effect of specific aspects, such as VCMs (if effective at all) is less clear [3,7,9-12]. Two patient studies that have evaluated the effect of DAs with several types of VCM compared to DAs without VCM or information only, did find that VCMs in the form of an explicit values clarification exer- cise (VCE) lead to a higher percentage of patients who made an informed decision that was in agreement with their personal values [3], a higher congruence between values and treatment [3], and lead to feeling better pre- pared for decision making [13]. Prior studies with healthy participants have found mixed results [7,10]. When comparing explicit with implicit VCM [7,12], ex- plicit VCM were more effective in healthy participants [7], but no improvements were found in patient popula- tions [12]. Additionally, in theory, deliberation (with Page 3 of 13 Garvelink et al. BMC Medical Informatics and Decision Making 2014, 14:68 http://www.biomedcentral.com/1472-6947/14/68 Garvelink et al. The decision d For example, patients with monitoring coping styles have been found to ask more questions in the con- sultation, and to prefer more detailed information [27]. Moreover, it has been suggested that patients with a more neurotic personality prefer less participation in de- cision making about treatment, while more conscien- tious patients prefer more participation and deliberation [28]. We therefore hypothesized that having a monitor- ing coping style or a more conscientious personality would be associated with more extensive use of the DA and VCE, less DC, and more knowledge after viewing the DA. Blunting coping styles and neurotic personalities were thought to be associated with less use of the DA and VCE, more DC and less knowledge after viewing the DA. • What is important to me? - Wait and see indicated on two VAS scales a) whether the statement is considered to be an advantage or disadvantage to the FP option, and b) the importance of the statement [9] (Figure 1). Additionally, patients have the option to add arguments and rate these as well. After rating the im- portance of the separate statements, the DA generates a summary that provides an overview of patients’ answers in descending order from most important to least important (as indicated by the patient) (Figure 2). Moreover, patients Figure 1 Example of a statement in the value clarification exercise (cryopreservation of ovarian tissue). For each statement in the value clarification exercise, patient rate whether it is an advantage (green; right side of the figure) or disadvantage (red; left side of the figure) and the extent to which the statement is considered important in decision making about FP (by lengthening or shortening the bar) [18]. Figure 1 Example of a statement in the value clarification exercise (cryopreservation of ovarian tissue). For each statement in the value clarification exercise, patient rate whether it is an advantage (green; right side of the figure) or disadvantage (red; left side of the figure) and the extent to which the statement is considered important in decision making about FP (by lengthening or shortening the bar) [18]. Page 4 of 13 Garvelink et al. BMC Medical Informatics and Decision Making 2014, 14:68 http://www.biomedcentral.com/1472-6947/14/68 Garvelink et al. BMC Medical Informatics and Decision Making 2014, 14:68 http://www.biomedcentral.com/1472-6947/14/68 Figure 2 Example of the summary of given ratings. Experiment 1 The primary outcome measure was DC. This was measured with a Dutch translation of the decisional con- flict scale (DCS) (including the subscales values clarity, informed decision making, effective decision making, de- cision making support, decision making uncertainty) [29]. The total scale consists of 16 items measured on a 5 point Likert scale ranging from 0 (totally disagree) 4 (totally agree). A total DC score is obtained by adding up the scores on the items, dividing them by the number of items and rescoring them from 0–100. A higher score on the DCS, or one of its subscales, indicates more DC. Participants Participants (N = 140) were healthy women between 18– 36 years old (M = 20.8, SD = 3.4), who had sufficient understanding of the Dutch language. Participants were invited by advertisements at universities, in libraries and on websites (including social media). Participants participated in exchange for either money (location 1; 8 euros) or course credits (location 2). Participants at location 1 had to actively approach the researcher and had to make an appointment to participate. Participants at location 2 could easily subscribe through an online sys- tem. All participants gave their informed consent before participating. In order to test the above mentioned hypotheses, two experiments were performed with healthy participants making hypothetical decisions about FP. In order to make participants more similar to patients, we induced with neutral, sad and anxious emotions in them. Whereas we are well aware of the limitations of including healthy par- ticipants instead of patients, we chose for healthy partici- pants to be able to include enough participants to reach sufficient power. Additionally, we thought it would be un- ethical to test these specific hypotheses in a patient popu- lation, before they were tested in non-patients. In experiment 1 we studied the effect of type of DA (information only versus information + VCE) on DA-use, DC, and knowledge. Additionally we assessed the effect of VCE-use on DC and knowledge. In experiment 2 we assessed associations between several personality charac- teristics and information seeking styles with the extent to which the DA was used and on DC and knowledge. The decision d The red box in the column with disadvantages (left side of the figure) represents the extent to which the rated disadvantage is important in the decision whether or not to opt for a certain FP option (in this case cryopreservation of embryos), as indicated by the patient herself in the previous step (Figure 1). The green box in the column with advantages (right side of the figure) represents the extent to which the rated advantage is important in the decision whether or not to opt for a certain FP option (in this case cryopreservation of embryos), as indicated by the patient herself in the previous step (Figure 1) [18]. Figure 2 Example of the summary of given ratings. The red box in the column with disadvantages (left side of the figure) represents the extent to which the rated disadvantage is important in the decision whether or not to opt for a certain FP option (in this case cryopreservation of embryos), as indicated by the patient herself in the previous step (Figure 1). The green box in the column with advantages (right side of the figure) represents the extent to which the rated advantage is important in the decision whether or not to opt for a certain FP option (in this case cryopreservation of embryos), as indicated by the patient herself in the previous step (Figure 1) [18]. Procedure M Measurements The study was completely computer- ized, outcomes were measured with questionnaires and web statistics. All measures were measured immediately after viewing the DA, except for knowledge which was measured both before and after viewing the DA. Methods experiment 1 Study design Emotion induction Emotions were induced by a com- bination of a short film fragment and background music during the entire experiment, two methods that have previously been found to be successful for inducing moods [30]. (ΔM = −2.0). No differences were observed between the three emotion induction conditions. Likely, the hypo- thetical script, which all participants had to read fol- lowing the emotion induction and before measurement of emotions, and the decision itself, may have evoked feelings of sadness and anxiety in all participants. Since no differences on perceived emotions were found be- tween emotion induction conditions, we controlled for emotion induction condition in all analyses but no fur- ther analyses were conducted with emotions. Directly after emotions were induced, respondents read a neutral, sad or anxious hypothetical script (subtly adapted to the induced emotions with words related to how somebody feels) in which they were asked to im- agine that they were at a consultation with their oncolo- gist and just received the diagnosis of breast cancer, for which they would be treated with chemotherapy. Since chemotherapy might influence their fertility, they are of- fered the chance to preserve their fertility before under- going chemotherapy. At the end of the script women were referred to a DA website to prepare them for mak- ing a decision. Respondents were then actually referred to the DA, using the following text: “by clicking on the link below you are referred to a decision aid about fertil- ity preservation for breast cancer patients. You are asked to make a decision whether or not you want to preserve your fertility, and if so, how”. They were instructed to spend as much time, and view as many pages on the DA as they thought was necessary to make a decision, there was no minimum or maximum. Methods experiment 1 Study design The study was a 2 (type of DA: DA with information only or DA with information and a VCE) by 3 (emotion: neutral, anxious, or sad) between subjects factorial de- sign, stratified by location (Leiden University – location 1, Tilburg University – location 2). For the randomization we used a block randomization scheme with variable blocks sizes containing all 6 possible combinations of emotions and type of decision aid randomization per block, developed by the department of medical statistics of the LUMC. The DA with information only consists of textual information (consisting of 20 separate webpages; for lay out see Figure 3b) and the DA with VCE addition- ally consists of a VCE for each FP option (consisting of six separate webpages) (for lay out see Figure 3a). All partici- pants gave their informed consent before participating. The experiment has been performed in accordance with the Declaration of Helsinki. Experiment 1 was primarily conducted at location 2, where no formal ethical approval was required. Other outcomes were knowledge about FP, measured with 10 statements about FP options with the answer cat- egories “true”, “false”, or “I do not know”. Furthermore, we measured preferred FP option (5 categories: wait and see (not undergoing a fertility sparing treatment), cryopreser- vation of embryo’s, oocytes, ovarian tissue, do not know), socio-demographic characteristics (age, child wish, parity, experience with (breast) cancer in relatives and peers, rela- tional status, cohabiting, education, ethnicity, religious af- filiation), and web statistics such as total time spent on the DA and number of informational- and VCE-pages viewed. Page 5 of 13 Garvelink et al. BMC Medical Informatics and Decision Making 2014, 14:68 http://www.biomedcentral.com/1472-6947/14/68 Garvelink et al. BMC Medical Informatics and Decision Making 2014, 14:68 http://www.biomedcentral.com/1472-6947/14/68 Figure 3 The two types of DAs: a) DA with information (blue buttons) and the VCE (red button); b) DA with information only (blue buttons). and the VCE (red button); b) DA with information only (blue buttons). A with information (blue buttons) and the VCE (red button); b) DA with information only (blue buttons). Emotion induction Emotions were induced by a com- bination of a short film fragment and background music during the entire experiment, two methods that have previously been found to be successful for inducing moods [30]. Statistics Analyses were conducted with SPSS 20.0. Differences be- tween the DAs in continuous outcomes with only one measurement moment (e.g. DC) were tested with one-way ANOVAs with DA-type (VCE +/−) as between-subjects factor. Differences in knowledge scores at baseline and after viewing the DA were tested with a General Linear Model (GLM) for repeated measures, with DA-type (VCE +/−) as between-subjects factor. Analyses were subdivided in primary (intention to treat) and secondary analyses (based on actual use of the DA and VCE). Since not all participants randomized to in- formation plus VCE actually used the VCE, we conducted secondary analyses with a new grouping variable, consist- ing of three arms: information only (VCE-), information plus a VCE which was not used (VCE +−), and informa- tion plus a VCE which was used (VCE ++). This variable (three groups) replaced the fixed variable “DA-type” in the ANOVA and GLM for repeated measures as described above. Post hoc analyses were conducted to check for specific group differences. All the analyses were done, while controlling for the effect of emotion induction condition and location. In order to test whether the emotion induction was successful, participants were asked before (pre induc- tion - I), immediately after emotion induction and after reading the script (post induction - II), and after view- ing the DA (post DA - III), to what extent they felt happy, anxious and sad at that moment on a 7-point Likert scale (i.e. “to what extent do you feel happy at this moment?”). This emotion manipulation check indi- cated that all participants felt more sad (ΔM = 2.1) and anxious (ΔM = 2.1) after induction, and less happy Page 6 of 13 Garvelink et al. BMC Medical Informatics and Decision Making 2014, 14:68 http://www.biomedcentral.com/1472-6947/14/68 correcting for desire for children. Results of the additional analyses were similar to the above mentioned results. Participants and socio-demographic characteristics One-hundred fifty-one women participated. We ex- cluded 11 women because of incomplete data on main outcomes due to problems with internet or the question- naire. The total population used for data analyses con- sisted of 140 participants, 39 in location 1, and 101 women in location 2. Posthoc analyses indicated that women who used the VCE, and women who received information only (who were not able to use a VCE), reported significantly better (lower) scores on DC (F(2,122) = 6.4, p < .01), values clarity (F(2,122) = 9.4, p < .001), decisional support (F(2,122) = 3.4, p < .05), and informed decision making (F(2,122) = 3.2, p < .05) than women who were able to but did not use the VCE. Furthermore, women who had used the VCE reported better (lower) scores on effective decision making (F(2,122) = 4.4, p < .05) than those who did not use it (Table 2). At baseline there were no differences in socio- demographic characteristics between the locations (data not shown). Furthermore, randomized conditions (DA- types) were comparable on most socio-demographic characteristics. With regard to future desire for children we found that women in the information only condition somewhat less often had a child wish than women in the VCE + conditions (χ2 = 7.17, p < .05; Table 2). Effect of type of DA on decision making, DA use, decisional conflict, knowledge (Primary analyses) Of the total population, 114 women (81%) were able to make a decision whether or not to preserve fertility: 24 women (21%) wanted to wait and see, and 90 women (79%) chose to cryopreserve either embryos (n = 45), oo- cytes (n = 34) or ovarian tissue (n = 11). Conclusion experiment 1 Experiment 1 showed no difference in knowledge or DC between women who received a DA with or without a VCE. Secondary analyses revealed less DC for women who used the VCE compared to those who chose not to use it. However, there was no difference in DC between the VCE-users and the women who received a DA with information only (without VCE). There were no effects of DA-type (information with or without VCE) on time spent on the DA or number of pages viewed (Table 2). Mean number of pages viewed for the total group was 13.4 (SD = 7.7) and mean time spent on the DA was 8.9 minutes (SD = 7.9). The correl- ation between time spent on DA and pages viewed was high (r = .75, p < .001), therefore we chose to use only “time spent” in further analyses. Power calculation A sample size of 64 participants per treatment arm was considered sufficient to analyze main effects on DC with a power of 0.8 (Cohen’s d = 0.5; β = 0.2; α = 0.05). Within the two DA-conditions respondents were equally ran- domized among the three different emotion conditions. Effect of using the VCE on total DA use, decisional conflict (Secondary analyses) Of the women in the VCE + condition (n = 70), only 33 women (47%) had viewed the VCE (VCE++, Table 2). These women spent on average 2.5 minutes (range 10 seconds – 8 minutes) on the VCE. There was a significant difference in time spent on the DA between women who did or did not use the VCE, but not with women who were not able to use the VCE (F(2,123) = 9.2, p < .001). Women who had used the VCE spent more time on the DA than women who had not. Experiment 2 p Experiment 1 showed less DC for women using a VCE compared to women who chose not to use it, but no dif- ference compared to women who received information only, so we were interested in finding explanations for this difference. Since there was no difference between the VCE-users and the women who received a DA with information only (without VCE) this might be an effect of VCE-use in a self-selected group (for example related to personality characteristics), and is not likely an effect of the VCE per se. Therefore, in experiment 2 personality characteristics were measured to investigate whether DA- and VCE-use and effectiveness of DA- and VCE-use were associated with certain personality characteristics. There were no significant differences in DC (including scores on all subscales) or knowledge between women who received the DA with information only (VCE-) or with information and a VCE (VCE+) (Table 2). In both conditions, the DA led to a significant increase in know- ledge (F(1,127) = 264.96, p < .001). At baseline, mean knowledge score for the total group was 4.2, after view- ing the DA it was 7.6; a relative increase of 81%. More- over, after adjustment for baseline knowledge there was a significant positive relation between knowledge after viewing the DA and time spent on the DA (r = .33 p < .001). In the first experiment, only a minority of respondents who received a DA with VCE, accessed the VCE. Since no emphasis was put on the availability of the VCE in their DA, it is possible that some did not see the VCE. Therefore, to increase the number of VCE-users in Experiment 2, we Since there was a significant difference in desire for chil- dren between the groups, we have repeated all the analyses Garvelink et al. BMC Medical Informatics and Decision Making 2014, 14:68 http://www.biomedcentral.com/1472-6947/14/68 Garvelink et al. Experiment 2 BMC Medical Informatics and Decision Making 2014, 14:68 http://www.biomedcentral.com/1472-6947/14/68 Page 7 of 13 Page 7 of 13 Table 2 Socio-demographic characteristics, differences in decision-making, decisional conflict, knowledge and DA use between women who received a DA with information only or a DA with information and an explicit VCE (subdivided by whether they used the VCE or not), controlled for emotion induction condition DA with information only (VCE-) DA with information plus VCE (VCE+) Primary analysis (A vs B) Secondary analysis (A vs C vs D) Total group VCE- (N = 70) Total group VCE + (N = 70) VCE used (VCE++) (n = 33) VCE not used (VCE + −) (n = 37) F-(condition) or χ2-value Post hoc F-(condition) or χ2-value Post hoc A B C D Age, mean (SD) 20.7 (3.3) 20.9 (3.5) 20.4 (3.5) 21.6 (3.5) NS NS Child wish, yes, n (%) 56 (80) 64 (91) 34 (91) 30 (91) 6.9* A < B 7.17* A < D = C Children, yes, n (%) 3 (4) - - - - - Partner, yes, n (%) 34 (49) 42 (60) 24 (65) 18 (55) NS NS Decisional conflict Total DCS, M (SD) 40.9 (11.6) 43.6 (14.2) 37.9 (15.7) 48.6 (10.6) NS 6.4 A = C < D Values clarity, M (SD) 27.7 (14.5) 32.0 (18.4) 22.7 (16.4) 40.3 (16.1) NS 9.4 A = C < D Decisional support, M (SD) 44.7 (14.2) 45.9 (16.7) 38.4 (18.2) 52.7 (11.9) NS 3.4* A = C < D Effective DM, M (SD) 32.8 (15.7) 33.6 (18.7) 27.3 (19.9) 39.2 (15.9) NS 4.4* D > C Uncertainty, M (SD) 36.6 (17.8) 40.3 (16.8) 40.9 (20.3) 39.8 (13.2) NS 0.97 NS Informed DM, M (SD) 65.2 (22.6) 69.6 (22.9) 64.1 (26.6) 74.5 (17.9) NS 3.2* A = C < D Knowledge Knowledge post DA, M (SD) 7.3 (1.9) 7.2 (1.7) 7.4 (1.7) 6.9 (1.8) NS NS Time spent (minutes) Total time spent, M (SD) 8.5 (7.4) 9.3 (8.4) 14.3 (9.2) 4.9 (4.4) NS 9.2** D < A < C Time spent on information only, M (SD) 8.5 (7.4) 8.3 (7.3) 11.8 (8.0) 4.9 (4.4) NS 3.9* D < A < C Pages viewed Δ 12.5 (2–38) 14.4 (3–36) 21 (9–36) 8.5 (3–17) NS 27.61** D < A < C Made a decision, yes, n (%) 56 (80) 58 (82.8) NS NS * = p < 0.05 = p < 0.01, NS = not statistically significant; M = mean; SD = standard deviation; A = DA with information only (VCE-); B = DA with information and VCE (VCE+); C = DA with information and VCE, VCE used (VCE++). Experiment 2 D = DA with information and VCE, VCE not used (VCE + −). Δ some pages were viewed repeatedly by the respondents. Table 2 Socio-demographic characteristics, differences in decision-making, decisional conflict, knowledge and DA use between women who received a DA with information only or a DA with information and an explicit VCE (subdivided by whether they used the VCE or not), controlled for emotion induction condition Table 2 Socio-demographic characteristics, differences in decision-making, decisional conflict, knowledge and DA use between women who received a DA with information only or a DA with information and an explicit VCE (subdivided by whether they used the VCE or not), controlled for emotion induction condition added a third condition to the experiment: information plus VCE, with explicitly referring to the VCE. preserve your fertility and how”. Respondents who were specifically referred to the VCE additionally received the following text: “We would like to point out that the de- cision aid consists of both textual information about fer- tility preservation as well as the chapter “deciding about fertility preservation” which is meant to help you order your thoughts about fertility preservation and make a decision. Please use this chapter in making your own de- cision about fertility preservation”. The experiment has been performed in accordance with the Declaration of Helsinki. The study was approved by a local Ethics Com- mittee at Leiden University, from Tilburg University no additional formal ethical approval was required. Methods experiment 2 Study design Participants (N = 199) were randomly assigned to a DA with information only (VCE-), a DA with information and a VCE without referring to the VCE (VCE+), and a DA with information and a VCE with explicitly refer- ring to the VCE (VCE++), stratified by location (Leiden University – location 1, Tilburg University – location 2). For the randomization we used a block randomization scheme with variable blocks sizes containing all 3 pos- sible combinations of type of decision aid and referral to the VCE per block, developed by the department of medical statistics of the LUMC. Respondents were re- ferred to the DA with the following text: “By clicking on the link below you are referred to a decision aid about fertility preservation for breast cancer patients. You are asked to make a decision whether or not you want to Participants and socio-demographic characteristics Participants and socio-demographic characteristics Information seeking styles (Monitoring and Blunting) were measured with a short version of the Threatening Medical Situations Inventory (TMSI) of Miller [31], after the example of Ong et al. [27]. A monitoring information seeking style indicates cognitive confrontation; a person with this style tends to actively seek out and monitor information about the threatening event [31]. A blunting information seeking style indicates cognitive avoidance; a person with this style tends to seek cognitive distraction from the threatening event and psychologically blunts threat- relevant information [31]. Respondents were asked to read two hypothetical situations (1-vague suspicious headache complaints and 2-choosing for uncertain heart surgery) and complete three monitoring and three blunting items on a five point Likert scale ranging from 1–5 (not at all to strongly applicable to me) for each scenario. Total monitoring and blunting scores were calculated by adding up all relevant items. Personality traits (neuroticism and conscientiousness) were measured with the neuroticism (8 items) and conscientiousness subscales (9 items) of the Dutch translation of the Big Five Inventory [32]. A high score on neuroticism indicates that women are emotionally instable; a high score on conscientiousness indicates that women are well-organized and task- and goal- directed [33]. Participants were asked to rate their agreement with statements about their perception of themselves in varying situations, on a five-point Likert scale ranging from 1 (strongly disagree) to 5 (strongly agree). Total scores were calculated by adding up all relevant items, divided by the total number of items per scale. One hundred ninety-nine eligible women participated. Due to missing data on some questions, the total population used for data analyses consisted of 197 par- ticipants, 91 women in location 1, and 106 women in location 2. At baseline, there were no significant differences with regard to socio-demographic characteristics between conditions. Mean age of the respondents was 21.4 years old (range 18–32), 179 women (90%) had a future desire for children, and nobody had children. Participants Participants were healthy women between 18–32 years old (M = 21.4, SD = 2.3), with sufficient understanding of the Dutch language. Participants were invited by adver- tisements at the same universities as in experiment 1. Participants participated in exchange for either course Page 8 of 13 Garvelink et al. BMC Medical Informatics and Decision Making 2014, 14:68 http://www.biomedcentral.com/1472-6947/14/68 Garvelink et al. BMC Medical Informatics and Decision Making 2014, 14:68 http://www.biomedcentral.com/1472-6947/14/68 Garvelink et al. BMC Medical Informatics and Decision Making 2014, 14:68 http://www.biomedcentral.com/1472-6947/14/68 credits/hours or money (6 Euros) at both study loca- tions. All participants gave their informed consent before participating. continuous outcomes were tested with ANOVAs. Ana- lyses were subdivided in primary (intention to treat) and secondary analyses (based on actual use of the DA and VCE). Associations between personality characteristics and DA-use were studied with Pearson’s product mo- ment correlations (PPMC) and GLMs. Procedure The study consisted of two parts. Part I con- sisted of completing questions about personality and in- formation seeking style. Part II consisted of reading a neutral hypothetical script (see Experiment 1) after which respondents viewed a version of the DA (according their randomization) and completed questionnaires related to their decision making (process). Both parts were presented as independent studies of different researchers. All the analyses were done, while controlling for the effect of location. Effect of condition on decision making, DA use, decisional conflict, knowledge Primary analyses One hundred fifty-two women (77%) were able to make a decision whether or not to preserve fertility, of which 31 (20%) women wanted to wait and see, and 121 (80%) women chose to cryopreserve either em- bryos (n = 67), oocytes (n = 47) or ovarian tissue (n = 7). There were no differences between the 3 conditions in total time spent on the DA and the extent to which the informational pages were used (Table 3). However, we did find differences in the extent to which the VCE was used; women who were referred to the VCE signifi- cantly more often used the VCE (F(2,127) = 3.2 p < .05), viewed more VCE pages (F(2,127) = 8.8, p < .001), and spent more time on the VCE (F(2,127) = 5.1, p < .01) than women in the VCE + condition who were not referred. No significant differences were found between randomization conditions with regard to DC (or subscales of the DCS) (Table 3). Use of the DA lead to a relative increase in knowledge of 71% (M = 4.2 to M = 7.2) in the total popu- lation (F(1,191) = 24.5 p < .001). No differences in know- ledge were found between the randomization conditions. Moreover, after adjustment for baseline knowledge score there were significant positive relations between know- ledge after viewing the DA, time spent on the DA (r = .33 p < .001), time spent on the informational pages (r = .31, p < .001) and time spent on the VCE (r = .18, p < .05). Power calculation P i d Presuming a medium effect size (f = 0.25), we needed a total of 179 participants in three groups to reach a power of 0.8 (α = 0.05, β = 0.2, with 1 covariate). Measurements Measures were as in experiment 1, with addition of the following scales: Statistics ff Differences in knowledge scores at baseline and after viewing the DA were tested with a General Linear Model (GLM) for repeated measures. Differences in other Garvelink et al. Statistics ff BMC Medical Informatics and Decision Making 2014, 14:68 http://www.biomedcentral.com/1472-6947/14/68 Table 3 Differences in decision-making, decisional conflict, knowledge and DA use between women who received information only or information plus VCE (subdivided by referral to the VCE, and use of the VCE) (N = 197) Information only VCE- (N = 65) Information plus VCE (VCE+) (N = 132) A No referral to VCE (n = 66) With referral to VCE (n = 66) Primary analysis (A vs B vs E) Secondary analysis (H vs I) Total (no referral) (N = 66) VCE not used (VCE + −) (n = 31) VCE used (VCE++) (n = 35) Total (with referral) N = 66 VCE not used (VCE + −) (n = 17) VCE used (VCE++) (n = 49) Total VCE not used (C + F) (n = 48) Total VCE used (D + G) (n = 84) F- value F- value Post hoc analysis B C D E F G H I Time spent (min) 7.7 (5.6) 8.9 (6.6) 6.4 (6.5) 11.2 (6) 9.4 (6.9) 4.8 (5.1) 11.1 (6.8) 5.8 (6.0) 11.1 (6.4) NS 15.6 H < I Time on informational pages 7.7 (5.6) 7.8 (5.93) 6.4 (6.5) 8.9 (5.2) 7.1 (5.6) 4.8 (5.1) 7.9 (5.6) 5.8 (6.0) 8.3 (5.4) NS 4.3* H < I Pages viewed (incl vce pages) 13.3 (8.7) 16.1 (9.7) 11.1 (4.8) 20.4 (10.9) 17.4 (11.4) 7.7 (3.6) 20.7 (11.5) 9.9 (4.6) 20.5 (11.2) NS 20.9 H < I Informational pages 13.3(8.7) 13.2 (6.1) 11.1 (4.8) 15.1 (6.6) 11.9 (6.9) 7.7 (3.6) 13.3 (7.3) 9.9 (4.6) 14.1 (7.0) NS 7.1 H < I Knowledge After viewing the DA 7.5 (1.6) 7.1 (1.9) 7.3 (1.9) 6.9 (2.0) 7.2 (1.8) 6.5 (2.4) 7.5 (1.5) 7.0 (2.1) 7.2 (1.8) NS NS Decisional conflict Total DCS M (SD) 44.1 (12.3) 43.6 (11.4) 41.8 (10.1) 45.2 (12.3) 41.6 (9.5) 44.5 (7.6) 40.6 (9.9) 42.8 (9.3) 42.5 (11.2) NS NS Values clarity M (SD) 32.9 (14.7) 34.2 (15.2) 33.6 (13.3) 34.7 (16.8) 30.6 (13.7) 37.7 (12.2) 28.1 (13.5) 35.1 (12.9) 30.9 (15.2) NS NS Decisional support M (SD) 45.2 (14.4) 45.3 (14.5) 43.3 (12.4) 47.1 (16.0) 43.3 (12.4) 48.5 (12.6) 41.5 (11.9) 45.1 (12.6) 43.9 (13.5) NS NS Effective DM M (SD) 37.3 (16.7) 34.2 (14.3) 31.6 (12.9) 36.6 (15.2) 32.3 (13.3) 32.7 (16.7) 32.1 (12.1) 32.0 (14.2) 34.0 (13.6) NS NS Uncertainty M (SD) 41.5 (16.7) 40.9 (16.7) 41.1 (17.2) 40.7 (16.5) 39.6 (14.6) 37.3 (13.8) 40.5 (14.9) 39.7 (16.1) 40.6 (15.5) NS NS Informed DM M (SD) 66.0 (21.4) 66.5 (18.2) 62.9 (20.4) 69.7 (15.6) 65.4 (19.9) 70.6 (15.9) 63.6 (21.0) 65.6 (19.1) 66.2 (19.1) NS NS Personality traits, high M (SD) Neuroticism 3.1 (0.53) 3.0 (0.61) 2.9 (0.57) 3.1 (0.64) 3.1 (0.54) 3.1 (0.62) 3.1 (0.52) 2.9 (0.58) 3.1 (0.57) NS NS Conscientiousness 3.5 (0.54) 3.6 (0.64) 3.6 (0.59) 3.5 (0.69) 3.5 (0.63) 3.4 (0.82) 3.5 (0.56) 3.5 (0.68) 3.5 (0.61) NS NS Monitoring 19.7 (4.59) 19.9 (4.31) 20.1 (4.36) 19.7 (4.30) 19.7 (4.1) 19.3 (4.36) 19.9 (4.08) 19.8 (4.34) 19.8 (4.15) NS NS Blunting 18.6 (2.9) 17.8 (3.11) 18.1 (2.58) 17.5 (3.53) 18.1 (3.2) 18.1 (3.49) 18.1 (3.17) 18.0 (2.88) 17.9 (3.31) NS NS p < 0.05; *p < 0.001, NS = not significant; min = minutes; M = mean; SD = standard deviation; DA = decision aid; VCE = values clarification exercise. eviation; DA decision aid; VCE values clarification exercise. A DA with information only (VCE ); B DA with information and VCE, not used; D = DA with information and VCE, without referral to VCE, VCE used; E = DA with information and VCE, without referral to information and VCE with referral to VCE VCE used Statistics ff A = DA with information only (VCE-); B = DA with information and VCE, without referral to VCE; C = DA with information and VCE, without referral to VCE, VCE not used; D = DA with information and VCE, without referral to VCE, VCE used; E = DA with information and VCE, without referral to VCE; F = DA with information and VCE, with referral to VCE, VCE not used; G = DA with information and VCE, with referral to VCE, VCE used. Page 10 of 13 Page 10 of 13 Garvelink et al. BMC Medical Informatics and Decision Making 2014, 14:68 http://www.biomedcentral.com/1472-6947/14/68 Garvelink et al. BMC Medical Informatics and Decision Making 2014, 14:68 http://www.biomedcentral.com/1472-6947/14/68 Garvelink et al. BMC Medical Informatics and Decision Making 2014, 14:68 http://www.biomedcentral.com/1472-6947/14/68 Secondary analyses Of the women in the VCE + condi- tions (with and without referral, n = 132), 84 viewed the VCE (63%). Women who made use of the VCE spent more time on the total DA (F(2,128) = 15.6 p < .001), and on the informational pages of the DA (F(2,128) = 4.3, p < .01) and viewed more informational pages (F(2,128) = 7.1, p < .001) than those who did not, indicating that they used the whole DA more thoroughly. Within VCE + (with and without referral), there were no significant differ- ences in DCS or any of the subscales between women who did (VCE++) or did not use the VCE (VCE + −), indicating that VCE-use was not related to differences in DC between the conditions (Table 3). No differences in knowledge were found between women who did or did not use the VCE. whether DA- and VCE-use and effectiveness were affected by personality characteristics. Experiment 2 confirmed that there was no association between VCE-use and DC or knowledge, and showed that information seeking style affected DA use (number of pages viewed), but not VCE- use. Personality traits were to some extent associated with aspects of DC. In both experiments there was a large knowledge increase of both DAs, indicating that the infor- mation in the DA is beneficial with regard to knowledge, especially for women who use the DA more thoroughly, highly conscientious women and women with more moni- toring information seeking styles. Statistics ff g g y Since quality criteria for DAs anticipate on the addition of a VCM to DAs [32,34], but the results between studies on the effectiveness of VCM vary from beneficial to no (significant) effects [3,7,11-13], we thought it was import- ant to study the effect of our DA plus VCE before imple- menting it in patient care. However, it seems that not all patients or participants tend to use a VCE when avail- able. In both our experiments there were women who had used the information on the DA, but not the VCE. Al- though active referral to the VCE increased use of the VCE, independent of personality or information seeking style, still 17 women (15%) who were referred to the VCE did not use it (experiment 2). In the condition without referral about half of the women used the VCE in both experiments. A study with patients who were actually fa- cing the decision to undergo FP found even lower per- centages of patients (23%) that used their VCE [35,36]. Although VCE-use does not have to take much extra time (in our experiments: ±5 minutes), it is an extra effort in the already short time patients have to get informed and make a decision, so it should be considered whether active referral is appropriate. The hereby conducted exper- iments did not show a direct beneficial effect of VCE-use with regard to knowledge or DC. Therefore, we found no obvious reason to recommend increasing VCE-use by actively referring patients to it. Since other VCM were not always beneficial either, quality criteria should perhaps be more cautious regarding VCM recommendation as well [37]. Effect of personality characteristics and information seeking style on DA use, decision making, decisional conflict and knowledge Personality characteristics and information seeking styles were equally distributed (Table 3). Blunting (with regard to information seeking) was as- sociated with viewing less informational pages (r = −.38, p < .001) and less total pages (r = −.29, p < .001). None of the personality traits were significantly associated to the extent to which the DAs were used (time spent, pages viewed). With regard to DC, being more neurotic was associated with more decision making uncertainty (r = .18 p < .01), and decision making support (r = .15, p < .05) and being more conscientious was associated with less deci- sion making uncertainty (r = −.15, p < .05). None of the in- formation seeking styles were associated with aspects of DC. Knowledge after viewing the DA was associated with a more conscientious personality (r = .15, p < .05) and a more monitoring information seeking style (r = .15, p < .05) (cor- rected for baseline knowledge). Discussion Sometimes, deci- sion making may improve when the decision is made after distraction, due to the so-called unconscious thought effect [11,49]. These results have to be interpreted with caution due to some limitations. The DA used in this study was ori- ginally designed for patients, who make the decision in consultation with a physician, not directly after viewing the DA, so results of a healthy population making the decision by themselves, directly after viewing the DA may not be completely generalizable to patients that are actually facing this decision. Moreover, effects were mea- sured immediately after decision making, but it is pos- sible that a DA has more effect on DC and preparation for decision making sometime after the decision is made [13]. Despite randomization, there was a significant dif- ference in desire for future children between women who received a DA with VCE and those who received in- formation only in experiment 1. Although all respon- dents had to imagine that they had a “hypothetical desire for children” for the future as part of the hypo- thetical script, their actual desire for children could have influenced decision making about fertility preservation. Therefore, all analyses were repeated while controlling for whether or not women had a desire for children. As the results of these analyses were very comparable to the results reported here, we may conclude that the results of experiment one are not critically dependent on baseline levels of desired children. In experiment 1, fewer women than expected used the VCE, which re- duced our power. Therefore we added a third condition to the second experiment, in which women were actively referred to the VCE. In these experiments, levels of DC were relatively high (worse) compared to other studies with patients [12,42-44] and healthy participants [10], but comparable to studies with healthy students as participants [7,45]. Possibly, in contrast to what we would have expected, not actually fa- cing the decision made decision making harder. Moreover, most studies which assessed DC in patients studied pri- mary treatment decisions, which are different decisions than the decision to undergo FP or not, which is an “extra” decision that has to be made in an emotionally challenging period between diagnosis and start of the oncologic treat- ment [46,47]. Discussion It is possible that our healthy participants did not really ex- perience the threat, or did not have a desired emotion (which should follow from decision making), which may have affected their information seeking style and their de- cision making process. Also, it is likely that actual patients are sadder than healthy participants, and therefore elabor- ate more on information [40,41]. However, in the current experiments we were not able to study this properly. It is possible that participants in experiment 1 were more simi- lar to patients because of the sad emotions that were in- duced with them. Moreover, all participants in experiment 1 felt more sad and anxious after the induction with happy, sad or anxious emotions. The most plausible ex- planation therefore is that besides the three different mood induction techniques that were used in the study (a movie, music and suggestions in the script) all partic- ipants had to read a relatively sad hypothetical script and make a difficult (hypothetical) decision, which may have overruled the effect of the other mood induc- tion techniques. Unfortunately, this precluded us from analyzing the DA effectiveness in different emotional states. may therefore approach the decision more analytically compared to patients from the general population which may increase DC scores. Interestingly, other studies with actual patients [3,13] more often find beneficial effects of VCEs than studies with healthy participants [7,10]. This may also be related to discrepancies between the way DAs are designed and how they are used in healthy partici- pants. It should be noted that the DA as used in the ex- periments was originally designed for patients, who use the DA in preparation for a consultation with a physician in which a final decision is made about FP. This consult- ation is often within a few days after diagnosis (and DA use). In the experiments, respondents had to decide dir- ectly after viewing the DA, without support from a phys- ician. Hence, both the limited amount of available time and the lack of interaction about the decision may have influenced decision making for our participants. It is likely that in the experiments decisions were made consciously since they were made directly after viewing the DA. Ac- tual patients may make more intuitive decisions, since they are distracted in the time between using the DA and visiting the physician to decide. Discussion In the above mentioned experiments we assessed the effectiveness of a DA with information only or with additional VCE with regard to knowledge and DC, and the effect of personality characteristics on DA use and effectiveness. Additionally, in secondary analyses we assessed differences in effect between women who did or did not use the VCE. Experiment 1 showed no difference in knowledge or DC between DAs with or without a VCE. Secondary analyses revealed less DC for women who used the VCE compared to those who did not use the VCE, but it was unlikely that the VCE had caused this difference, since there was no difference in DC between women who received information plus VCE and used the VCE and women who received information only. In experiment 2 personality characteristics were measured to investigate We did find a beneficial effect of both DAs (with or without VCE) on knowledge, since use of the DA lead to a relative knowledge increase of 71-81% com- pared to baseline (experiment 2 and 1 respectively), and time spent on the DA was related to knowledge increase after using the DA. It is likely that the in- crease in knowledge is mostly related to the informa- tional pages. None of the personality characteristics or information seeking styles were associated with VCE-use; information seeking styles were only associated with DA-use in gen- eral, and personality was only associated with DC. How- ever, effect sizes were small (<.3). Consistent with the Page 11 of 13 Page 11 of 13 Garvelink et al. BMC Medical Informatics and Decision Making 2014, 14:68 http://www.biomedcentral.com/1472-6947/14/68 literature, women with more blunting coping styles viewed less pages on the DA website [27,38]. More neurotic women reported to be more uncertain about the decision. However, Case et al. [39] mention that information seek- ing style does not only depend on personality, but also on the threat and controllability that is experienced, and on the desired effect of the information [39]. I.e., information can be used to do something about a potential threat, or to be reassured that there is no threat [39]. Additionally, anticipated emotions that are imagined with potential out- comes of decision making may affect the decision [26]. Author details 1 1Department of Gynecology, Leiden University Medical Center (LUMC), Mail zone VRSP, P/O Box 9600, 2300 RC Leiden, the Netherlands. 2Department of Medical Decision Making, LUMC, Leiden, the Netherlands. 3Tilburg Institute for Behavioral Economics Research (TIBER), Department of Social Psychology, Tilburg University, Tilburg, the Netherlands. 19. Iyengar SS, Wells RE, Schwartz B: Doing better but feeling worse. Looking for the “best” job undermines satisfaction. Psychol Sci 2006, 17(2):143–150. 20. Schwartz B, Ward A, Monterosso J, Lyubomirsky S, White K, Lehman DR: Maximizing versus satisficing: happiness is a matter of choice. J Pers Soc Psychol 2002, 83(5):1178–1197. Discussion For patients it is often a decision between their chances for survival, and the extent of their desire for children taken into account their possibilities for FP (related to personal characteristics such as their age, or whether they have a partner) [48]; factors that often ex- clude some FP options and therefore might facilitate decision making. Likely, our healthy participants who were not actually facing the decision of FP did not take these factors into account which may have increased their DC scores. Additionally, students are highly educated and Page 12 of 13 Garvelink et al. BMC Medical Informatics and Decision Making 2014, 14:68 http://www.biomedcentral.com/1472-6947/14/68 Garvelink et al. BMC Medical Informatics and Decision Making 2014, 14:68 http://www.biomedcentral.com/1472-6947/14/68 Conclusions 7. Abhyankar P, Bekker HL, Summers BA, Velikova G: Why values elicitation techniques enable people to make informed decisions about cancer trial participation. Health Expect 2011, 14(1):20–32. doi:10.1111/j.1369- 7625.2010.00615.x. 7. Abhyankar P, Bekker HL, Summers BA, Velikova G: Why values elicitation techniques enable people to make informed decisions about cancer trial participation. Health Expect 2011, 14(1):20–32. doi:10.1111/j.1369- 7625.2010.00615.x. The above mentioned experiments indicate that our DA about FP for breast cancer patients seems beneficial with regard to knowledge increase, but that the VCE does not seem to improve knowledge or DC. However, nor did use of the VCE seem to cause any harm, other than the time involved in completing it (which was acceptable). Additionally, it is important to understand that personal- ity characteristics and information seeking style may be important factors in determining the extent to which DAs are used and helpful for women. It is of utmost im- portance that these findings are assessed in patients as well, since results may be different when actually facing the decision to preserve fertility. 8. Llewellyn-Thomas H: Values clarification. In Shared Decision Making in Health Care: Achieving Evidence Based Patient Choice. 2nd edition. Edited by Elwyn G, Edwards A. Oxford: Oxford University Press; 2009:123–133. 8. Llewellyn-Thomas H: Values clarification. In Shared Decision Making in Health Care: Achieving Evidence Based Patient Choice. 2nd edition. Edited by Elwyn G, Edwards A. Oxford: Oxford University Press; 2009:123–133. 9. Feldman-Stewart D, Brennenstuhl S, Brundage MD, Roques T: An explicit values clarification task: development and validation. Patient Educ Couns 2006, 63(3):350–356. doi:10.1016/j.pec.2006.04.001. 9. Feldman-Stewart D, Brennenstuhl S, Brundage MD, Roques T: An explicit values clarification task: development and validation. Patient Educ Couns 2006, 63(3):350–356. doi:10.1016/j.pec.2006.04.001. 10. Sheridan SL, Griffith JM, Behrend L, Gizlice Z, Jianwen C, Pignone MP: Effect of adding a values clarification exercise to a decision aid on heart disease prevention: a randomized trial. Med Decis Making 2010, 30(4):E28–E39. doi:10.1177/0272989X10369008. 10. Sheridan SL, Griffith JM, Behrend L, Gizlice Z, Jianwen C, Pignone MP: Effect of adding a values clarification exercise to a decision aid on heart disease prevention: a randomized trial. Med Decis Making 2010, 30(4):E28–E39. doi:10.1177/0272989X10369008. 11. de Vries M, Fagerlin A, Witteman HO, Scherer LD: Combining deliberation and intuition in patient decision support. Patient Educ Couns 2013, 91(2):154–160. doi:10.1016/j.pec.2012.11.016. 12. 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W2058160371.txt	https://europepmc.org/articles/pmc2010035?pdf=render	en		Erythropoiesis and iron metabolism in Hodgkin's disease	British journal of cancer	1,979	cc-by	2,973	Br. J. Cancer (1979) 40, 365 ERYTHROPOIESIS AND IRON METABOLISM IN HODGKIN'S DISEASE S. AL-ISMIAIL, I. CAVILL, I. H. EVANSt, A. JACOBS, C. RICKETTS, D. TREVETT* AND J. A. WHITTAKER Froom the Department of Haematology, Welsh National School of Medicine, University Hospital of Wales, and the t Velindre Hospital, Cardiff Received 21 Marchl 1979 Accepted 14 May 1979 Summary.-Recently developed techniques for the investigation of iron kinetics were used to study the disturbance of iron metabolism in 19 untreated patients with Hodgkin's diseases (HD). The erythroid abnormality in newly diagnosed HD appears to be confined to those patients with systemic symptoms of weight loss, night sweats and fever, and consists of depression of marrow erythroid activity. These patients had a significantly lower haemoglobin and serum iron concentration and a higher serum ferritin concentration, both when compared to normal subjects and to those patients with HD who lacked systemic symptoms. Ineffective erythropoiesis and redcell destruction were not significantly increased. The present findings confirm that HD patients with systemic symptoms have a depression of erythropoiesis, and that in these patients the marrow fails to respond to the stimulus of mild anaemia. THE MECHANISM of anaemia in Hodgkin's disease (HD) is unclear. Abnormalities of iron utilization have been noted on many occasions in the past (Giannopoulos & Bergsagel 1959; Haurani et al., 1963) and ineffective erythropoiesis (Cline & Berlin 1963). Beamish et al. (1972) suggested that impairment of iron release from the reticuloendothelial (RE) system with a consequent failure of iron delivery to the marrow was common in HD. Many of the patients investigated in these earlier studies had already undergone treatment with radiation and chemotherapy. In addition, the ferrokinetic methods used did not provide a quantitative assessment of effective and ineffective red-cell production (Cavill & Ricketts, 1974). The purpose of the preesnt study was to re-evaluate and define the abnormalities of iron metabolism and the possible mechanism of anaemia in patients presenting with HD. Ferrokinetic methods allowing quantitative estimates of erythropoiesis were used (Ricketts et al., 1975). 25 PATIENTS Nineteen newly diagnosed untreated patients with HD and 14 healthy adults were studied. Fully informed consent was obtained in all cases. There were 14 males and 5 female patients and their ages ranged from 17 to 69 years with a mean of 43 years. None of the patients was receiving any form of medical treatment at the time of investigation. The diagnosis of HD was based on lymphnode biopsy in all patients, except one in w%hom the diagnosis xvas made after splenectomy. Routine haematological and biochemical investigations, marrow aspiration and trephine biopsy, chest X-ray and hilar tomography, radiological skeletal survey and bipedal lymphangiography were carried out in all patients. The stage of the disease was assessed before laparotomy according to the Ann Arbor International Convention (Carbone et al., 1971). All patients except those with biopsyproven Stage IV disease then underwent laparotomy and splenectomy with open liver biopsy. The histological classification wias according to Lukes & Butler (1966) as modified at Rye (Lukes et al., 1966) and Ann Arbor (Rapport et al., 1971) International Conven- 366 S. AL-ISMAIL ET AL. tions. The patients were classified as Type A or B on the basis of the absence or presence of systemic symptoms of unexplained fever (more than 38°C), wNeight loss of more than 10% during the 6 months preceding the diagnosis, or night sweats (Carbone et al., 1971). AIETHODS Haemoglobin concentration and inean corpuscular vollumlle (MCV) w-ere measured with a Coulter Counter Model S. Serum iron concentration and total iron-binding capacity wNere measured according to the method of Young & Hicks (1965) as modified by Babson & Kleinmen (1967). The method of Jones & Worwood (1975) wsas used to measure the serum ferritin concentration. Radioisotope studies wAere carried out at presentation, before laparotomy. A sample of each subject's plasma transferrin was specifically labelled Mith 59Fe (Cavill, 1971) and the plasma 59Fe clearance curve wNas definied over 10-14 days by the method of Cavill et atl. (1976). The results wxAere analysed as described by Ricketts et al. (1975) and the following parameters wNere calculated: total erythroid marrow iron turnover (MIT), effective red-cell iron turnover (RCIT), and ineffective iron turnover (IIT). The mean cell lifespan and mean red-cell production were calculated from red-cell iron turnover. Ineffective iron turnover wNas expressed as a percentage of MIT. The differences between means wiere assessed using a one-way analysis of variance, wNhilst correlation between parameters wras assessed using the Pearson product-moment correlation coefficient. RESULTS The number of patients showing different stages of the disease, histological classes and the presence or absence of symptoms is shown in Table I. The haematological and iron status of the subjects are summarized in Table II. Only 5 of the patients were anaemic (haemoglobin concentration < 13 g/dl for men or < 12 g/dl for women) but 9 patients showed a microcytic blood picture (MCV < 84 fl). There was no significant difference in the haemoglobin concentration or MCV between the 4 stages of the disease, nor did TABLE I. The distribution of 19 untreated patients w(ith Hodgkin's disease (HD) in relation to syMpto0mP,s, staye of disease and histological class8fication Stage of (lisease Histological type I 11 Lymphocytic pre(l ominaiit 2 1 2 . 1 4 _ 4 6 2 4 4 TTI IV Noduilar sclerosing M\lixed celluilarityv Lymphocyte (leplete(l Type A Type B 2 they differ from the control group. However, patients with systemic symptoms had lower haemoglobin concentrations and lower MCVs compared both to the normal subjects and to the patients without such symptoms (P < 0.005). There was a significant correlation between MCV' and serutm iron concentration (r=0 58, P)< 001) and sideropenia was predominantly associated with the presence of systemic symptoms (Table III). Patients with systemic symptoms had a mean serum iron concentration wlhich was significantly lower (P<00005) than both that of the patients without such symptoms and that of the normal group. Although serum iron varied between the 4 stages of the disease, there was no clear relationship with advancing stage. Serum ferritin was significantly higher in the patients with HD than in the control group, and significantly higher concentrations (P < 0 01) were found with advancing stages of the disease (Table III). There was a significant negative correlation between the serum iron concentration and serum ferritin concentration (r= -057, P < 0 02); patients with low serum iron concentrations had high ferritin concentrations. This appears to be related both to the presence of anaemia and systemic symptoms. The mean serum ferritin concentration in the non-anaemic patients was 185 (Lg/l and the mean value in the anaemic patients was 776 Mug/l (P < 0.01); the mean serum ferritin con- ERYTHROPOIESIS IN HODGKIN S DISEASE 367 r I eC_ _0 CO 0 C CO_ 00C;O o~~~~~~~~x C) >H 00 0 C) ho = 00 CO H- L_ --~m _" ¢ CO N0 - I tCAr X C6 8 ." _q_0 00 0 O O 4- H N - _~ _c Cto _ _ CO 000 00 -- cE CO 0 ° 00 OCO' 0 I) H-~ 0 -4 -0 q 000 ajCw CO - oo - - CE 0 0 0 N- N- 00 H - C) CO C1)n C) CO 10es 4-4 C2) Ca COD ho 00 rh CO C) 0 0 ON 00 - CACO0 4 C) _° 00 oo _0 - 0 hoQ - 00 to 000t 00 H --00_ C:c>tQt o H N-_> 00- hoo4 01 000 ~00COX ho 00 to 0 N 1 _ 00 C - 0 _ 4-.: 0 V - "~I oo I lOi S L 0 V 00 0 _1 -, ^ m Cw 4 00 -4 0 - N N- _ V; - -0 N S ho- 01 Nb ' hoc < _ 00 N 0 S o m01000 1b EV; 0D 00 0 0J .° o ~ ~ 0 ~~~> O C. C) O) : 0 O- hd 0 ° 0 JE o°E B 2t$ $ 0 C) H H =~~C 368 S. AL-ISMAIL ET AL. centration in patients with systemic symptoms was 795 Htg/l and in those without such symptoms 122 itg/l (P < 0.005). The serum ferritin in Type A patients was not significantly different from normal. The mean values for marrow iron turnover, red-cell iron turnover, ineffective erythropoiesis and red cell lifespan and tissue iron turnover are summarized in Table III. Total MIT and RCIT at each stage of the disease were similar, and not significantly different from normal (P> 0.05). Neither the percentage of IIT nor the red-cell lifespan differed between each stage of the disease and normal. However, patients with systemic symptoms had a lower mean MIT and RCIT than both normal subjects and Type A patients (P < 0 01), whereas there were no differences between patients without symptoms and normal subjects (Table III). This correlation of depressed erythropoiesis and systemic symptoms was particularly evident in patients with Stage III disease. The 4 patients with systemic symptoms Type B had significantly lower MIT (P < 0 05) and RCIT (P < 0 01) than the 4 T'ype A patients. DISCUSSION The principal mechanisms proposed to account for anaemia in HD are haemolysis and abnormalities in utilization of iron for erythropoiesis, characterized by ineffective erythropoiesis (Cline & Berlin, 1963) and a reticuloendothelial block in iron release (Beamish et al., 1972). Zarabi et al. (1977) have suggested that relative failure of erythropoiesis rather than an RE block may be primarily responsible for the anaemia of chronic disease. The extent to which these factors contribute to the anaemia in HD is difficult to assess, because most studies have been carried out on patients with advanced disease, many of whom had already been treated. In a study of 23 untreated patients with HID at presentation Beamish et al. (1972) found that the disturbance in iron metabolism and erythropoiesis was related to the stage of the disease. However, the staging and the division of patients into those with localized and generalized disease was based on clinical observation only. Whittaker et al. (1978) found that 24/60 patients (400%) changed their clinical stage after laparotomy and splenectomy. This emphasizes the importance of laparotomy and splenectomy in disease staging and in relating the stage of the disease to the disturbance of iron metabolism. A reduced serum iron concentration in the presence of stainable iron in the marrow is characteristic of the anaemia of HD, and serum iron concentration was significantly lower than normal in our patients. However, no coiisistent decrease in the level of serum iron with advaincing pathological stage was observed. The most striking difference in serum iron concentration was between the low levels in patients with systemic symptoms and the levels in symptom-free patients. Five of our patients showed a hypochromic microcytic anaemia. Ultmann et al. (1966) found such anaemia in 10% of patients with HD at presentation. None of our patients haad iron deficiency as judged from stainable iron in the marrow or the level of serum ferritin. The latter was higher than in the normal subjects, and increasing concentrations were found with advancing stages of the disease. Significantly higher eoncentrations were found in patients withl systemic symptoms. These were similar to those of Jones et al. (1973) and Jacobs et al. (1976), who showed that the increased serum ferritin concentration was related to the activity and spread of HD. The increased serum ferritin concentration in HD are probably related partly to anaemia and partly to the nonspecific and poorly understood changes known to occur in RE cells of all cancer patients (Cartwrigbt & Lee, 1971). Liver involvement may be a factor contributing to high serum ferritin in Stage IV disease. Red-cell lifespan was not significantly reduced in our patients. Two of 8 untreated patients with HD described by ERYTHROPOIESIS IN HODGKIN S DISEASE Beamish et al. (1972) had a shortened redcell lifespan as measured by 51Cr. However, in generalized disease, haemolysis seemed to be more common, and occurred in 70-90% of patients (Ultmann, 1958; Najean et al., 1967; Cline & Berlin, 1963; Giannopoulos & Bergsagel, 1959). Ineffective erythropoiesis has been suggested as a possible mechanism contributing to the anaemia of HD (Cline & Berlin, 1963), although this could only be inferred from the data at that time. Measurement of ineffective erythropoiesis in our patients showed this to be at normal levels. The most striking erythroid abnormality in our patients was a depression of total marrowiron turnover and conisequently of effective red-cell iron turnover in those patients with systemic symptorns. This association of systemic symptoms with depressed erythropoiesis was seen in patients who share the same pathological stage of the disease. In the 8 patients in Stage III, MIT aind RCIT were lower in the 4 patients with Type B disease. Our results suggest that an abnormality of erythropoiesis is not necessarily linked directly to an abnormality of iron metabolism. Zarabi et al. (1977), using an animal model, suggested that relative failure of erythropoiesis may be primarily responsible for the anaemia of chronic disease, and our results are consistent with this hypothesis. The cause of this depression remains unclear however. Ward et al. (1971) reported a decrease in plasma erythropoietin levels in patients with lymphoma, as well as in patients with chronic inflammation and infection. Zucker et at. (1974), however, found that serum erythropoietin levels are elevated in the anaemia of malignancy but elicit a decreased marrow response. The production of catabolic tumour products, the secretion of physiological inhibitors of erythropoiesis, or some form of metabolic competition, have been previously postulated as possible causes of failure of marrow response (Bowdler & Prankered, 1962; Field et atl., 1968). 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https://openalex.org/W4313498975	https://aip.scitation.org/doi/pdf/10.1063/5.0135575	English	null	Analytical solutions for vortex flow at the tangential inlet of a vertical dropshaft	Physics of fluids	2,023	cc-by	6,510	Analytical solutions for vortex flow at the tangential inlet of a vertical dropshaft Wangru Wei (卫望汝) ; Lu Chang (常璐)  Physics of Fluids 35, 015160 (2023) https://doi.org/10.1063/5.0135575 Articles You May Be Interested In Numerical study on scroll vortex intake with non-uniform approach flow conditions 24 October 2024 05:23:12 I. INTRODUCTION geysers. A vortex flow forms inside the dropshaft when the flow diver- sion system is designed according to possible rainfall events (Jain and Ettema, 1984). The hydraulic performance of vortex–flow intake is good with appropriate energy dissipation and air entrainment (Toda and Inoue, 1999; Zhao et al., 2006; and Dai and Jin, 2022). Recently, the transition from plunging flow to swirling flow in the dropshaft was studied using a physical model and numerical simulations (Chang and Wei, 2022a; 2022b). Transition criteria between the two types of flow in the tangential intake structures have been proposed based on the velocity and pressure distributions. Urbanization increases urban flood risk by decreasing the perme- able area and increasing runoff. In addition, global climate change has led to more extreme rainfall events. A flood diversion scheme that routes upstream stormwater to underground systems is an effective and economic method of flood control (Li et al., 2022; Arega et al., 2008; Lee et al., 2008; and Li et al., 2021). Tangential intake structures are often used in flood diversion schemes to drive surface runoff to a vertical vortex drop shaft (Jain and Ettema, 1987; Vischer and Hager, 1995). The performance of the flood diversion system depends on the hydraulic design of the intake structure. The hydraulic design of a vortex dropshaft, including flow char- acteristics and geometry, is important for the appropriate operation of flood diversion systems. Extensive experiments and theoretical studies on tangential intake structures have been conducted (Jain and Kennedy, 1983; Jain and Ettema, 2017; Huang et al., 2016; and Kadia et al., 2022). The analytical solution of the head-discharge relationship of the vortex flow in a dropshaft was given by Binnie and Hookings (1948). The tangential vortex intake used in the San Diego ocean out- fall was studied by developing a comprehensive model (Brooks and Blackmer, 1962). A physical tangential intake model for flood The interior flow pattern of a dropshaft is determined by the type of flow and structure geometry, including vortex flows and plunging flows (Ma et al., 2016; Yu and Lee, 2009). For plunging flows, incom- ing flow from the tangential intake impinges the dropshaft wall, entraining a large amount of air (Ervine and Ahmed, 1982; Ye et al., 2022). Backflow phenomena are observed for some operation condi- tions, such as flow choking and the submergence of the dropshaft inlet (Ervine and Falvey, 1987; Sallam et al., 2002). AFFILIATIONS 1State Key Laboratory of Hydraulic and Mountain River Engineering, Sichuan University, Chengdu 610065, China 2Department of Civil and Environmental Engineering, The Hong Kong University of Science and Technology, Hong Kong, China a)Electronic mail: weiwangru@scu.edu.cn b)A th t h d h ld b dd d l h @ t hk 1State Key Laboratory of Hydraulic and Mountain River Engineering, Sichuan University, Chengdu 610065, China 2Department of Civil and Environmental Engineering, The Hong Kong University of Science and Technology, Hong Kong, China a)Electronic mail: weiwangru@scu.edu.cn b) g @ )Author to whom correspondence should be addressed: luchang@ust.hk b)Author to whom correspondence should be addressed: luchang@ust.hk ABSTRACT Vertical dropshafts with tangential intake structures are often used in urban drainage systems to route surface flow into underground systems. Vortex flow driven by a tangential inlet is the typical flow pattern in the vertical dropshaft. However, analytical solutions of the key hydraulic transition from chute flow to vortex flow have not been investigated, and theoretical guidelines of the vortex dropshaft are not available. In the present study, a series of theoretical analytical solutions are proposed to determine the circumferential flow generated at the vertical dropshaft inlet. The contraction ratio of the tapering chute, dropshaft size, and flow depth at the joint section determines the vortex flow performance in the vertical dropshaft. Based on the free surface drop height, theoretical criteria are proposed to describe the spiral movement intensity in the vertical dropshaft. Systemic experiments for different vortex dropshaft models are tested, and the experimental observations agree with the theoretical analysis. The present investigation provides a general reference for a tangential dropshaft inlet design that can smoothly route rotational flow down a vertical dropshaft. 24 October 2024 05:23:12 V C 2023 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http:// creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/5.0135575 Physics of Fluids ARTICLE Analytical solutions for vortex flow at the tangential inlet of a vertical dropshaft Analytical solutions for vortex flow at the tangential inlet of a vertical dropshaft Cite as: Phys. Fluids 35, 015160 (2023); doi: 10.1063/5.0135575 Submitted: 21 November 2022 . Accepted: 4 January 2023 . Published Online: 27 January 2023 Cite as: Phys. Fluids 35, 015160 (2023); doi: 10.1063/5.0135575 Submitted: 21 November 2022 . Accepted: 4 January 2023 . Published Online: 27 January 2023 I. INTRODUCTION The high amount of air can lower the drainage capacity of the stormwater and cause explosive 35, 015160-1 Phys. Fluids 35, 015160 (2023); doi: 10.1063/5.0135575 V C Author(s) 2023 35, 015160-1 Phys. Fluids 35, 015160 (2023); doi: 10.1063/5.0135575 V C Author(s) 2023 Physics of Fluids scitation.org/journal/phf ARTICLE scitation.org/journal/phf the tapering and annular section. The water discharge Q from the approach chute enters the tangential tapering section within a chute width B. The length of the tapering chute is L with a constant bottom slope b. The vertical dropshaft is a circular conduit with a diameter size of D. The joint section of the dropshaft inlet is a circumferential cross-section connected to the tangential chute. The cross-section width is e, the related arc length is l, and the related central angle is a. The contraction ratio of e/D is introduced as the design parameter rep- resenting the relative chute width to the dropshaft size. At the initial cross-section of the tapering chute, the flow depth is h0 and hA is the flow depth at the joint section A. The angle of the water flowing into the dropshaft is b, and the average flow velocity is Vx. As the water clings to the annular wall, the vortex flow begins and rotates down the dropshaft. After turning 360, the annular cross-section is defined as section B. The drop height of the free surface is Dh. The flow swirls as an annular jet inside the dropshaft, which can be approximated as an irrotational vortex (Jain and Ettema, 1987). interception was built by Lee et al. (2005); the hydraulic features inside the shaft were found to be heavily dependent on the physical design of the model. The instabilities of the intercepted flow were caused by flow blocking and choking in the tapering channel. A stable design cri- terion for tangential intake was proposed based on the experimental results and a one-dimensional model (Yu and Lee, 2009). The choking conditions inside the plunging flow dropshafts were reported to be controlled by the flow discharge and the dropshaft size (Camino et al., 2015). However, most of design guidelines were obtained based on experimental studies. Theoretical analyses should describe the flow pattern transition from chute flow to vortex flow, providing better insight into the flow transition in the complex structure. I. INTRODUCTION In this study, the flow transition criteria inside a vertical drop- shaft are investigated by analytical methods, and the tangential inlet designs of the vortex dropshaft are discussed. The analytical solution considers two important observations. First, the gradual flow depth variation in the tangential taper section determines the flow depth and related velocity at the joint section of the dropshaft. Second, the free surface drop height along the vertical dropshaft represents the vortex flow performance and the rotation movement intensity. The theoreti- cal analyses regarding the analytical solution are presented below, and detailed effects of flow and geometry characteristics on the flow rota- tion assessment are discussed. The experiment is designed according to the free drainage condi- tion (Yu and Lee, 2009) for which the approach water flow is stable throughout the chute and dropshaft. There is no backup flow, such as sudden swell flow and hydraulic jump flow, re-entering the taper sec- tion; the free surface variation is completely due to the streamwise movement and sidewall contraction. The hydraulic tunnel model is composed of polymethyl methacrylate glass. The approach chute width B is 24 cm, with a horizontal bottom floor. The taper section length is L ¼ 35 cm, with b ¼ 13. The vertical dropshaft D is 24 cm. Systemic flow conditions with different contraction ratios of e/D are conducted for experimental tests. The water flow discharge Q is 5–35 l/s, and the e/D is 0.10–0.60. For e/D >0.6, the flow pattern in the dropshaft transitions to plunging flow, and the flow rotation disap- pears (even with a tangential intake design). The experiment setup is composed of a water supply, an approach chute, and a vertical dropshaft with a tangential inlet. The water is supplied from a water tank, flowing into a rectangular approach channel. The approach flow is under the subcritical condition with the flow Froude number Fr0 ¼ V0/(gh0)0.5 ranging from 0.14 to 0.95, where V0 is the approach A. Flow depth calculation along the tangential tapering chute For a relatively short flow distance Ds, the differential form of the equation is For a relatively short flow distance Ds, the differential form of the equation is The tangential tapering chute is an open channel with a bottom slope of i ¼ sin b and a gradually reduced width from B to e. The flow regime in the channel is a non-uniform gradient flow. For a steady approach flow in a rectangular channel of uniform cross section, the basic differential equation for non-uniform gradient flow can be given from the energy equation, for which the flow discharge is constant without necessary branching and convergence (Chanson, 2004). It is given as Ds ¼ DEs i J ¼ Esd Esu i J : (6) (6) Equation (6) is the basic formula for calculating the water surface pro- file of a non-prismatic open channel, which can be solved by the step- by-step trial method. In Eq. (6), DEs is the specific energy difference from the upstream to downstream sections with the length of Ds. Esd and Esu represent the downstream- and upstream-specific energy. The average hydraulic gradient of the flow section Ds is generally as ids ¼ dh þ a0 þ f ð Þd v2 2g ! þ Q2 K2 ds; (1) (1) J ¼ 1 2 JU þ JD ð Þ; (7) J ¼ 1 2 JU þ JD ð Þ; (7) (7) where s is the per unit streamwise distance along the sloping chute and h is the streamwise flow depth. v2/(2g) is the kinetic energy head of the fluid. For the gradually varied flow in the tapering chute, the stream- wise resistance head loss is weak, and the local head loss is neglected, coefficient is assumed to be f ¼ 0, and the flow velocity distribution coefficient is assumed to be a0 ¼ 1. The coefficient K is the flow dis- charge modulus K ¼ ACR0.5, where A is the cross-sectional area, C is the Chezy coefficient, and R is the hydraulic radius. It represents the effects of channel geometries and wall resistance on the flow discharge capacity (Streeter and Wylie, 1981). Then, Eq. (1) can be rewritten as where JU and JD are the hydraulic gradients at the upstream and down- stream sections (Wu, 2004). II. TYPICAL FLOW PATTERNS AT THE DROPSHAFT INLET 24 October 2024 05:23:12 For a vertical dropshaft with a tangential intake, the typical flow pattern is shown in Fig. 1. A slope taper structure is used as the joint section to drive the approach chute flow and generate rotation. A side- wall gradually contracts along the streamwise direction, and the chute width decreases until reaching the annular wall of the vertical drop- shaft. The flow depth varies in the tapering section, and vortex flow is generated by the deflection of the tangential and annular walls. The swirling flow free surface drops down the vertical shaft with an air core in the center. The geometry of the joint structure is shown in Fig. 2. The horizontal plane is X and Z directions, and Y is the vertical direction, where the origin point is the joint point of tangency between FIG. 1. Flow features in the joint section of the vertical dropshaft inlet. FIG. 1. Flow features in the joint section of the vertical dropshaft inlet. Phys. Fluids 35, 015160 (2023); doi: 10.1063/5.0135575 35, 015160-2 Phys. Fluids 35, 015160 (2023); doi: 10.1063/5.0135575 35, 015160-2 V C Author(s) 2023 FIG. 2. The flow inside the inlet structure for the theoretical analysis. Physics of Fluids ARTICLE scitation.org/journal/phf Physics of Fluids FIG. 2. The flow inside the inlet structure for the theoretical analysis. FIG. 2. The flow inside the inlet structure for the theoretical analysis. FIG. 2. The flow inside the inlet structure for the theoretical analysis. dEs ds ¼ i Q2 K2 ¼ i J; (3) flow velocity deduced by flow discharge and water depth and g is the gravity acceleration. The flow Reynolds number Re0 ¼ V0h0/ ranges from 1.80 104 to 1.68 105, where is the water viscosity. The flow discharge is controlled by a thin-plate weir at the downstream of the dropshaft. The channel floor and sidewalls are made of Perspex glass for optical access. The averaged flow depth along the tapering chute and the surface location in the dropshaft are measured by a point gauge. III. THEORETICAL SOLUTIONS (5) II. TYPICAL FLOW PATTERNS AT THE DROPSHAFT INLET (3) where Es is the cross-sectional specific energy, which can be calculated by the following equation: where Es is the cross-sectional specific energy, which can be calculated by the following equation: Es ¼ h þ V2 2g ¼ h þ Q2 2gA2 : (4) (4) J is the hydraulic slope J ¼ Q2 K2 ¼ h þ V2 RC2 : (5) A. Flow depth calculation along the tangential tapering chute The flow characteristics of the wall-adhering water flow inside the shaft are relatively complex, and the flow can be approxi- mated as a free irrotational flow (Jain and Ettema, 1987). A control volume is selected for the theoretical analysis in this flow range, at inlet section A and outlet section B. The flow enters horizontally from inlet section A and flows out vertically from outlet section B. According to the conservation of angular momentum and ignoring the friction of the wall, the angular momentum change of flow passing through the control volume is 0, ð cs r V ð Þ V dA ð Þ ¼ 0; (9) (9) where r is the radial coordinate, V is the velocity vector, and dA is the outward normal vector of the control face. Based on the conservation of angular momentum in the control volume and the zero- momentum flux at the solid wall (V dA ¼ 0), the angular momentum flux in the horizontal plane that enters section A (UA) is the same with that of the control volume from outlet section B (UB), FIG. 3. Comparison of calculated and measured water depths along the tangential tapering section. flow enters the dropshaft, there are two controls both at the upstream tapering section and at the arc area of the junction. The channel width, sidewall contraction, and sloping floor of the tapering chute are hydraulic factors resulting in a variable free surface trend. UA ¼ UB: (10) (10) By neglecting wall friction, this momentum theorem applies to a con- trol volume with a transition flow section from the tapering inlet chan- nel to the vertical dropshaft. The swirling flow is considered to be symmetrical, and the swirling velocity in the horizontal plane is pro- vided by the inlet velocity component at the x-direction Vx. Thus, the angular momentum flux of the inlet section A (UA) can be expressed as A. Flow depth calculation along the tangential tapering chute According to the given approach flow conditions, the water depth at the end of the tapering chute can be obtained at the start of the inlet section using the trial method. The results of the theoretical model calculations are compared with the experimental data for different e/D and Q conditions (Fig. 3). The gradually varied flow depth trends agree with the experimental data, both for the gradual increasing and decreasing free surface pro- files. Thus, the gradually varied flow depth analytical solution is avail- able for the tangential chute flow calculation. For all e/D and Q conditions, the calculated hA values were compared favorably with the tested data in Fig. 4(a). Well-predicted results of hA make it possible to obtain the average flow velocity at the arc area of the joint cross sec- tion. Moreover, the depth-discharge relation from the measurements is suggested as hA ¼ 40(0.6 e/D)Q, as shown in Fig. 4(b). As the d h þ v2 2g ! ¼ i Q2 K2 ds (2) (2) (2) or or 35, 015160-3 Phys. Fluids 35, 015160 (2023); doi: 10.1063/5.0135575 V C Author(s) 2023 35, 015160-3 Phys. Fluids 35, 015160 (2023); doi: 10.1063/5.0135575 V C Author(s) 2023 scitation.org/journal/phf Physics of Fluids ARTICLE scitation.org/journal/phf FIG. 3. Comparison of calculated and measured water depths along the tangential tapering section. Vy is the vertical velocity component at the junction point. Dt is the time needed for the first 360 turn. Vt is the tangential velocity at the junction. The flow characteristics of the wall-adhering water flow inside the shaft are relatively complex, and the flow can be approxi- mated as a free irrotational flow (Jain and Ettema, 1987). A control volume is selected for the theoretical analysis in this flow range, at inlet section A and outlet section B. The flow enters horizontally from inlet section A and flows out vertically from outlet section B. According to the conservation of angular momentum and ignoring the friction of the wall, the angular momentum change of flow passing through the control volume is 0, Vy is the vertical velocity component at the junction point. Dt is the time needed for the first 360 turn. Vt is the tangential velocity at the junction. B. Free surface drop height in the dropshaft 24 October 2024 05:23:12 At the dropshaft inlet section A, the water flow clings to the shaft as an annular jet. After the first rotation of 360 along the shaft wall, the water surface in the dropshaft drops a distance Dh to section B. Accordingly, a theoretical analysis model is established by taking the shaft inlet area as the research object. The limiting condition of “free drainage” inside the inlet channel can be described by the first-order approximation UA ¼ ðe 0 Vx D 2 r Vx hA cos b dz ¼ eðD eÞ 2 hA cos b V2 x; (11) (11) where r is the horizontal distance perpendicular to the draft wall at the entrance and hA/cos b is the water level of section A. Substituting Eq. (11) into Eq. (9), the flow rate can be simplified as Dh ¼ VyDt ¼ Vy pD Vt : (8) (8) FIG. 4. Comparison of calculated and measured water depths hA at the dropshaft inlet section: (a) theoretical calculated results and (b) depth-discharge relation. of calculated and measured water depths hA at the dropshaft inlet section: (a) theoretical calculated results and (b) depth-discharge relation. FIG. 4. Comparison of calculated and measured water depths hA at the dropshaft inlet section: (a) theoretical calculated results and (b) dep Phys. Fluids 35, 015160 (2023); doi: 10.1063/5.0135575 V C Author(s) 2023 35, 015160-4 Phys. Fluids 35, 015160 (2023); doi: 10.1063/5.0135575 V C Author(s) 2023 Physics of Fluids UA ¼ QVx D e 2 : (12) (12) sin ðb þ bÞ ¼ y0 cos b þ L tan b h0= cos b L= cos b hA tan b : (17) (12) (17) Similar to inlet section A, the angular momentum flux of outlet section B (UB) can be expressed as Consequently, the drop height of the flow free surface Dh in the first 360 turn can be expressed as UB ¼ DVt 2 ðD 2 Ra Vy2prdr ¼ VtD 2 Vyp D 2 2 R2 a " # ; (13) Dh ¼ k tan b pD 1 e=D : (18) (18) (13) According to Eq. (18), the drop height of the water surface Dh is only related to the bottom slope b, inlet width e, and shaft diameter D. B. Free surface drop height in the dropshaft Based on the measured results of Dh, a fitting trend about the relation- ship between k and e/D is obtained, which follows exponential expres- sion k ¼ 0.5(e/D)1. According to the experimental results in Fig. 5, with an increase in e/D, the free surface drop height gradually decreases. Thus, the drop height of the flow free surface can be calcu- lated as where Ra is the radius of the air core at section B. Meanwhile, the flow rates through sections A and B should be identical to obey mass con- servation. The relationship between the tangential velocity component Vt and the x-direction velocity component Vx of the inlet flow can be obtained as Vt ¼ Vx 1 e D : (14) (14) Dh ¼ pD tan b 2 e D 1 e D : (19) As the inclined water flows to the taper chute outlet, the angle b (Fig. 2) can be used to establish the relationship between the component veloci- ties in the x direction (Vx) and the component velocity in the y direction (Vy) as As the inclined water flows to the taper chute outlet, the angle b (Fig. 2) can be used to establish the relationship between the component veloci- ties in the x direction (Vx) and the component velocity in the y direction (Vy) as (19) The comparison of calculated Dh with the experimental data is shown in Fig. 6. The analytical predictions can differ from the experiments by up to 20%–30%. The fluctuating flow surface occurs in the junction section, in which the water flow transfers from the chute pattern to the vortex pattern. Due to the arch boundary of the tapered outlet, the water depth is slightly non-uniform compared to the theoretical pre- diction, which may cause the difference in elevation among the flow lower surfaces. Moreover, for small discharges, the tapering flow is supercritical, and these uncertainty effects are apparent. The overall flow patterns are well predicted by the semi-empirical method for dif- ferent flow discharge and tapering structure conditions. For large e/D designs and small flow rates at the dropshaft inlet, the thin swirling flow layer may not be sufficiently resolved by the conservation of angular momentum, which may be affected by the wall impact conver- sion of energy. C. Assessment of the flow rotation performance in the dropshaft inlet Introducing the coefficient g, Introducing the coefficient g, g ¼ Q VAp D 2 2 ; (23) g ¼ Q VAp D 2 2 ; (23) g arccos 1 2ðe=DÞ ½ tan b cos b ðe=DÞ 1 e=D ð Þ : (24) At the vortex dropshaft inlet with a tangential tapering chute, the surface drop within the swirling water is a typical flow pattern. Considering the approach flow depth at the joint section, the flow sur- face elevation can be used to normalize the flow depth Dh. Based on the experimental results in Fig. 7, the surface drop is significant for small flow discharge Q<10 l/s, and values of Dh/(hA/cos b) are mostly larger than 4.0. Moreover, for relatively large e/D >0.30, the flow drop height can exceed over ten times the approach flow depth. With the increase in Q, the drop height of the flow surface decreases and the Dh/(hA/cos b) values are smaller than 4.0. For relatively larger flow dis- charge (Q> 20 l/s), experiments result in Dh/(hA/cos b) 1.0 for all e/D conditions. These findings indicate that the vortex intensity is mainly affected by the coupling of approach flow and tangential con- traction ratio at the dropshaft inlet. According to the model photos, (23) 24 October 2024 05:23:12 g arccos 1 2ðe=DÞ ½ tan b cos b ðe=DÞ 1 e=D ð Þ : (24) (24) For the left side of this equation, g represents the flow conditions com- pared to the dropshaft inlet structure design, and a large flow discharge indicates a high vortex movement capacity. The right side consists of the structure design parameters, including the tapering chute and the contraction ratio. For moderate and weak conditions, similar criterion relationships can be obtained. For the left side of this equation, g represents the flow conditions com- pared to the dropshaft inlet structure design, and a large flow discharge indicates a high vortex movement capacity. The right side consists of the structure design parameters, including the tapering chute and the contraction ratio. For moderate and weak conditions, similar criterion relationships can be obtained. Using u for the right side of Eq. (24), a systemic assessment for all present experiments is shown in Fig. 8. B. Free surface drop height in the dropshaft Vy Vx ¼ k tan b: (15) (15) Considering the non-uniform velocity distribution at the junction section due to the taper contraction and flow variation effects, the coefficient k is introduced which is determined by the contraction ratio of the taper section. The value of b can be deduced by the geometric relationship among the water flow (h0 and hA) and taper type (L and b) factors. In the case of decreasing flow depth varia- tion, the equation is expressed as 24 October 2024 05:23:12 tan ðb bÞ ¼ h0 hA L= cos b hA tan b : (16) (16) For the case of increasing flow depth variation, the equation is FIG. 5. Variation of the drop height of the free surface: (a) effects of e/D and (b) coefficient k trend. FIG. 5. Variation of the drop height of the free surface: (a) effects of e/D and (b) coefficient k trend. 35, 015160-5 scitation.org/journal/phf Physics of Fluids ARTICLE scitation.org/journal/phf FIG. 6. Comparisons of calculated Dh with tested data. the flow rotation performance is improved when accompanied by weak flow down along the annular wall. Thus, a simple criterion for the flow rotation assessment in vortex dropshafts can be proposed. The flow surface drop height during the first 360 rotation is one to four times greater than that of the water surface elevation at the dropshaft inlet. The swirling flow can be classi- fied as (1) strong rotation for Dh/(hA/cos b) 1, (2) moderate rotation for 1 Dh/(hA/cos b) 4, and (3) weak rotation for Dh/(hA/cos b) 4. The relationship of the flow characteristics at the dropshaft inlet is described as hA cos b ¼ Q lvx cos b ; (20) l ¼ D 2 arccos 1 2 e D : (21) (20) (21) The strong rotation condition can be rewritten as FIG. 6. Comparisons of calculated Dh with tested data. 2Q VADarccos 1 2 e D pD tan b cos b 2 e D 1 e D : (22) (22) C. Assessment of the flow rotation performance in the dropshaft inlet For the geometric shape design, a flat tapering bottom, sharp contraction of the tangential chute, and a small dropshaft diameter size can be considered potential ways to improve the vortex capacity. While, the present results indi- cate that the small flow discharge (e.g., Q <10 l/s) may reduce the vor- tex pattern for a certain geometric design. These relations can be used for the vortex dropshaft design. For the vertical dropshaft with a tan- gential intake, other guidelines also affect the flow discharge operations in engineering applications, such as the flow discharge capacity, energy dissipation, and free drainage conditions (Slisskii et al., 1980; Hager, 1990; Toda and Inoue, 1999; Qiao et al., 2013; and Chan et al., 2018). Combined with the present analytical solutions, the flow pattern at the joint structure of the vortex dropshaft can be fully assessed to improve the hydraulic design in urban drainage systems. FIG. 7. Effects of the flow discharge and tangential contraction ratio on the drop height of the flow surface in the dropshaft. According to the flow behavior in the tangential intake of the ver- tical vortex dropshaft, the changes in free surface profile from the chute flow pattern to the rotation flow pattern can be predicted. The parameter ranges of the present analytical solutions that can be applied for the tangential inlet of the vertical dropshaft includes 0.14 Fr0 0.95 and free drainage condition. This is helpful to improve the hydraulic design of the underground tunnel system, where flow intake FIG. 7. Effects of the flow discharge and tangential contraction ratio on the drop height of the flow surface in the dropshaft. FIG. 7. Effects of the flow discharge and tangential contraction ratio on the drop height of the flow surface in the dropshaft. Phys. Fluids 35, 015160 (2023); doi: 10.1063/5.0135575 V C Author(s) 2023 35, 015160-6 35, 015160-6 Phys. Fluids 35, 015160 (2023); doi: 10.1063/5.0135575 Phys. Fluids 35, 015160 (2023); doi: 10.1063/5.0135575 V C Author(s) 2023 V C Author(s) 2023 Physics of Fluids scitation.org/journal/phf ARTICLE FIG. 8. Assessment of flow rotation performance in a vortex dropshaft: (a) rotation capacity variation and (b) effect of flow discharge rate. energy equation of non-uniform gradient flow. As the chute flow enters the dropshaft, the conservation of angular momentum is used for the drop height calculation of the swirling free surface. C. Assessment of the flow rotation performance in the dropshaft inlet A series of equa- tions are obtained, and the analytical solution results are in agreement with the experiments. Moreover, a criterion from the relative drop height to the approach flow surface elevation is proposed for the flow rotation capacity assessment. The coupling of the three-dimensional geometry parameter and the specific flow discharge operations deter- mine the vortex flow performance in the dropshaft, varying from strong to weak rotation. The present investigation provides a general reference for a tangential dropshaft inlet design that can lead to smooth rotational flow down a vertical dropshaft. REFERENCES FIG. 8. Assessment of flow rotation performance in a vortex dropshaft: (a) rotation capacity variation and (b) effect of flow discharge rate. Arega, F., Lee, J. H. W., and Tang, H. W., “Hydraulic jet control for river junction design of Yuen Long Bypass Floodway, Hong Kong,” J. Hydraul. Eng. 134(1), 23–33 (2008). Arega, F., Lee, J. H. W., and Tang, H. W., “Hydraulic jet control for river junction design of Yuen Long Bypass Floodway, Hong Kong,” J. Hydraul. Eng. 134(1), 23–33 (2008). design of Yuen Long Bypass Floodway, Hong Kong,” J. Hydraul. Eng. 134(1), 23–33 (2008). Binnie, A. M. and Hookings, G. A., “Laboratory experiments on whirlpools,” Proc. R. Soc. London, Ser. A 194, 398–415 (1948). structures are vital to the success of these flood diversion schemes. The present possible criterion for the design of tangential intakes can ensure to give a reliable hydraulic performance of the vortex flow in the dropshaft with respect to flow discharge capacity and air entrain- ment control. It should be noted that the present analytical solution is established for the smooth flow control without overflow, energy loss, and flow instability. These limitations may occur for some geometry designs of the intake structure. Further specific considerations for the effects of inlet junctions on the three-dimensional vortex flow pattern should be taken in a long vertical dropshaft into the tunnel system. structures are vital to the success of these flood diversion schemes. The present possible criterion for the design of tangential intakes can ensure to give a reliable hydraulic performance of the vortex flow in the dropshaft with respect to flow discharge capacity and air entrain- ment control. It should be noted that the present analytical solution is established for the smooth flow control without overflow, energy loss, and flow instability. These limitations may occur for some geometry designs of the intake structure. Further specific considerations for the effects of inlet junctions on the three-dimensional vortex flow pattern should be taken in a long vertical dropshaft into the tunnel system. Brooks, N. H. and Blackmer, W. H., “Vortex energy dissipator for San Diego ocean outfall—laboratory investigations,” Report No. KH-R-5 (California Institute of Technology, Pasadena, California, 1962). gy Camino, G. A., Zhu, D. Z., and Rajaratnam, N., “Flow observations in tall plung- ing flow dropshafts,” J. Hydraul. Eng. 141(1), 06014020 (2015). Chan, S. N., Qiao, Q. S., and Lee, J. Author Contributions 24 October 2024 05:23:12 Wangru Wei: Conceptualization (equal); Formal analysis (equal); Methodology (equal); Writing – original draft (equal); Writing – review & editing (equal). Lu Chang: Conceptualization (equal); Methodology (equal); Writing – original draft (equal); Writing – review & editing (equal). Wangru Wei: Conceptualization (equal); Formal analysis (equal); Methodology (equal); Writing – original draft (equal); Writing – review & editing (equal). Lu Chang: Conceptualization (equal); Methodology (equal); Writing original draft (equal); Writing g q g p q Methodology (equal); Writing – original draft (equal); Writing – review & editing (equal). The authors have no conflicts to disclose. The authors have no conflicts to disclose. REFERENCES H., “On the three-dimensional flow of a stable tangential vortex intake,” J. Hydro-Environ. Res. 21, 29–42 (2018). tangential vortex intake,” J. Hydro-Environ. Res. 21, 29– Chang, L. and Wei, W., “Numerical investigation of the plunging and vortex- flow regimes occurring in drop shafts with a tangential intake,” J. Irrig. Drain. Eng. 148(8), 04022026 (2022a). Chang, L. and Wei, W., “Numerical study on the effect of tangential intake on vortex dropshaft assessment using pressure distributions,” Eng. Appl. Comput. Fluid Mech. 16(1), 1100–1110 (2022b). DATA AVAILABILITY The data that support the findings of this study are available from the corresponding author upon reasonable request. ACKNOWLEDGMENTS The reported research was supported by the National Natural Science Foundation of China (Grant No. 51979183) and the Open Funding of the State Key Laboratory of Hydraulics and Mountain River Engineering of Sichuan University (Grant No. SKHL2104). IV. CONCLUSIONS In the present study, theoretical solutions are provided for the hydraulic design of a vertical dropshaft with a tangential chute. The approach flow characteristics at the joint section between the tangen- tial tapering chute and the vertical dropshaft can be obtained by the Chanson, H., The Hydraulics of Open Channel Flow: An Introduction (Butterworth Heinemann, Elsevier Ltd., Oxford, UK, 2004). Dai, S. and Jin, S., “Numerical investigations of unsteady critical flow conditions over an obstacle using three models,” Phys. Fluids 34(2), 025103 (2022). 35, 015160-7 35, 015160-7 Phys. Fluids 35, 015160 (2023); doi: 10.1063/5.0135575 V C Author(s) 2023 Phys. Fluids 35, 015160 (2023); doi: 10.1063/5.0135575 V C Author(s) 2023 Physics of Fluids ARTICLE scitation.org/journal/phf Li, C., Xiao, H., Jiang, R., and Ye, X., “Effect of wall slip on vertical film drainage in presence of soluble surfactants,” Phys. Fluids 33(4), 042113 (2021). Ervine, D. A. and Ahmed, A. 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J., Sun, S., and Ling, Y., “Evolution of supersonic corner vortex in a hypersonic inlet/isolator model,” Phys. Fluids 28(12), 126101 (2016). p Sallam, K. A., Dai, Z., and Faeth, G. M., “Liquid breakup at the surface of turbu- lent round liquid jets in still gases,” Int. J. Multiphase Flow 28(3), 427–449 (2002). Jain, S. C., Tangential vortex-inlet, J. Hydraul. Eng. 110(12), 1693–1699 (1984). Jain, S. C. and Ettema, R., “Vortex-flow intakes,” in Swirling Flow Problems at Intakes, IAHR Hydraulic Structures Design Manual, edited by J. Knauss (Balkema, Rotterdam, The Netherlands, 1987), pp. 125–137. Slisskii, S. M., Kuznetsov, E. B., and Akhmedov, T. IV. CONCLUSIONS K., “Multistage whirlpool shaft spillway,” Hydrotech. Constr. 14(9), 892–895 (1980). Streeter, V. L. and Wylie, E. B., Fluid Mechanics (McGraw-Hill Press, Singapore, 1981). Jain, S. C. and Ettema, R., “Vortex-flow intakes,” in Swirling Flow Problems at Intakes (Routledge, 2017), pp. 125–137. Toda, K. and Inoue, K., “Hydraulic design of intake structures of deeply located underground tunnel systems,” Water Sci. Technol. 39(9), 137–144 (1999). Jain, S. C. and Kennedy, J. F., Vortex-Flow Drop Structures for the Milwaukee Metropolitan Sewerage District Inline Storage System (Iowa Institute of Hydraulic Research, The University of Iowa, 1983). Vischer, D. L. and Hager, W. H., “Vortex drops,” in Energy Dissipaters, IAHR Hydraulic Structures Design Manual 9, edited by D. L. Vischer and W. H. Vischer, D. L. and Hager, W. H., “Vortex drops,” in Energy Dissipaters, IAHR Hydraulic Structures Design Manual 9, edited by D. L. Vischer and W. H. Hager (Balkema, Rotterdam, The Netherlands, 1995), pp. 167–181. Kadia, S., R€uther, N., Albayrak, I., and Pummer, E., “Reynolds stress modelling of supercritical narrow channel flows using OpenFOAM: Secondary currents and turbulent flow characteristics,” Phys. Fluids 34, 125116 (2022). Wu, C. G., The Hydraulics of Open Channel Flow: An Introduction (Butterworth- Heinemann, Elsevier Ltd., Oxford, UK, 2004). Lee, J. H. W., Chan, C. H. C., Kuang, C. P., Clark, P., Townsend, N., and Shiu, W. Y., “Hydraulic model study of the Tai Hang Tung storage scheme,” J. Hydraul. Res. 46(1), 11–23 (2008). Heinemann, Elsevier Ltd., Oxford, UK, 2004). Ye, F., Xu, W., and Wei, W., “Air entrainment mechanism of chute bottom aera- tors in high-speed chute flows,” Phys. Fluids 34(11), 115113 (2022). Lee, J. H. W., Chan, H. C., Yu, D. Y., and Choi, D. K. W., “Physical hydraulic model tests for Hong Kong West Drainage Tunnel in Northern Hong Kong Island-intake structure,” Final Report, Croucher Laboratory of Environmental Hydraulics, The University of Hong Kong, 2005. Yu, D. and Lee, J. H., “Hydraulics of tangential vortex intake for urban drainage,” J. Hydraul. Eng. 135(3), 164–174 (2009). Zhao, C. H., Zhu, D. Z., Sun, S. K., and Liu, Z. P., “Experimental study of flow in a vortex drop shaft,” J. Hydraul. Eng. 132(1), 61–68 (2006). 24 October 2024 05:23:12 35, 015160-8 35, 015160-8 Phys. Fluids 35, 015160 (2023); doi: 10.1063/5.0135575 V C Author(s) 2023
W4229969828.txt	http://www.nomos-elibrary.de/10.5771/0506-7286-1971-1-109.pdf	de		Strukturprobleme der Entwicklungsländer	Verfassung und Recht in Übersee	1,971	cc-by	1,070	wenn auch die Kulturleistungen dieser Regionen nicht unterschätzt werden dürfen. Aber in diesen Regionen beruht das gesamte Erziehungssystem auf der Mission, während die anderen asiati schen und ein Teil der afrikanischen Na tionen ein islamisches oder buddhisti sches Erziehungssystem besaßen, das den westlichen Einfluß verarbeiten und überstehen konnte. Der starke Einfluß, den die traditionellen Gruppen auf den einzelnen haben, erschwert das Erwach sen eines gemeinsamen Nationalgefühls, das für die Errichtung eines größeren Staatsgebildes unerläßlich ist. In Neu Guinea wird versucht, durch Erziehung und durch Förderung der Mobilität eine Bevölkerung zu schaffen, die in der La ge ist, Probleme unter gesamtstaatlichen Aspekten zu lösen. Daneben wird ver sucht, durch Beteiligung der Bevölke rung an den politischen Entscheidungen, für die Erlangung der Unabhängigkeit eine Grundlage für die Formulierung der politischen Willens äußerungen zu schaffen. Ob in Neu-Guinea gelingt, was auch in Afrika erhofft wurde, dort aber durch die sich überstürzenden Ereignis se nicht mehr verwirklicht werden konnte, bleibt abzuwarten. Der vorliegende Band gibt eine gute Einführung in die Probleme neuer Staa ten besonders für den, der sich mit den Problemen im ostasiatischen Raum noch nicht beschäftigt hat. Die Kürze der Darstellung und die Weite des Themas führen sicher h:iufig zu Vereinfachun gen, möglicherweise auch zu Verfäl schungen, die Verfasser bemühen sich aber alle um eine möglichst neutrale unparteiische Darstellung, so daß das Buch Anfängern im Studium der über seeischen Welt wärmstens empfohlen werden kann. Henning v. Wedel Strukturprobleme der Entwicklungsländer hrsg. v. Institut für Internationale Soli darität der Konrad-Adenauer-Stiftung, Schriftenreihe Bd. 5 v. Hase und Koehler Verlag Mainz 1 969, 88 S. Dieser vom Institut für Internationale Solidarität der Konrad-Adenauer-Stif tung herausgegebene Band mit vier Ein zelbeiträgen soll, wie das Vorwort be tont, mithelfen, eine Diskussionsgrund lage für die drängenden Probleme der Entwicklungsländer zu geben. Diesem Anspruch werden die Beiträge kaum gerecht. Der erste Beitrag von Raupach, der das Verhältnis der Entwicklungsländer zwi schen Ost und West behandelt, gibt außer einigen allgemein bekannten Ar gumenten für die Notwendigkeit von Entwicklungshilfe nur eine kurze Dar stellung des Verhältnisses der Sowjet union zu den Entwicklungsländern. Der Verfasser weist mit Recht darauf hin, daß die Größe der Probleme und die Schnelligkeit, mit der sie sich infolge des Bevölkerungswachstums zuspitzen, keine Zeit für lange politische Diskus sionen lassen. Zur schwierigen Lage der Entwicklungsländer, die einerseits dar auf achten müssen, keine der beiden Sei ten durch zu offene Anlehnung an die andere zu verschrecken, andererseits aber häufig Hilfe nur unter politischen Vorbedingungen erhalten, nimmt der Verfasser keine Stellung. Der zweite Beitrag von Dürr will Al ternativen der Wachstumsplanung auf zeigen. Er bedient sich dazu der her kömmlichen wirtschaftspolitischen Mo delle. Er lehnt eine direkte Wachstums planung ab mit dem Hinweis darauf, daß eine solche Planung außerordentlich schwierig sei, weil der marginale Kapi talkoeffizient kaum richtig prognosti ziert werden könne. Darauf untersucht er die Planung der Wachstumsbedin gungen. Er meint, daß unbedingte Vor aussetzung eines Wachstums eine soziale Marktwirtschaft sei. Der Möglichkeit der Bildung eines Kapitalmonopols glaubt der Verfasser durch eine straffe 1 09 https://doi.org/10.5771/0506-7286-1971-1-109, am 29.06.2024, 07:04:55 Open Access – - https://www.nomos-elibrary.de/agb Steuerpolitik begegnen zu ·können. Um das Sparen attraktiver zu machen, schlägt der Verfasser die Förderung von Sparkassen außerhalb der Städte und von Kreditgenossenschaften vor. Um die Kapitalflucht einzudämmen, soll eine Po litik der Geldwertstabilisierung betrie ben werden. Es braucht wohl kaum ge sagt zu werden, daß diese Vorschläge dermaßen unrealistisch sind, daß sie kei ner weiteren Erörterung bedürfen. Schließlich meint der Verfasser, daß es unbedingt notwendig sei, politische Sta bilität herbeizuführen, um die wirt schaftliche Entwicklung nicht zu hem men. Es sei die Entscheidung für eine klare ordnungspolitische Konzeption er forderlich, die für den Verfasser not wendig eine Entscheidung zu einer Lei stungsgesellschaft mit sozialer Markt wirtschaft ist. Der Beitrag von von Blanckenburg be schäftigt sich mit der Landwirtschaft als Grundlage der Industrialisierung. Der Verfasser kommt zu dem Ergebnis, daß eine Industrialisierung ohne eine lei stungsfähige Landwirtschaft nicht mög lich sei. Leider diskutiert er das Phäno men des steigenden Pro-Kopf-Einkom mens bei steigendem Anteil der Land wirtschaft am Sozialprodukt und der Erwerbstätigen, wie es sich in Indien zeigt, nicht. Der letzte Beitrag von Paul befaßt sich mit dem Analphabetentum. Nach einer allgemeinen Einleitung befaßt die Ver fasserin sich mit den Problemen der Alphabetisierung, wobei sie besonders auf die Sprachenvielfalt in einem Staate, das teilweise völlige Fehlen einer Schrift und die Probleme bei der Schaffung von Lehrmaterial und der Ausbildung von Lehrern hinweist. Die Hauptschwie rigkeit sieht die Verfasserin aber zu Recht in der Schaffung einer Motivation zum Lernen und der Schaffung der Möglidlkeiten, schon jetzt den Neu-Al phabeten ausreichende Anwendung des neuen Wissens zu ermöglichen. Wenn auch die beiden letzten Beiträge wenigstens teilweise zu wirklichen Strukturproblemen Stellung nehmen, so erfüllt der Band weder die durch den Titel noch die durch das' Vorwort ge weckten Erwartungen. Henning v . We de l D IETER EHRHARDT D e r Be griff des Mikrostaats im Völker r echt und in der internationalen Ord nung Scientia Verlag, Aalen 1 970, 1 15 S., 22,- DM. ,Mit der Dekolonisation auch kleinster Gebiete wie Barbados, Malediven so wie Nauru und ihrer Entlassung in die politische Unabhängigkeit ist die bereits vor dem 2. Weltkrieg in der iVölkerrechtswissenschaft erörterte Fra :ge nach der Staatseigenschaft politischer Kleinstverbände erneut aufgeworfen worden. War dieses Problem wegen der 'Verhältnismäßig geringen Zahl der da 'm als bestehenden Ministaaten - Liech tenstein, Monaco und San Marino weitgehend nur für den Völkerrechtler von theoretischem Interesse, so hat es gegenwärtig darüber hinaus auch erheb liche politische Bedeutung erhalten. Das wird deutlich, vergegenwarugt man sich, daß nach dem Bericht des 24er Ausschusses der UN in absehbarer Zu kunft mit einer Entlassung von weite ren 48 Gemeinwesen in die Unabhän gigkeit zu rechnen ist, von denen je des weniger als 300 000 Einwohner hat. Eine Aufnahme dieser politischen Ge bilde als Staaten in die UN würde das bereits zugunsten der Kleinstaaten be stehende Stimmgewicht so verschieben, daß die Repräsentanten von 4 v. H . der Weltbevölkerung sich z u einer Zweidrittelmehrheit in der Volksver sammlung finden könnten. Trotz der politischen und völkerrecht lichen Aktualität dieses Themenberei ches ist bisher eine eingehende Unter suchung vor allem der völkerrechtli chen Probleme solcher kleinster unab hängiger Territorien nicht erfolgt. Zwar hat bereits F a r r a n 1 960 auf die Notwendigkeit einer solchen Analyse hingewiesen ; sein Appell hatte jedoch keinen durchgreifenden Erfolg. Es ist deshalb verdienstvoll, daß Verfasser die sen Fragenkomplex aufgegriffen, schrift- 110 https://doi.org/10.5771/0506-7286-1971-1-109, am 29.06.2024, 07:04:55 Open Access – - https://www.nomos-elibrary.de/agb
https://openalex.org/W2621932845	https://bmcmededuc.biomedcentral.com/track/pdf/10.1186/s12909-017-0929-9	English	null	“On the same page”? The effect of GP examiner feedback on differences in rating severity in clinical assessments: a pre/post intervention study	BMC medical education	2,017	cc-by	5,548	Sturman et al. BMC Medical Education (2017) 17:101 DOI 10.1186/s12909-017-0929-9 Sturman et al. BMC Medical Education (2017) 17:101 DOI 10.1186/s12909-017-0929-9 Open Access * Correspondence: n.sturman1@uq.edu.au 1Primary Care Clinical Unit, Faculty of Medicine, The University of Queensland, Brisbane, Queensland, Australia 2Royal Brisbane & Women’s Hospitals, Level 8, Health Sciences Building, Herston, QLD, Australia Full list of author information is available at the end of the article “On the same page”? The effect of GP examiner feedback on differences in rating severity in clinical assessments: a pre/post intervention study Nancy Sturman1,2* , Remo Ostini3, Wai Yee Wong4, Jianzhen Zhang5 and Michael David6 Background the 1970s have evaluated various examiner training and feedback strategies (including didactic teaching about cognitive error and biases, and rating practice using videotaped performances with feedback of expert rat- ings and facilitated discussion of inter-examiner differ- ences) and found weak, if any, evidence of their effectiveness [13–15]. Several authors have speculated that “physician raters” might be “impervious to train- ing”, and suggested that outlier examiners are best managed by excluding them from further participation in assessments [14] or using equating or adjusting strat- egies to compensate for severity differences [6, 16]. However it remains plausible that more effective exam- iner interventions may yet be identified. One long recognised challenge in clinical assessment is minimising differences in student grades which are due to inconsistencies in examiner ratings. Whether this challenge is addressed from a psychometric perspective, for example generalizability theory [1], or from social cognition frameworks [2–5], student grades should de- pend as little as possible on who examines them. Inconsistencies in examiner ratings are related to a number of factors, including differences in examiner se- verity. “Examiner severity” is an examiner’s position on a spectrum from very lenient to very stringent, reflecting a consistent tendency to use a particular part of the rele- vant rating scale in their ratings [6]. Examiners with dif- ferences in severity assign different scores to examinees (although they may rank them and/or diagnose their strengths and weaknesses similarly). Several studies have shown that clinician examiners manifest different levels of severity, and that this has a significant impact on examinee grades and assessment decisions across a range of clinical skills assessments. These include work- based assessments, oral examinations and OSCEs [5–8]. Although extreme differences in examiner severity are probably relatively uncommon [9] modest differences in examiner severity may make important differences to student grades and pass-fail decisions. In this study we investigated the effect of an examiner feedback intervention on the severity of examiner rat- ings. We provided general practitioner examiners with written feedback comparing all examiners’ ratings over the preceding 18 months of general practice clinical case examinations. The intervention was intended to assist examiners to “calibrate” their rating judgements with those of their examiner colleagues, and reduce dif- ferences in examiner severity. Abstract Background: Robust and defensible clinical assessments attempt to minimise differences in student grades which are due to differences in examiner severity (stringency and leniency). Unfortunately there is little evidence to date that examiner training and feedback interventions are effective; “physician raters” have indeed been deemed “impervious to feedback”. Our aim was to investigate the effectiveness of a general practitioner examiner feedback intervention, and explore examiner attitudes to this. Methods: Sixteen examiners were provided with a written summary of all examiner ratings in medical student clinical case examinations over the preceding 18 months, enabling them to identify their own rating data and compare it with other examiners. Examiner ratings and examiner severity self-estimates were analysed pre and post intervention, using non-parametric bootstrapping, multivariable linear regression, intra-class correlation and Spearman’s correlation analyses. Examiners completed a survey exploring their perceptions of the usefulness and acceptability of the intervention, including what (if anything) examiners planned to do differently as a result of the feedback. Results: Examiner severity self-estimates were relatively poorly correlated with measured severity on the two clinical case examination types pre-intervention (0.29 and 0.67) and were less accurate post-intervention. No significant effect of the intervention was identified, when differences in case difficulty were controlled for, although there were fewer outlier examiners post-intervention. Drift in examiner severity over time prior to the intervention was observed. Participants rated the intervention as interesting and useful, and survey comments indicated that fairness, reassurance, and understanding examiner colleagues are important to examiners. Conclusions: Despite our participants being receptive to our feedback and wanting to be “on the same page”, we did not demonstrate effective use of the feedback to change their rating behaviours. Calibration of severity appears to be difficult for examiners, and further research into better ways of providing more effective feedback is indicated. © 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. Sturman et al. BMC Medical Education (2017) 17:101 Page 2 of 7 Page 2 of 7 Background y The study was designed to test the following hypotheses: The study was designed to test the following hypotheses: 1) Examiner self-perceptions of rating severity (“severity self-estimates”) will be more accurate following the intervention Differences in examiner severity are poorly understood. Lenient examiners may tend to be “candidate–centred”, whereas stringent examiners may be more focused on maintaining high clinical standards [10]. Differences in se- verity may be linked to different examiner conceptions of standards, and of academic failure [11]. Severity may also be conceptualised as a type of sensitivity-specificity trade- off: more stringent examiners may prioritise detecting (and failing) incompetent students more highly (sensitiv- ity) than avoiding failing competent ones (specificity), and indeed there is some evidence that sensitivity and specifi- city correspond to two different metacognitive processes associated with calibration [12]. Attempts to understand examiner severity are also complicated by the discrepancy that can exist between public ratings and private judge- ments about examinees, including a reluctance to fail underperforming students [2]. 2) Ratings of more stringent examiners will tend to become more lenient, and ratings of more lenient examiners will tend to become more stringent, following the intervention We also wished to investigate the stability of examiner severity over time, and examiner perceptions of the ac- ceptability and usefulness of the intervention. Methods To test Hypotheses 1, Spearman’s correlations between examiner severity self-estimates, and examiner mean overall ratings for the two examinations preceding these estimates, were performed, and pre- and post- interven- tion correlations were compared. Examinations were conducted five times a year in Brisbane, Queensland. All examiners were practising general practitioners with active involvement in practice-based teaching and/or tutorial teaching. The examination cases were selected from a database of ap- proximately forty recently developed standardised cases, and students were allocated 18 min to complete each case (including perusal time). Students were assigned to examiners predominantly on a consecutive alphabetical basis, although a small number of manual adjustments were made, for student convenience or to avoid examiners examining students who were particu- larly well known to them. To test Hypothesis 2, we compared the number of outlier examiners (defined here as the number of exam- iners whose individual mean rating was more than three Standard Errors from the overall examiner mean rating) pre and post intervention. A non-parametric bootstrapping method using 10,000 iterations [17] was also used to compare the direction of change in exam- iner ratings following the intervention, between those examiners who were initially more lenient than the me- dian examiner in their pre-intervention ratings and those examiners who were initially more stringent. A multivariable linear regression was also performed on average examiner ratings, controlling for differences in individual Case difficulty by including overall average Case rating as a variable. The variable of interest in this analysis was the change in examiner average ratings post-intervention. See Fig. 1 for a summary of study timelines, and analyses of effectiveness of intervention. Prior to the intervention, the examiner participants provided basic demographic information, and indicated their previous examiner experience. They were invited to “rate yourself in terms of your leniency or stringency as an examiner in recent general practice clinical case examinations” on a visual analogue scale, from “very lenient” to “very stringent”. Intra-class coefficients were calculated to identify the historical relationship between 2012 and 2013 examiner ratings (prior to the intervention) and the relationship between examiner ratings pre and post-intervention. The intervention was administered in July 2013, and consisted of the provision of a written summary of indi- vidual examiner ratings over the preceding 18 months (see Table 1). The same information was presented to all examiners, in a de-identified, coded format. Methods All examiners who had examined in any general practice clinical case examinations, in the 18 months prior to the intervention, were invited to participate in this study. In these examinations, third year University of Queensland medical students completing their general practice place- ments were examined by general practitioner examiners on two standardised clinical cases using standardised pa- tients, a Diagnostic Case focused on diagnostic skills and a Management Case focused on patient management. Each case was rated using a standard marking rubric with four criteria rated from 1 (unsafe) to 7 (exemplary); these four item level ratings were averaged to give a final score out of seven on each case, and the passing standard was set at 4. Both the Diagnostic and Management Cases assessed stu- dents on communication skills. The Diagnostic Case also assessed students on history-taking, physical examination Little is also known about examiner perceptions of their own severity. A poor correlation (0.11) was found between family medicine OSCE examiners’ self-perceived severity and the severity of their ratings [7], and stringent exam- iners were unaware of their “hawkishness” in another study [9]. Little is known about the willingness and/or ability of examiners to change their severity after receiving feedback. Although Harasym et al. reported that “feedback to examiners on their stringency/leniency did not change their behaviour”, they included very little information about the nature of this feedback [7]. Studies dating from Sturman et al. BMC Medical Education (2017) 17:101 Page 3 of 7 Page 3 of 7 Following the intervention, a further two examinations were conducted, after which participants were invited to complete a second retrospective severity self-estimate based on these most recent examinations. and formulating differential diagnoses. The Management Case also assessed students on the content of their man- agement, consultation structure, and management of an ethical or professionalism issue. Examiners from the examiner pool examined in a variable number of sessions, working with various standardised patients, over the study period. and formulating differential diagnoses. The Management Case also assessed students on the content of their man- agement, consultation structure, and management of an ethical or professionalism issue. Examiners from the examiner pool examined in a variable number of sessions, working with various standardised patients, over the study period. Methods Examiners were supplied only with their own code, in order to en- able them to identify their own data but not identify other examiners in the data provided. Post-intervention survey data were analysed using de- scriptive statistics and a content analysis. Two investigators (NS and AW) independently coded and categorised the free form survey responses descriptively prior to meeting to identify emerging themes by consensus [18]. Examiners were invited to complete a survey exploring their perceptions of the usefulness and acceptability of the intervention feedback. The survey included free form responses to questions about what was learned from the feedback, which aspects were useful, and what (if any- thing) examiners planned to do differently as a result of the feedback. Results Sixteen of the seventeen examiners who examined during the study period consented to participate in the project; the non-consenting examiner ceased examining in August 2012. One consenting examiner did not return any survey responses. Participant demographic information and rele- vant experience are shown in Table 2. Table 1 Feedback to examiners on Case Ratingsa 1 Overall mean rating, Standard Deviation (SD), and range of each examiner’s Diagnosticb Case ratings 2 Mean differences between each examiner’s mean rating and the mean ratings of every other examiner, highlighting significant differences 3 Overall mean rating and SD for each Diagnosticb case 4 Mean ratings, SDs, and range of ratings for each examiner on each Diagnosticb case examined aInformation for the 2012 and 2013 examinations was presented separately bThe equivalent information was also presented for Management Cases Table 1 Feedback to examiners on Case Ratingsa 1 Overall mean rating, Standard Deviation (SD), and range of each examiner’s Diagnosticb Case ratings 2 Mean differences between each examiner’s mean rating and the mean ratings of every other examiner, highlighting significant differences 3 Overall mean rating and SD for each Diagnosticb case 4 Mean ratings, SDs, and range of ratings for each examiner on each Diagnosticb case examined aInformation for the 2012 and 2013 examinations was presented separately bThe equivalent information was also presented for Management Cases A total of 240 ratings from the two examinations imme- diately pre-intervention and 240 ratings from the two examinations post-intervention were analysed; there were no missing ratings for these examinations. Participating examiners marked between 1 and 8 different cases, and between 8 and 60 individual students over these four examinations. Of the 15 participants who examined in these examinations in 2013, eight examined both pre- and post-intervention. Sturman et al. BMC Medical Education (2017) 17:101 Page 4 of 7 a b Fig. 1 Data collection timelines (a) and analysis schema (b) correlation post-intervention (Spearman correlation −0.52 for diagnostic case and −0.80 for management case). Summary descriptive statistical data for participating examiner ratings are presented in Table 3. The distribu- tion of ratings approximated to a normal distribution. g g The number of outlier examiners was lower after the intervention than before the intervention, for both Diagnostic and Management Cases. Results BMC Medical Education (2017) 17:101 Page 5 of 7 Table 4 Changes in examiner ratings (a) Analysis of Effectiveness of Intervention Outlier Analysis Diagnostic Case Type Management Case Type Pre-intervention 8 (out of 11 examiners) 5 (out of 9 examiners) Post-intervention 7 (out of 13 examiners) 4 (out of 13 examiners) Bootstrapping Analysis Diagnostic Case Type Management Case Type Change in lenient examiner ratings −0.18 (95%CI −0.52 – +0.17) −0.18 (95%CI −0.79 – +0.34) Change in stringent examiner ratings +0.37 (95%CI +0.14 – +0.28) +0.17 (95%CI −0.35 – +0.64) Difference in change between lenient and stringent examiners +0.55 (95%CI +0.05 – +0.68) +0.35 (95% CI −0.37 – +1.07) Multivariable Linear Regression Diagnostic Case Type Management Case Type Full model F (4,3) = 4.96; p > 0.11 F (5,1) = 0.52; p > 0.77 Intervention effect t = 0.54; p > 0.62 t = −0.16; p > 0.90 (b) Analysis of Historical Stability of Examiner Severity Intra-class Correlation Analysis Diagnostic Case Type Management Case Type Intra-examiner correlation between average ratings 2012 and pre- intervention 2013 0.420 0.179 Intra-examiner correlation between average ratings pre- and post- intervention 2013 0.665 0.578 Table 5 Examiner attitudes to examiner feedback (N = 14) Survey question Rated from Strongly disagree(1) to Strongly agree(5) Average score (range) The comparative examiner marking feedback was useful to me in informing me about my leniency or stringency as an examiner 4.3 (3–5) The comparative examiner marking feedback was easy to understand 3.6 (1–5) I am interested in receiving comparative examiner marking feedback in future 4.4 (3–5) Comparative marking feedback is effective in improving the reliability of our examinations 4.0 (3–5) Survey comments on the usefulness and uses of the marking feedback Key themes and illustrative responses Fairness “to ensure that we are on the same page and that students are being examined as fairly as possible” P3 “Helps to identify whether I am “in the ball park” or too extreme, as these are judgements and need to be calibrated” P8 Reassurance “I thought I was being to (sic) lenient, but my mind has been put at ease” P14 “Also supports decisions to fail students which are hard, if I can see that other examiners are prepared to do this too” P8 Understanding “(to) see what other examiners find important in the students’ performance and to get an understanding of what level the students are expected to be at” P14 “Comparing marks with other examiners helps align your marks with your peers but also helps understand other examiners’ approach to Table 5 Examiner attitudes to examiner feedback (N = 14) Table 5 Examiner attitudes to examiner feedback (N = 14) Survey question Average score (range) Table 5 Examiner attitudes to examiner feedback (N = 14) Survey question Rated from Strongly disagree(1) to Strongly (5) Average score (range) Table 5 Examiner attitudes to examiner feedback (N = 14) Understanding “(to) see what other examiners find important in the students’ performance and to get an understanding of what level the students are expected to be at” P14 “Comparing marks with other examiners helps align your marks with your peers, but also helps understand other examiners’ approach to exams and situations and examining” P4 of the feedback, seven (out of fifteen) participants indi- cated that they did not plan to make any changes. Results Free text comments from two relatively lenient examiners who did plan to change included plans to “be more aware generally of where I feel students should sit on the marking scale” (P3) and “be careful not to mark up or avoid poor marks without a clear reason” (P12). A relatively stringent examiner planned to “pay more strict attention to the marking criteria on the marking sheet” (P11). Another relatively stringent examiner (P4) commented that the feedback “accords with my self- belief that I am not particularly lenient”, adding that “I doubt that I will change my examining practice as a re- sult of seeing where I sit”. Another examiner (P9) com- mented that “we may all come to mark more uniformly (or perhaps not) based on this information, but perhaps we need more information to know if this makes our marking any more valid.” lenient examiners. However this was found only for Diagnostic Cases, and the ratings of more lenient ex- aminers did not decrease significantly for either Case type. No significant effect of the intervention for ei- ther Case type was found in the Multivariable Linear Regression analysis. The intra-class correlation ana- lysis (see Table 4) found that intra-examiner ratings were more consistent pre and post intervention than they were historically between 2012 and 2013. These results are summarized in Table 4. Findings from the post-intervention survey (presented in Table 5) indicated that the marking feedback inter- vention was perceived as interesting and useful. The key emerging themes from the free text comments were fairness of examinations, reassurance for examiners, and understanding other examiners (see illustrative quotations in Table 5). Results The bootstrapping analysis showed a significant increase in the ratings of more stringent examiners post-intervention, and a significant difference between the direction of change in ratings post-intervention comparing stringent and Participant severity self-estimates ranged from 3 to 7 on a visual analogue scale, from 1 (very lenient) to 10 (very stringent). Severity self-estimates had a median of 6 and a mean of 5.3. No participant demographic fac- tors were significantly correlated with average overall ratings. There was a weak or modest correlation between sever- ity self-estimates and average overall ratings pre- interven- tion (Spearman correlation 0.29 for diagnostic case and 0.67 for management case). There was a negative Table 3 Examiner overall ratings Pre-Intervention N = 120 Post-Intervention n = 120 Diagnostic Case Mean 5.035 5.129 Diagnostic Case Standard Deviation 0.941 0.881 Diagnostic Case Range 3.00–7.00 2.5–7.00 Number of outlier examiners in Diagnostic Case Examinationsa 8 (out of 11 examiners) 7 (out of 13 examiners) Management Case Mean 4.874 4.990 Management Case Standard Deviation 1.104 0.954 Management Case Range 2.75–6.75 3.75–7.00 Number of outlier examiners in Management Case Examinationsa 5 (out of 9 examiners) 4 (out of 13 examiners) aExaminers whose mean rating was more than 3 standard errors from the overall mean Table 3 Examiner overall ratings Table 3 Examiner overall ratings Table 2 Demographics and Experience of General Practitioner Examiner Participants (N = 15) Gender Female 9: Male 6 General practice experience (range in years) 1.5–35 Medical student teaching experience (range in years) 0.5–33 Number of general practice clinical case examinations previously examined by examiner (range) 2–30 Previous participation in informal discussions about assessment with other examiners Yes:15 No: 0 Previous participation in an examiner training session Yes: 13 No: 2 Previous experience of co-marking with another examiner Yes: 12 No: 3 Table 2 Demographics and Experience of General Practitioner Examiner Participants (N = 15) Sturman et al. Discussion We found no evidence that our examiner intervention, intended to reduce differences in examiner severity, was effective. This is in keeping with previous literature, in which well-intentioned examiner training and feedback In answer to the survey question about any changes examiners planned to make to their marking as a result Page 6 of 7 Page 6 of 7 Sturman et al. BMC Medical Education (2017) 17:101 has not proven effective. Significant differences in exam- iner severity were present both pre and post interven- tion, as shown by the number of examiners whose average rating was more than 3 standard errors from the overall mean (see Table 3). There did appear to be fewer of these outlier examiners post-intervention, but this is of uncertain significance. Our examiners were unable to estimate their own severity accurately, and indeed their severity self-estimates were less accurate after they were provided with our marking feedback. There are well- known methodological problems with research on the accuracy of self-assessment [19] including assumptions that participants are “measuring the same dimensions of performance using the scale in the same way”. The valid- ity of using a visual analogue scale anchored from leni- ent to stringent for self-assessment of examiner severity could be contested. Indeed, the concept of examiner se- verity itself probably warrants further discussion in the literature. Our new finding of reduced self-assessment accuracy post-intervention should be tested in further research. A limitation of our study is that it was not pos- sible to disentangle student ability, case difficulty, stan- dardised patient factors, examiner severity, and their interactions. This was due to the disconnected design of our examinations, including the nesting of examiners in Cases, which precluded the use of a generalizability study [20]. We investigated differences in examiner se- verity using a number of other analyses, however. examinations [23]. However, in other studies severity indi- ces for standardised patient and clinician OSCE examiners drifted substantially from their initial value after 3 or 4 months, particularly on generic rating scales of interper- sonal and communication skills [24], and a small number of standardised patient examiners drifted significantly even within a three month time period [25]. Hemmer found that examiners became more stringent after group discussion with other examiners [26]. This instability merits further investigation, and suggests caution in using examiner equating or adjustment strategies based on pre- vious examiner severity. Conclusion Although investigations of examiner severity in authentic settings are difficult conceptually and methodologically, and no intervention to reduce differences in severity has been proven effective to date, it is generally agreed that defensible clinical assessments should minimise differ- ences in student grades which are due to inconsistencies in examiner ratings, including differences in examiner se- verity. Although increasing the number of assessment nodes, contexts and/or examiners may even out these inconsistencies [1], it remains important to calibrate ex- aminers as effectively as possible, especially if this increase is not feasible. Although our intervention did not appear to be effective, our findings do not suggest that clinician examiners are “impervious” to feedback about their sever- ity, as has been suggested previously in the literature. On the contrary, they were interested in the feedback, and ac- knowledged the importance of being “on the same page” as other examiners. Examiner drift may be related in part to ongoing examiner attempts to calibrate their ratings. Calibration however appears to be difficult for examiners, and the impacts of examiner self-efficacy and examiner compliance may also complicate calibration. Much re- mains to be understood about clinical examiner judg- ments, including examiner severity, and examiner self- monitoring and meta-cognition. In the interim, we would argue that examiners should be provided with the most informative and useful data possible about their rating be- haviours. The rating feedback in our own intervention may have been sub-optimal, and further research is indi- cated to explore optimal feedback strategies. We included survey data and free form responses which enabled us to explore examiner attitudes to the intervention. Our findings suggest that examiners were committed to fair and reliable examinations, and inter- ested in receiving marking feedback and engaging in fur- ther discussion with other examiners. Some participants were cautious about using the marking feedback we pro- vided to calibrate their rating behaviours, partly because they were open to the possibility that outlier examiners (including themselves) were making valid judgements. Other participants indicated that they would attempt to re-calibrate their severity, but appeared to be unsuccessful in this attempt. We also found that examiner severity was unstable even in the absence of an examiner feedback intervention, particularly for our Management Cases. This inherent instability may complicate studies of examiner training strategies. Conclusion Previous findings from investigations of intra-rater consistency in clinical assessment have been conflicting, and often have difficulty distinguishing drifts in examiner severity from drifts in task difficulty (even the same task may be easier for a better prepared examinee cohort, for example). Several studies have found that examiner severity was relatively stable over time and examinees, in clinical long cases [21] OSCE stations [10], short answer questions [22] and oral Discussion The findings that severity may be more stable on some items, and for some examiners, than others, suggests that item-level and individual examiner- level analyses may further inform this area. Acknowledgements Acknowledgements The authors acknowledge the examiner participants from the Discipline of General Practice, University of Queensland, for embracing and evaluating the project. Funding No funding was received for this project. Funding f Funding No funding was received for this project. Funding No funding was received for this project. Page 7 of 7 Page 7 of 7 Sturman et al. BMC Medical Education (2017) 17:101 Ethics approval and consent to participate 17. Mooney CZ, Duval RD. Bootstrapping a nonparametric approach to statistical inference. London: SAGE; 1993. Ethics approval was obtained from the University of Queensland Human Research Ethics Committee 2,013,001,120. Consent was obtained from participating examiners. Examiners were provided with rating data irrespective of their consent to participate in the study, and no student data were identified in the study. 17. Mooney CZ, Duval RD. Bootstrapping a nonparametric approach to statistical inference. London: SAGE; 1993. 18. Leavy P. The Oxford handbook of qualitative research. Oxford New York: Oxford University Press; 2014. 19. Ward M, Gruppen L, Regehr G. Measuring self-assessment: current state of the art. Adv Health Sci Educ Theory Pract. 2002;7(1):63–80. 20. Bloch R, Norman G. Generalizability theory for the perplexed: a practical introduction and guide: AMEE guide no. 68. Med Teach. 2012;34(11):960–92. Availability of data and materials 10. McManus IC, Thompson M, Mollon J. Assessment of examiner leniency and stringency ('hawk-dove effect') in the MRCP(UK) clinical examination (PACES) using multi-facet Rasch modelling. BMC Med Educ. 2006;6:42. Datasets analysed during the study are available from the corresponding author on reasonable request, recognising that examination results and examiner ratings are sensitive data. 11. Rees CE, Knight LV, Cleland JA. Medical educators' metaphoric talk about their assessment relationships with students: 'you don't want to sort of be the one who sticks the knife in them'. Assessment Eval Higher Educ. 2009; 34(4):455–67. Consent for publication Not applicable. 16. Raymond MR, Harik P, Clauser BE. The impact of statistically adjusting for rater effects on conditional standard errors of performance ratings. Appl Psychol Meas. 2011;35(3):235–46. 16. Raymond MR, Harik P, Clauser BE. The impact of statistically adjusting for rater effects on conditional standard errors of performance ratings. Appl Psychol Meas. 2011;35(3):235–46. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 21. Fleming PR, Manderson WG, Matthews MB, Sanderson PH, Stokes JF. Evolution of an examination: M.R.C.P. (U.K.). Br Med J. 1974;2(5910):99–107. 22. Wass V, Wakeford R, Neighbour R, Van der Vleuten C. Royal College of general P. Achieving acceptable reliability in oral examinations: an analysis of the Royal College of general practitioners membership examination's oral component. Med Educ. 2003;37(2):126–31. Authors’ contributions NS conceived and led the project, contributed to data collection, analysis and interpretation, and drafted and revised the article. RO led the data analysis, contributed to interpretation, and revised the paper. WW contributed to conceiving the project, data collection and analysis. JZ contributed to data collection and analysis, and revised the paper. MD contributed to data analysis. All authors read and approved the final manuscript. 12. Schraw G, Kuch F, Gutierrez AP. Measure for measure: calibrating ten commonly used calibration scores. Learn Instr. 2013;24:48–57. 12. Schraw G, Kuch F, Gutierrez AP. Measure for measure: calibrating ten commonly used calibration scores. Learn Instr. 2013;24:48–57. 13. Cook DA, Dupras DM, Beckman TJ, Thomas KG, Pankratz VS. Effect of rater training on reliability and accuracy of mini-CEX scores: a randomized, controlled trial. J Gen Intern Med. 2009;24(1):74–9. 14. Newble DI, Hoare J, Sheldrake PF. The selection and training of examiners for clinical examinations. Med Educ. 1980;14(5):345–9. 14. Newble DI, Hoare J, Sheldrake PF. The selection and training of examiners for clinical examinations. Med Educ. 1980;14(5):345–9. Author details 1 1Primary Care Clinical Unit, Faculty of Medicine, The University of Queensland, Brisbane, Queensland, Australia. 2Royal Brisbane & Women’s Hospitals, Level 8, Health Sciences Building, Herston, QLD, Australia. 3Rural Clinical School Research Centre, Faculty of Medicine, The University of Queensland, Brisbane, Queensland, Australia. 4Institute for Teaching and Learning Innovation, The University of Queensland, Brisbane, Queensland, Australia. 5Centre for Chronic Disease, Faculty of Medicine, The University of Queensland, Brisbane, Queensland, Australia. 6School of Public Health, The University of Queensland, Queensland, Australia. p 23. Weingarten P, Tabenkin K. Variations among examiners in family medicine residency board oral examinations. Med Educ. 2000;34(1):13–7. 24. Harik P, Clauser BE, Grabovsky I, Nungester RJ, Swanson D, Nandakumar R. An examination of rater drift within a Generalizability theory framework. J Educ Meas. 2009;46(1):43–58. 25. McKinley DW, Boulet JR. Detecting score drift in a high-stakes performance- based Assessment. Adv Health Sci Educ. 2004;9(1):29–38. 25. McKinley DW, Boulet JR. Detecting score drift in a high-stakes performance- based Assessment. Adv Health Sci Educ. 2004;9(1):29–38. 26. Hemmer AP, Hawkins LR, Jackson NJ, Pangaro NL. Assessing how well three evaluation methods detect deficiencies in medical Studentsʼ professionalism in two settings of an internal medicine clerkship. Acad Med. 2000;75(2):167–73. Received: 22 September 2016 Accepted: 11 May 2017 Received: 22 September 2016 Accepted: 11 May 2017 Competing interests Competing interests The authors declare that they have no competing interests. 15. Noel GL, Herbers JJE, Caplow MP, Cooper GS, Pangaro LN, Harvey J. How well do internal medicine faculty members evaluate the clinical skills of residents? Ann Intern Med. 1992;117(9):757. 15. Noel GL, Herbers JJE, Caplow MP, Cooper GS, Pangaro LN, Harvey J. How well do internal medicine faculty members evaluate the clinical skills of residents? Ann Intern Med. 1992;117(9):757. References 1. Brennan RL. Performance assessments from the perspective of Generalizability theory. Appl Psychol Meas. 2000;24(4):339–53. 1. Brennan RL. Performance assessments from the perspective of Generalizability theory. Appl Psychol Meas. 2000;24(4):339–53. 2. Berendonk C, Stalmeijer RE, Schuwirth LW. Expertise in performance assessment: assessors' perspectives. Adv Health Sci Educ Theory Pract. 2013;18(4):559–71. 3. Govaerts MJ, Van de Wiel MW, Schuwirth LW, Van der Vleuten CP, Muijtjens AM. Workplace-based assessment: raters' performance theories and constructs. Adv Health Sci Educ Theory Pract. 2013;18(3):375–96. 4. Gingerich A, Regehr G, Eva KW. Rater-based assessments as social judgments: rethinking the etiology of rater errors. 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Harasym PH, Woloschuk W, Cunning L. Undesired variance due to examiner stringency/leniency effect in communication skill scores assessed in OSCEs. Adv Health Sci Educ. 2008;13(5):617–32. 8. Williams RG, Klamen DA, McGaghie WC. Cognitive, social and environmental sources of bias in clinical performance ratings. Teach Learn Med. 2003;15(4):270–92. 9. Bartman I, Smee S, Roy M. A method for identifying extreme OSCE examiners. Clin Teach. 2013;10(1):27–31.
https://openalex.org/W4320147826	https://journals.ufs.ac.za/index.php/jch/article/download/5731/4693	English	null	Making and unmaking bonds: Humanitarianism, local politics and peace building in Southeastern Zimbabwe, 1988 to 1992	Journal for contemporary history/Joernaal vir eietydse geskiedenis	2,022	cc-by	10,261	30 30 EMAIL: kundaimanamere@gmail.com DOI: https://dx.doi. org/10.38140/sjch. v47i2.5731 ISSN 0258-2422 (Print) ISSN 2415-0509 (Online) Southern Journal for Contemporary History 2022 47(2):30-50 PUBLISHED: 31 December 2022 kundaimanamere@gmail.com DOI: https://dx.doi. org/10.38140/sjch. v47i2.5731 ISSN 0258-2422 (Print) ISSN 2415-0509 (Online) Southern Journal for Contemporary History 2022 47(2):30-50 PUBLISHED: 31 December 2022 AFFILIATION: 1 Post-Doctoral Research Fellow, International Studies Group, University of the Free State, South Africa 1 Post-Doctoral Research Fellow, International Studies Group, University of the Free State, South Africa Keywords: Humanitarianism, social cohesion, host differentiation, refugees, peacebuilding, narratives, Chilonga, Zimbabwe MAKING AND UNMAKING BONDS: HUMANITARIANISM, LOCAL POLITICS AND PEACEBUILDING IN SOUTHEASTERN ZIMBABWE, 1988 TO 1992. AUTHOR: Kundai Manamere1 AFFILIATION: 1 Post-Doctoral Research Fellow, International Studies Group, University of the Free State, South Africa ABSTRACT For a long time, perspectives of governments, civil societies and humanitarian organisations have overshadowed the voices of host communities during humanitarian emergencies. In a few instances where literature mentions host communities, they are often portrayed as homogenous groups that share similar views and attitudes towards those in need of assistance. In this article, I draw from host-refugee interactions to argue for the incorporation of local voices in civil society and humanitarianism studies and to illustrate the need to disaggregate host communities and pay attention to local politics during interventions. The influx of thousands of migrants from different countries and ethnic groups, changes the environment of the host community positively and negatively. Often, initial benevolence gives way to hostility as resource scarcities and insecurities arise. When this happens, it creates an environment of suspicion, blame, and stigma, which negatively impacts relations and cohesion between the two groups. Paying attention to local residents’ diverse perspectives during humanitarian emergencies may contribute to conflict prevention and peacebuilding. Keywords: Humanitarianism, social cohesion, host differentiation, refugees, peacebuilding, narratives, Chilonga, Zimbabwe Keywords: Humanitarianism, social cohesion, host differentiation, refugees, peacebuilding, narratives, Chilonga, Zimbabwe Manamere / Making and Unmaking Bonds 31 1 BE Harrell-Bond, Imposing aid: Emergency relief to refugees (Oxford: Oxford University Press, 1986); L Malkki, Purity and exile: Violence, memory, and national cosmology among Hutu refugees in Tanzania (Chicago: Chicago University Press, 1995); L Malkki, “Speechless emissaries: refugees, humanitarianism, and dehistoricization”, Cultural Anthropology 11 (3), 1996, pp. 377-404; L Malkki, The need to help: The domestic arts of international humanitarianism (Durham: Duke University Press, 2015); KL Jacobsen, “Making design safe for citizens: A hidden history of humanitarian experimentation”, Citizenship Studies 14 (1), 2010, pp. 89-103; G Coleman , “Hacker politics and publics”, Public Culture 23 (3), 2011, pp. 511-616. 1 BE Harrell-Bond, Imposing aid: Emergency relief to refugees (Oxford: Oxford University Press, 1986); L Malkki, Purity and exile: Violence, memory, and national cosmology among Hutu refugees in Tanzania (Chicago: Chicago University Press, 1995); L Malkki, “Speechless emissaries: refugees, humanitarianism, and dehistoricization”, Cultural Anthropology 11 (3), 1996, pp. 377-404; L Malkki, The need to help: The domestic arts of international humanitarianism (Durham: Duke University Press, 2015); KL Jacobsen, “Making design safe for citizens: A hidden history of humanitarian experimentation”, Citizenship Studies 14 (1), 2010, pp. 89-103; G Coleman , “Hacker politics and publics”, Public Culture 23 (3), 2011, pp. 511-616. 2 BE Harrell-Bond, Imposing aid, p. xii. 3 M Ticktin, “Transnational humanitarianism,” Annual Review of Anthropology 43, 2014, p. 276. 4 Malkki, “Speechless emissaries”, p. 384. 5 J Robbins, “Beyond the suffering subject: Toward an anthropology of the good”, Journal of the Royal Anthropological Institute, 19, 2013, pp. 447-462. p g p M Ticktin, “Transnational humanitarianism,” Annual Review of Anthropology 43, 2014, p. 276. Malkki, “Speechless emissaries”, p. 384.f J Robbins, “Beyond the suffering subject: Toward an anthropology of the good”, Journal of the Royal Anthropological Institute, 19, 2013, pp. 447-462. 1. INTRODUCTION Scholars such as Feldman have stressed the need to mainstream service delivery and ensure that the host population benefits significantly from the services that humanitarian organisations provide.7 This argument is more discernible in refugee studies which claim that such an approach harnesses the potential of refugees, discourages parallel service delivery and encourages social cohesion.8 In eastern Zimbabwe, for instance, this approach has resulted in, “cordial relations, peaceful co-existence and cooperation among Tongogara camp refugees and their hosts”.9 Veronika Fajth et al have shown that economic interaction fostered trust between Congolese refugees and their hosts in Rwanda.10 In Cameroon, the health delivery system improved with the presence of refugees.11 However, I argue that despite the existence of these works, less attention has been paid to host communities’ own voices on how they comprehend various challenges resulting from humanitarian emergencies. The majority of humanitarianism works that discuss host communities’ needs and challenges largely present a technocratic and romanticised view of the community as a single unit, homogenous, equal, undifferentiated, and accommodative of refugees. and around humanitarian spaces.6 Using their work as intellectual scaffolding, this paper extends the argument that refugee camps are not exceptional and isolated humanitarian spaces, set apart from the ordinary spaces of life or host communities. Their set-up and function, in some cases, unintentionally create social and political complexities as well as hostile environments in and around them. As such, there has been a notable scholarly shift in literature, which reveals an increased interest in the welfare of host populations of late. Scholars such as Feldman have stressed the need to mainstream service delivery and ensure that the host population benefits significantly from the services that humanitarian organisations provide.7 This argument is more discernible in refugee studies which claim that such an approach harnesses the potential of refugees, discourages parallel service delivery and encourages social cohesion.8 In eastern Zimbabwe, for instance, this approach has resulted in, “cordial relations, peaceful co-existence and cooperation among Tongogara camp refugees and their hosts”.9 Veronika Fajth et al have shown that economic interaction fostered trust between Congolese refugees and their hosts in Rwanda.10 In Cameroon, the health delivery system improved with the presence of refugees.11 However, I argue that despite the existence of these works, less attention has been paid to host communities’ own voices on how they comprehend various challenges resulting from humanitarian emergencies. 6 M Agier, “Humanity as an identity and its political effects (A note on camps and humanitarian government)”, Humanity 1 (1), 2010, pp. 29-45; I Feldman, “The humanitarian condition: Palestinian refugees and the politics of living”, Humanity 3 (2), 2012, pp. 155-172. 7 S Feldman, “Development assisted integration: A viable alternative to long-term residence in camps?” PRAXIS, The Fletcher Journal of Human Security 12, 2007. 8 I Idris, Effectiveness of various refugee settlement approaches, K4D Helpdesk Report 223 (Brighton: Institute of Development Studies, 2017). 9 J Mhlanga and GA Muchinako, “Burdens or benefits: A critical analysis of the nexus between refugees and host communities in Zimbabwe”, Human Ecology 60, 2017, pp. 2-3. 10 V Fajth et al, “How do refugees affect social life in host communities? The case of Congolese Refugees in Rwanda,” Comparative Migration Studies, 7 (33), 2019. 11 L Tatah et al., “Impact of refugees on local health systems: A difference in differences analysis in Cameroon”, PLoS One 11 (12), 2016. 6 M Agier, “Humanity as an identity and its political effects (A note on camps and humanitarian government)”, Humanity 1 (1), 2010, pp. 29-45; I Feldman, “The humanitarian condition: Palestinian refugees and the politics of living”, Humanity 3 (2), 2012, pp. 155-172. g g ( ) L Tatah et al., “Impact of refugees on local health systems: A difference in differences analysis in Cameroon”, PLoS One 11 (12), 2016. g p g y ( ) pp S Feldman, “Development assisted integration: A viable alternative to long-term residence in camps?” PRAXIS, The Fletcher Journal of Human Security 12, 2007.f 8 I Idris, Effectiveness of various refugee settlement approaches, K4D Helpdesk Report 223 (Brighton: Institute of Development Studies, 2017). i J Mhlanga and GA Muchinako, “Burdens or benefits: A critical analysis of the nexus between refugees and host communities in Zimbabwe”, Human Ecology 60, 2017, pp. 2-3.f V Fajth et al, “How do refugees affect social life in host communities? The case of Congolese Refugees in Rwanda,” Comparative Migration Studies, 7 (33), 2019.ff 1. INTRODUCTION Using narratives and newspapers, this article explores the views and interpretations of host communities following a disease outbreak that killed nearly 200 Mozambican refugees in Chambuta camp, located on Zimbabwe’s southeastern border with Mozambique, in August 1992. Since the 1980s, humanitarianism literature, particularly that on refugees and displacement, has largely focused on the suffering of refugees.1 Barbara Harrell Bond’s book especially explored the Ugandan refugee assistance in Southern Sudan and showed how relief organisations failed to, “listen to refugees who were progressively understood in literature as victims unable to fend for themselves”.2 In the 1990s, Liisa Maalki’s work on Hutu refugees from Burundi living in Tanzania similarly indicated how refugees suffered in silence. Narratives of refugees suffering were further strengthened by medical humanities scholars between the 1980s and 1990s as new forms of media and technology made certain kinds of suffering visible during the famine in Ethiopia and the Rwandan genocide.3 As Maalki demonstrated, “the suffering body of the refugee held particular importance in the camps because it was seen by humanitarians as providing a more reliable account of experience than a refugee’s stories or words”.4 The refugee’s suffering body became a prime object of investigation aimed at influencing more effective interventions in humanitarian spaces, especially in refugee camps.5 This way of seeing put more emphasis on the refugees and significantly undermined the negative and unintended consequences of humanitarian interventions on host communities. Over the past decade, scholars such as Michel Agier and Ilana Feldman have embarked on understanding the unintended consequences of humanitarianism as well as teasing out new meanings of the political in SJCH 47(2) \| December \| 2022 SJCH 47(2) \| December \| 2022 32 and around humanitarian spaces.6 Using their work as intellectual scaffolding, this paper extends the argument that refugee camps are not exceptional and isolated humanitarian spaces, set apart from the ordinary spaces of life or host communities. Their set-up and function, in some cases, unintentionally create social and political complexities as well as hostile environments in and around them. As such, there has been a notable scholarly shift in literature, which reveals an increased interest in the welfare of host populations of late. 7 S Feldman, Development assisted integration: A viable alternative to long term residence in camps?” PRAXIS, The Fletcher Journal of Human Security 12, 2007. 8 I Idris, Effectiveness of various refugee settlement approaches, K4D Helpdesk Report 223 (Brighton: Institute of Development Studies, 2017). 9 J Mhlanga and GA Muchinako, “Burdens or benefits: A critical analysis of the nexus between refugees and host communities in Zimbabwe”, Human Ecology 60, 2017, pp. 2-3. 10 V Fajth et al, “How do refugees affect social life in host communities? The case of Congolese Refugees in Rwanda,” Comparative Migration Studies, 7 (33), 2019. 11 L Tatah et al., “Impact of refugees on local health systems: A difference in differences 10 V Fajth et al, “How do refugees affect social life in host communities? The Refugees in Rwanda,” Comparative Migration Studies, 7 (33), 2019.f 1. INTRODUCTION The majority of humanitarianism works that discuss host communities’ needs and challenges largely present a technocratic and romanticised view of the community as a single unit, homogenous, equal, undifferentiated, and accommodative of refugees. g qf g This paper challenges this view by examining how a disease outbreak at Chambuta refugee camp in southeastern Zimbabwe in 1992 created Manamere / Making and Unmaking Bonds 33 an enduring resentment of refugees in the surrounding host communities. Beth Elise Whitaker calls for a disaggregation of the host community based on gender, class and age.12 Updating Whitakers’ concept of host community heterogeneity, this article makes an empirical and methodological contribution by considering the importance of incorporating local voices in humanitarianism studies. I emphasise why it is essential to disaggregate the host community and pay attention to local politics. More significantly, I reflect on how incorporating local voices reframes debates on humanitarianism. By focusing on local narratives of host communities’ experiences, perspectives and attitudes towards refugees and epidemic disease, this article addresses the fundamental assumptions about relations between affected populations and their hosts as well as humanitarian interventions in emergency situations. I further argue that paying attention to local residents’ diverse perspectives during humanitarian emergencies may contribute to conflict prevention and peacebuilding between refugees and host communities. This research places people at the centre of analysis by drawing from their narratives of disease to demonstrate the importance of understanding local politics during humanitarian crises. I carried out 18 in-depth interviews with villagers, village heads, health personnel and former refugees in Chilonga between September and December 2020. My informants in Chilonga have been threatened with eviction by the government, which intends to displace them for a Lurcene grass irrigation project since 2019.13 Therefore, interviews were conducted in a tense environment with heavy intelligence and military presence. This affected the recruitment of participants as some people feared for their lives. However, in other instances, the imminent eviction evoked memories about the earlier displacement of locals when the refugee camp was established and therefore yielded more information for this study. Although plagued by memory loss and exaggeration, in-depth interviews proved critical in showing variations in what hosts think about the refugees, displacement, diseases and drought. Most interestingly, interviews also reveal a differentiated and unequal host community, divided along clan and socio- economic lines. 12 BE Whitaker, “Refugees in Western Tanzania: The distribution of burdens and benefits among local hosts”, Journal of Refugee Studies 15 (4), 2002. 13 New Zimbabwe, 7 June 2020. 1. INTRODUCTION I also consulted newspaper reports on Mozambican refugees at the Herald House Library in Harare during the same period. The various newspaper reports provide the Zimbabwean government’s perspectives on the refugees as well as details on camp construction, administration, refugee influx, disease outbreaks, public health interventions, foreign aid, alleged corruption and misuse of donated goods by government officials. Ideally, SJCH 47(2) \| December \| 2022 SJCH 47(2) \| December \| 2022 34 archival material produced by the Department of Social Welfare would have yielded more information on administration, logistics, refugee statistics and epidemiology, but government documents from the 1990s are closed and, therefore, inaccessible to the public. Furthermore, the Ministry of Health’s public health annual reports for the period under study are surprisingly silent on the public health situation in refugee camps. I, therefore, rely on newspaper reports for statistics regarding the inflow of refugees and disease epidemiology which makes it difficult to establish solid demographic and epidemiological patterns. Notwithstanding these limitations, a comparison of interviews and newspapers reveals different stories about the disease outbreak. The government and its partners viewed the disease outbreak as a purely public health crisis occurring during the emergency phase of a humanitarian crisis. The link between refugee conditions and the threat of diseases is well established in historical, medical and anthropological literature. Scholars have explored, at length, how population movements are related to disease occurrence among refugee communities, which have emerged because of socio-political factors such as wars, civil strife and famine. Refugees and internally displaced populations are particularly vulnerable to the risk of disease as the camps in which they live are often poor and a result of complex humanitarian emergencies.14 Cholera, measles and meningitis account for 75 per cent of epidemics in refugee camps, as conflict and displacement concomitantly increase population vulnerability and reduce the system’s response capacity. Overcrowding, poor water and sanitation conditions, lack of vaccines, delayed diagnosis and reduced access to treatment often lead to the increased occurrence, severity and case fatality of infectious disease. On the other hand, local residents’ comprehension of public health remains characterised by complex intersections between biomedical and indigenous notions of disease, the body and the environment. Local residents identify both sanitary and spiritual causes of the disease outbreak. 14 M Rowland and F Nosten, “Malaria epidemiology and control in refugee camps and complex emergencies”, Annals of the Tropical Medicine and Parasitology 95 (8), 2001. 1. INTRODUCTION Some blame the refugees for poor sanitation, while others view the diseases as a curse from the ancestors over the unjustified displacement of local families paving the way for the construction of the refugee camp. In addition, some community members also believe that their ancestors were unhappy with the presence of the Barwe refugees, an ethnic group that they believed to be made up of cannibals and witches. By using narratives and perceptions of both refugees and host communities, this article reflects on the politics of voice and representation Manamere / Making and Unmaking Bonds 35 in history. It allows us to understand local interpretations of displacement, the occurrence of diseases, and interventions, thus informing humanitarianism policies from below. The policy implications are clear. Policymakers and practitioners need to be aware of these perspectives and establish alliances with local communities. For instance, while there are significant efforts to understand the disease problems of refugees, awareness campaigns regarding diseases associated with refugees should be included in health education among host communities to prepare them and allay unnecessary anxieties and avert blame and othering. Although this may not immediately eliminate “false” notions, it may at least bridge the various explanatory models and encourage cohesion between the two groups. Furthermore, I emphasise the need to pay attention to cleavages within host communities, be they ethnic, political, clan or socio-economic. Governments tend to narrowly consult villagers and local authorities, if they do at all, prior to launching intervention strategies. Yet, the success of interventions hinges on the attitude of local communities. Local voices are indispensable in intervention processes.i The first section of the article shows that initially, the host community and the refugees established mutual relations based on a shared ethnic identity and refugee experiences despite the subsequent displacement of several families from the campsite. The penultimate section discusses the refugee influx that led to the disease outbreak at the Chambuta refugee camp. The last section examines how the disease outbreak changed the attitude of most members of the host community from initial kindness to hostility. It explores alternative causal explanations of the disease outbreak as understood by local residents and considers what the majority of the hosts view as long-term impacts of refugee presence on their land. 15 Interview: Author with Njakeni Zumo, Former Mozambican Refugee, Pfuveni Village, Chilonga, Zimbabwe, 15 October 2020. 16 L Hultman, “The power to hurt in civil war: The strategic aim of RENAMO violence”, Journal of Southern African Studies 35 (4), 2009; CC Kelso, “Turning the Refugee Tide”, Cultural Survival Quarterly Magazine, 17 (1), 2010, available on-line at https://www.culturalsurvival. org/publications/cultural-survival-quarterly/turning-refugee-tide, accessed 5 December 2022. For more information on the causes of the Mozambican civil war see, S Funada-Classen, The Origins of war in Mozambique history of unity And division (Tokyo: Ochanomizu Shobo Co Ltd, 2012). 17 Interview: Author with Zumo, 15 October 2020. 2. MAKING BONDS: SHARED IDENTITIES, EXPERIENCES, AND DEVELOPMENT From the early 1980s, Mozambican refugees became an integral part of Zimbabwean communities along the Zimbabwe-Mozambique border. Commonly identified by Zimbabweans as makarushu, a derogatory name derived from the cashew nuts plantations of Mozambique, the refugees self- settled in local villages and worked on farms along the scarcely populated border. Some refugees spontaneously settled among their Zimbabwean relatives along the border areas. Other refugees chose to live in camps strategically located along the border. Njakeni Zumo, a former Mozambican refugee, moved with her family to Zimbabwe and self-settled in Chikombedzi, SJCH 47(2) \| December \| 2022 SJCH 47(2) \| December \| 2022 36 a rural area on the southeastern border, in 1980. “I gave birth to my first child the year that Robert Mugabe came to power. I think others who followed from Mozambique came later when my child was four or five years old. People who came from Mozambique first settled in local villages for many years. This was just bush, so we just settled without seeking any approval from Zimbabwe government authorities”.15 Her family lived in Chikombedzi for many years. During the 1992 drought, most of the refugees left for the refugee camps where the Zimbabwean government promised to look after them. But some did not go into camps; they continued to live in the villages. The Zimbabwean government had established four refugee camps along the Mozambican border to accommodate asylum seekers fleeing from the Mozambican civil war, which had left houses burnt, food stolen, and people mutilated, kidnapped or dead.16 These camps were Tongogara, Nyangombe, Nyamakiti and Mazoe River Bridge. The steady stream of refugees surged in 1987 when the war intensified. The Zimbabwean government established a fifth camp in 1988 to accommodate the tens of thousands of refugees in transit to bigger camps such as Tongogara. This camp was Chambuta, located in the Chilonga communal area along the southeastern border with Mozambique. Njakeni and her family moved from Chikombedzi to Chambuta refugee camp in 1992. They heard that there were trucks fetching Mozambicans to refugee camps, and they decided to go to Chambuta, leaving their crops in the fields before harvesting.17 The Chambuta camp derives its name from the Chambuta family, who heads one of the several villages under the Masuamele chieftainship. 18 Kelso, “Turning the Refugee Tide”. 19 Interview: Author with Cephas Chambuta, Chambuta Village head and former worker at the camp, 63 years old, Chambuta Village, Chilonga, Zimbabwe, 20 September 2020. Cephas worked in the Camp under the Social Welfare Department of the Government of Zimbabwe from the time it was established. 2. MAKING BONDS: SHARED IDENTITIES, EXPERIENCES, AND DEVELOPMENT When searching for land on which to settle the refugees, government officials consulted the Chambuta village head, who then identified and allocated the piece of land where the camp was constructed. It is however important to note at this point that this piece of land was located on the boundary of the Chambuta village under the Masuamele chieftainship and the Velemu village of the Chilonga chieftainship. Officially, the land fell under the jurisdiction of the Velemu village head and the Chilonga chieftainship. Consequently, upon camp construction, several families from Velemu village were displaced while 17 Manamere / Making and Unmaking Bonds 37 Chambuta villagers were widely employed in the camp. The Zimbabwean government upgraded Chambuta to a permanent refugee camp in July 1990. The camp quickly gained a deadly reputation. With a holding capacity of 20 000, by mid-1992, the camp held over 25 000 people. A drought-induced influx between June and August 1992 introduced an estimated 11 500 refugees to the camp, and in no time, available resources failed to cope.18 The system at Chambuta collapsed, and a public health nightmare unfolded. Interviews with villagers and village heads in Chilonga show that initially, from the mid-1990 to the outbreak of diseases in 1992, they largely welcomed the Mozambican refugees who settled in the Chambuta camp. Factors that encouraged cohesion include a shared ethnic identity, language and experiences. Moreover, to the villagers, the construction of a refugee camp with a school, a police post and a clinic represented a positive step towards the development of their area, which until 1990 had none of these facilities. The creation of the Zimbabwe-Mozambique border in 1891 “separated” different ethnic groups, which easily and frequently traversed borders for political, economic and social reasons. Such ethnic groups include the Shangani, who occupy southern Mozambique and Zimbabwe. The Shangani were the major ethnic group in the Chambuta refugee camp. Since colonial times the Shangani maintained kinship and cultural ties across the border, and in times of crisis, they provided refuge to those in need. These ties contributed to a mutual relationship between the hosts and the refugees when the camp was set up. The hosts viewed Mozambican refugees as relatives, not refugees. Cephas Chambuta, a village head and former administrator in the camp, explained, We had a mutual relationship with refugees; as you know, we are related […] they are Chauke (a Shangani totem). 18 Kelso, “Turning the Refugee Tide”. 20 Interview: Author with Ingwani Madomba, Former worker at the camp and Local villager, 64 years old, Chambuta Village, Zimbabwe, 24 September 2020. 21 Interview: Author with John Murami, 69 years old, Former refugee, Machiloli Village, Chilonga, Zimbabwe, 21 September 2020. 22 Interview: Author with James Chitsange, Velemu villager, 50 years old, Velumu, Zimbabwe, 1 October 2020. 23 The Herald, 7 February 1995. The government strongly discouraged village heads and chiefs from informally settling Mozambicans in their areas. Ingwani Madomba of Chambuta village, who also worked at the camp, adde gwani Madomba of Chambuta village, who also worked at the camp, added, The authorities realised that these people were also Shangani; we were relatives separated by the border. Some were related through intermarriages. When they came, local people also got married to refugees. Most women from here left with their refugee husbands upon repatriation. Misunderstandings were settled by the traditional courts, and there was not so much conflicts and hatred.20 2. MAKING BONDS: SHARED IDENTITIES, EXPERIENCES, AND DEVELOPMENT They are our parents since our ancestors came from Mozambique; these were our relatives, our uncles. They freely came into our villages, and we had beer parties together and exchanged food items. Some would sleep over and returned to the camp the next morning since there was a 6pm curfew. We also had intermarriages, I have a sister who was married to a refugee, and she went to Mozambique.19 SJCH 47(2) \| December \| 2022 SJCH 47(2) \| December \| 2022 38 24 Interview: Author with Thomas Salani, Local Villager, 62 years old, Chambuta Village, Zimbabwe, 20 September 2020. 25 The Herald, 16 March 1992. 26 Interview: Author with Livison Chikutu, 46 years, Velemu Village Head, Velemu Village, near Chambuta Camp, Zimbabwe, 4 October 2020. 27 Interview: Author with Thomas Salani, 20 September 2020. 28 Interview: Author with Cephas Chambuta, 20 September 2020. A former refugee who lives in Machiloli village, John Murami, attests to the generous reception of the hosts, They exchanged their goats and vegetables for mealie meal, tinned fish, beans, dried fish and clothing with the refugees.26 The refugees also accessed water from the surrounding villages when their population surged and the water supply diminished during the 1992 drought. The close proximity of the hosts to the camp gave them an advantage in building relationships with the refugees, which in turn made it possible for them to survive the drought. Those who came from other places across the country looking for maize meal in the camp usually waited for weeks before they found suppliers. Evidence of cohesion on the ground includes social visits, beer parties, and intermarriages, sharing water and exchanging food items. Since the refugees received food aid, they bartered goods with local residents. Barter trading of food items, in particular, created cohesion. The refugees often smuggled food out of the camp to be sold on the black market in surrounding villages and nearby towns of Chiredzi and Masvingo.25 People from villages that surround the camp claim that during the 1992 drought, they did not suffer greatly. They exchanged their goats and vegetables for mealie meal, tinned fish, beans, dried fish and clothing with the refugees.26 The refugees also accessed water from the surrounding villages when their population surged and the water supply diminished during the 1992 drought. The close proximity of the hosts to the camp gave them an advantage in building relationships with the refugees, which in turn made it possible for them to survive the drought. Those who came from other places across the country looking for maize meal in the camp usually waited for weeks before they found suppliers. p y y pp A local villager, Thomas Salani, reminisces the refugee camp as a “town”, a place where they got everything they needed from clothing to food before and during the drought.27 The presence of the refugee camp not only saved the hosts from the drought but brought modernity closer to home. For decades, locals lived in this “forgotten” part of the country without access to retail stores, schools, clinics, roads, toilets and clean water. The refugees came with development. Village head Cephas Chambuta says, There were no roads here, no bridges; now we have roads; from Lundi bridge, there was no road from Makosiya to Chilonga, but now we have it because of the people from Mozambique. A former refugee who lives in Machiloli village, John Murami, attests to the generous reception of the hosts, A former refugee who lives in Machiloli village, John Murami, attests to the generous reception of the hosts, I married my wife when I moved to the camp. My in-laws accepted me because I was an honest person. From the time I was living in the camp, I built good relations. Now I am ageing, I am happy, I have a family and a home. I am very grateful to the local community for looking after me.21 Even members of the displaced families interacted positively with the refugees despite simmering grievances. “It was not the refugees’ fault. They had nowhere to go. They needed shelter, and as their relatives, we supported them”, said James Chitsange of Velemu village.22 These perspectives illustrate that a shared ethnic identity significantly contributed to the acceptance of refugees by the majority of the host community. In addition, shared experiences of crisis and refuge seeking considerably influenced good relations between the hosts and the refugees. Having been refugees in Mozambique during the Zimbabwe war of liberation, Zimbabweans understood the need for refuge during conflict and showed appreciation for their stay in Mozambique. As indicated earlier, from the early 1980s, when Zimbabwe attained independence, chiefs and village heads along the borders settled Mozambican refugees who sought refuge during the civil war.23 According to Thomas Salani of Chambuta village, When refugees settled here, the Mozambique National Resistance (Resistência Nacional Moçambicana, RENAMO) militia followed and killed people in the villages accusing Zimbabweans of harbouring refugees. They would not go into the camp but killed people in the villages. Sometimes people slept in the bush, hiding from RENAMO. It was painful, and we wondered why we were dying to protect refugees, Manamere / Making and Unmaking Bonds Manamere / Making and Unmaking Bonds 39 but since we were once refugees in Mozambique during our own war of liberation, we were giving back.24 Evidence of cohesion on the ground includes social visits, beer parties, and intermarriages, sharing water and exchanging food items. Since the refugees received food aid, they bartered goods with local residents. Barter trading of food items, in particular, created cohesion. The refugees often smuggled food out of the camp to be sold on the black market in surrounding villages and nearby towns of Chiredzi and Masvingo.25 People from villages that surround the camp claim that during the 1992 drought, they did not suffer greatly. 29 Kelso, “Turning the Refugee Tide”. 30 The Herald, 20 June 1992. 31 Center for Disease Control (CDC), “Mortality among Newly Arrived Mozambican Refugees— Zimbabwe and Malawi, 1992”, Morbidity and Mortality Weekly Report (MMRV) 42 (24), 1993, available online at https://www.cdc.gov/mmwr/preview/mmwrhtml/00020997.htm, accessed 5 December 2020. A former refugee who lives in Machiloli village, John Murami, attests to the generous reception of the hosts, The secondary school that we now have, we inherited it from the Mozambicans. We also have grocery stores, a clinic and a police station which we didn’t have.28 These sentiments of appreciation are widely shared across the host villages. Despite these positive perceptions, however, when disease struck the camp in 1992, elements of suspicion, blame and othering emerged as hosts blamed refugees for the disease outbreak. SJCH 47(2) \| December \| 2022 40 32 P Spiegel, ‘Health programmes and policies associated with decreased mortality in displaced people in post emergency phase camps: A retrospective study,’ Lancet 360 (9349), 2002. 33 The Herald, 13 October 1992. 34 Interview: Author with Evelyn Hokela, Former Chambuta Refugee, Married to a local man, 65 years old, Hokela Village Hokela Village, Zimbabwe, 23 September 2020. 35 The Herald, 16 March 1992. 36 Interview: Author with Mr Makuyana, Environmental Health Technician, 66 years old, Chiredzi Hospital, Chiredzi, Zimbabwe, 23 September 2020. 37 Interview: Author with John Dzingai (pseudonym), 56 years old, Nurse, Chambuta Camp Clinic, Zimbabwe, 26 September 2020. 3. THE REFUGEE INFLUX AND THE MAKING OF A PUBLIC HEALTH NIGHTMARE AT CHAMBUTA At the end of 1992, when Mozambican president Joaquim Chissano signed an agreement with the RENAMO “rebel” leader Afonso Dhlakama which ended the 17-year civil war, the refugees’ numbers estimates were 230 000 for Zimbabwe, 25 000 for Zambia, 350 000 for South Africa and 1.3 million for Malawi (the highest population).29 An estimated 300 000 refugees spontaneously returned to Mozambique on their own, but the majority remained outside the country, fearing the threat of a renewed war. Besides the fear of civil war-related violence, the 1992 drought that hard hit Southern Africa presented a new threat to Mozambican refugees, whose estimated 2.3 million population already needed food assistance because of the induced displacements.l The severe drought forced thousands more to flee, creating emergencies in Zimbabwe’s refugee camps. When it opened as a transit camp in 1988, Chambuta had 338 people. In January 1992, 859 refugees had settled in the camp, and by May 1992, the refugee population had risen to 16500.30 In July 1992, Chambuta admitted more than 1000 refugees weekly. The refugee population in the camp swelled to 25 323 by October 1992, as the camp received an average of 4500 people monthly. These included some of the refugees who had initially self-settled in Zimbabwean villages. There were nearly 120 000 self-settled refugees along the Zimbabwean borders, and the 1992 drought forced some of them to go into camps where they expected to be fed. Consequently, in August 1992, 206 deaths were recorded, with crude mortality daily rates ranging between 3.5 and 8.2 deaths per 10 000 populations.31 Diarrhoea, dehydration, malnutrition and measles accounted for 75 per cent of all reported deaths. The official narrative drawn from various reports by the Zimbabwe Department of Social Welfare, the United Nations High Commission for Refugees (UNHCR) and other humanitarian organisations places the disease outbreak within the typical initial emergency phase following a refugee influx. During this period, refugee camps experience high mortality rates mostly due to preventable and treatable infections, often exacerbated by malnutrition, overcrowding, and poor access to water. 3. THE REFUGEE INFLUX AND THE MAKING OF A PUBLIC HEALTH NIGHTMARE AT CHAMBUTA The refugee influx at the camp also affected the food aid programmes as authorities failed to provide the daily World Food Programme SJCH 47(2) \| December \| 2022 SJCH 47(2) \| December \| 2022 42 stipulated 400g per individual.38 The 1992 drought diminished Zimbabwe’s food supplies and strained medical and social programmes. Several local and international non-governmental organisations intervened through the adoption of several medical interventions such as vaccinations, the construction of sanitation facilities, and humanitarian efforts like food distribution and supply of materials for shelter, among others. The Department of Social Welfare coordinated a sanitation and feeding programme as it partnered with non-governmental organisations through its Refugees Services Unit. The programme focused on preventive measures, primarily the provision of adequate food, safe water, toilets and shelter to curb the disease outbreak. The Red Cross, which was involved in assisting the refugees in moving from Mozambique to Zimbabwe and settling them in various camps, increased their monthly rations of maize meal, beans, seeds and clothing.39 The Red Cross also screened the refugees for malnutrition. Redd Barna, a Norwegian organisation which provided shelter to the refugees upon settlement, increased its funding by 30 per cent.40 The Catholic Development Commission (CADEC), which had begun work in Chambuta when it was used as a transit camp, provided supplementary feeding schemes, blankets and toilets to new arrivals. The UNHCR, in collaboration with the Baptist Church, sunk twelve boreholes and constructed a water purification and reservoir system for the camp.41 Medicines sans Frontiers, in collaboration with UNHCR, brought in more health personnel to assist Zimbabwean nurses. The Ministry of Health set up a satellite clinic at the Joachim Chissano School in order to increase health care services. All admissions were however made at the main clinic, which had a bed capacity of 32. Refugee women took turns cooking for the malnourished. There was a good ambulance system, and the Ministry of Transport repaired and maintained the road which was used to transport acute cases to Chiredzi Hospital.42 The Zimbabwean Ministry of Health initiated a massive measles vaccination programme. Environmental health technicians led sanitation and hygiene awareness programmes insisting on the importance of sanitary measures such as hand washing, covering of food, use of toilets, rubbish pits, buckets with lids, and safe water within the camp. 3. THE REFUGEE INFLUX AND THE MAKING OF A PUBLIC HEALTH NIGHTMARE AT CHAMBUTA Diarrhoea, measles, malaria, and Manamere / Making and Unmaking Bonds 41 respiratory tract infections rapidly spread across the refugee population, and the focus on healthcare during this period is on immediate lifesaving interventions within the camp.32 The reports maintain that it was a diarrheal disease outbreak resulting from malnutrition due to lack of food during the prolonged civil war, the displacement period as Mozambicans moved to Zimbabwe, and the 1992 drought that occurred across Southern Africa. The measles outbreak among refugee children was a result of restricted access to vaccines during wartime. g In March 1992, the Chambuta camp received an average of 50 new arrivals per day. This number increased between July and September 1992 to approximately 300 newcomers daily.33 Most of the newcomers at Chambuta were emaciated when they arrived at the camp. For many years they had failed to cultivate crops because of the war.34 The drought worsened their situation, and they spent days without food on their way to the camp. They required medical attention and put a strain on the already stretched health facilities. Nearly 300 to 400 refugees in the camp were treated daily for food deficiency-related diseases.35 Malnutrition, measles and diarrhoea were the major causes of death among children in the camp. At the camp clinic, the nursing staff failed to cope. Only eight nurses and four nursing aids worked around the clock.36 John Dzingai, a former nurse at the camp, remembers people dying as they waited to be treated during the influx period. He insists that more than half of the refugees were malnourished and developed diarrhoea when they began the feeding programme. He likens this to how overfed cattle develop diarrhoea after a prolonged dry season, “Some patients recovered, but some who were severely malnourished would get overwhelmed and die upon feeding. An average of 15 deaths per day occurred during the influx period from March 1992 to August 1992”.37 The soaring refugee population created sanitary problems as the existing facilities could not cope with the large numbers in the camp. At the same time, shelter materials were in short supply. The drought affected the availability of grass for roofing. 38 The Herald, 13 October 1992. 39 The Herald, 23 March 1992. 40 The Herald, 20 June 1992. 41 The Baptist Press, January 1990. 42 Interview: Author with John Dzingai, 26 September 2020. Manamere / Making and Unmaking Bonds 43 However, these public health interventions left out host communities and targeted refugees only. The hosts only had unrestricted access to curative health services at the refugee camp clinic. Preventive measures, such as sanitation and hygiene and vaccinations, in particular, were limited to refugees. Yet, despite the closed and isolated nature of the refugee camp as well as the perceived restriction of refugees’ movements in the camp, there was close interaction between refugees and host communities. Regarding infectious disease control programmes, for instance, it makes little epidemiological sense to become involved with one group and not with the other; the organisms which cause diseases are not so bureaucratically selective. Although John Dzingai, the former nurse at Chambuta camp, insists that the disease outbreak did not spread into surrounding villages, interviews with Velemu villagers claimed that the disease outbreak spread into their village and killed several people, as shall be shown below. These incidences triggered hate and stigma as Velemu villagers, particularly, blamed the refugees for causing the disease outbreak. The earlier established social cohesion waned. In contrast to the official narrative given by the government and international organisations, below, I unpack the different reasons why the majority of the hosts believed that the refugees were the source of diseases. 3. THE REFUGEE INFLUX AND THE MAKING OF A PUBLIC HEALTH NIGHTMARE AT CHAMBUTA Through these interventions, the Zimbabwean government and its partners reported that they had successfully contained the disease outbreak within the camp as morbidity and mortality cases dropped from September 1992. Manamere / Making and Unmaking Bonds 4. UNMAKING BONDS: POOR SANITATION, DISEASE OUTBREAKS AS A CURSE Drawn from interviews, the local narrative does not associate disease among refugees with the effects of war-induced famine, nor with malnutrition as a result of the 1992 drought. In their experience, refugees had food in abundance, and bartering food aid was a common survival strategy for local people during the drought. The explanation for the disease outbreak lies elsewhere. Depending on their village of origin, local residents associate the disease outbreak with overcrowding and poor sanitation, the anger of ancestral spirits or both. Both Chambuta and Velemu villagers widely identify the diarrheal disease as cholera which resulted from overcrowding and poor sanitation in the refugee camp. Those from Chambuta village, who mostly worked in the camp, for instance, Cephas Chambuta and Ingwani Madomba emphasise poor sanitation, while Velemu villagers, who were displaced as a result of the camp subscribe to the poor sanitation explanation but stress that the disease outbreak was a curse, a punishment from their ancestors following the displacement of several families from the campsite, to pave the way for refugees’ settlement. These alternative and diverse explanations of diseases are important in two ways. First, they show the heterogeneity of host SJCH 47(2) \| December \| 2022 SJCH 47(2) \| December \| 2022 44 communities, informed by clan and loss or benefits derived from the camp establishment. Second, they reflect an extra spiritual dimension to causal explanations of disease that are normally couched strictly in biomedical terms. It is these alternative explanations that determine the successful integration of refugees within host communities. The first alternative explanation shared across the two villages but mostly drawn from Chambuta villagers is that there was poor sanitation at the personal refugee level and in the camp. Despite the availability of toilets, some of the refugees preferred to use the bush, and there was human waste everywhere.43 At night someone would just go near the house and defecate, and flies would transmit the diseases. The clinic staff taught refugees about sanitary measures. They distributed soap for washing hands, buckets and other utensils. Non-governmental organisations provided disinfectants to put in toilet pits. They encouraged the refugees to eat warm food. They also taught them how to construct dish racks, but they were overwhelmed by the influx.44 It remained difficult for the government and non-governmental organisations to provide most of the basic necessities in the camp, especially shelter and sanitation. 43 Interview: Author with Ingwani Madomba, 24 September 2020. 44 Interview: Author with Evelyn Manyise, 53 years, Zimbabwean married to a Mozambican Former Chambuta Refugee, Muloveli village, Machindu, Zimbabwe, 13 October 2020. 45 Interview: Author with Cephas Chambuta, 20 September 2020. 46 Interview: Author with Cephas Chambuta, 20 September 2020. 47 Interview: Author with Phineas Chitsange, 48 years old, Velemu Village, Zimbabwe, 23 October 2020. 48 Interview: Author with James Chitsange, 1 October 2020. 49 Interview: Author with Lucas Velemu, 70 years old, Traditional healer/ Spirit medium, Changoma Village, Zimbabwe, 19 October 2020. 50 Interview: Author with John Dzingai, 26 September 2020. 51 FG McNeill, Aids, politics, and music in South Africa (Cambridge: Cambridge University Press, 2011). 52 A Ashforth, “An epidemic of witchcraft? The implications of aids for the post-Apartheid state”, African Studies 61 (1), 2002. 4. UNMAKING BONDS: POOR SANITATION, DISEASE OUTBREAKS AS A CURSE The influx overwhelmed the available resources. In addition, the multi-ethnic composition of the refugees meant that they were different people coming from different areas with different understandings of sanitation and hygiene. Amongst them were the Nyungwe, Barwe, Shangani, Sena, and Ndau. They were given soap but generally lacked sanitation practices.45 Some would not bathe or wash their clothes and dishes. Men particularly spent several days and nights going to beer parties within the camp and outside.46 These interviews highlight how difficult it was to achieve satisfactory sanitary conditions in the camp. Access to water, soap and other amenities did not guarantee the adoption of sanitary measures in the Chambuta camp. Personal observation shows that the pit toilets in the camp were too close to the houses. Therefore, coupled with overcrowding, occasional water shortages and incessant heat, disease transmission was difficult to contain. Velemu villagers argue that the “cholera” outbreak spread to their village due to close interaction with the refugees and killed many people, Mr Mawewe suffered from cholera, so did Rabhi, as well as the old man Mamombe. Peter and three more people got sick and were saved by the clinic. They had diarrhoea with blood. People in Base 1, particularly when they first settled, did not like using toilets. They used the bush, and as the camp expanded, there was a lot of dirt Manamere / Making and Unmaking Bonds Manamere / Making and Unmaking Bonds 45 and that led to diseases. The refugees were unhygienic; there was poor sanitation in the camp. We got to a point where we felt that it was better for the refugees to go back or be relocated elsewhere. In 1994 they were repatriated to Mozambique. We were happy that they went back, said Phineas Chitsange.47 and that led to diseases. The refugees were unhygienic; there was poor sanitation in the camp. We got to a point where we felt that it was better for the refugees to go back or be relocated elsewhere. In 1994 they were repatriated to Mozambique. We were happy that they went back, said Phineas Chitsange.47 Local residents believe that the food and beer they shared and exchanged with refugees were contaminated. They claim that initially, they thought this was a natural disease but later realised that this outbreak was a result of overcrowding. 4. UNMAKING BONDS: POOR SANITATION, DISEASE OUTBREAKS AS A CURSE “We even suspect that it came with some of the refugees who were coming from Tongogara and found favourable conditions in the overcrowded camp conditions and through interaction, the disease spread to local villages. Our people died as a result. We had never experienced diarrheal outbreaks before”, said James Chitsange of Velemu village.48 “Although the refugees were repatriated their cholera remained” Lucas Velemu lamented.49 In 1993, a diarrheal blood outbreak killed several people in Velemu and Makosiya villages, with between 16 to 20 people dying in Velemu alone. Official reports claim that it was enterohemorrhagic e.coli, a different type of diarrheal disease imported from South Africa by labour migrants.50 Although there were no cases among refugees, Velemu villagers link it to the 1992 diarrheal disease outbreaks in the camp. p The second alternative explanation emanating from Velemu village is that the disease outbreak was a punishment from their ancestors, who were angry over the Zimbabwean government’s failure to consult the correct traditional leadership, the instant displacement of locals from the campsite without compensation and the destruction of ancestral shrines during camp establishment. These ideas are not unique to the Shangani of southeastern Zimbabwe. Fraser McNeill demonstrates how biomedical understandings of AIDS have either been rejected or incorporated into local understandings of health, illness and death by the Venda of South Africa.51 Adam Ashforth observes similar ideas on how AIDS is associated with witchcraft and is framed as a “sent disease” in South Africa.52 These alternative causal explanations of disease are informed by a religious conviction that, SJCH 47(2) \| December \| 2022 SJCH 47(2) \| December \| 2022 46 There exists an invisible world, distinct but not separate from the visible world, that is inhabited by spiritual beings with which they can communicate and which they perceive to have an influence on their daily lives. This ‘spirit idiom’ governs relations both of one person to another, or of one person to a community, but also of people to the land.53 Therefore, similar to other regions of the world, religion has a marked influence on authority and legitimacy in African societies and as Stephen Ellis and Gerri ter Haar argue, African epistemologies should be incorporated into our understanding of contemporary Africa.54 Spirituality particularly is an important framework for constructing meanings around the disease. 53 S Ellis and G Ter Haar, “Religion and politics: Taking African epistemologies seriously”, The Journal of Modern African Studies 45 (3), 2007, p. 387. 54 Ellis and Ter Haar, “Religion and politics”, p. 387. 55 F Martineau et al., “Epistemologies of Ebola: Reflections on the experiences of the Ebola response anthropology platform”, Anthropological Quarterly 90 (2), 2017. S Ellis and G Ter Haar, “Religion and politics: Taking African epistemologies seriously”, The Journal of Modern African Studies 45 (3), 2007, p. 387. 54 Ellis and Ter Haar, Religion and politics , p. 387. 55 F Martineau et al., “Epistemologies of Ebola: Reflections on the experiences of the Ebola response anthropology platform”, Anthropological Quarterly 90 (2), 2017. ( ), , p 54 Ellis and Ter Haar, “Religion and politics”, p. 387. 4. UNMAKING BONDS: POOR SANITATION, DISEASE OUTBREAKS AS A CURSE In this article, I argue that these perspectives are fundamental in determining the successful integration of refugees and hosts. I show how these alternative views, attitudes and perceptions have a long-term impact on relations and peacebuilding between the two groups. Despite the mutual relationship that characterised the interaction between the hosts and the refugees, there remained simmering unexpressed grievances among members of displaced families in Velemu village. Nearly three decades after the disease outbreak, the blame lingers long after the refugees left. Velemu villagers blame recent spates of drought on refugee presence and argue that even the dead should have been repatriated. They claim that their ancestors withheld the gift of rain because of the burial of refugees on their land. The anger and hate towards refugees that is revealed in these narratives developed after the disease outbreak, as interviews reveal that during the initial stages of camp set-up, the host community interacted positively with the refugees.l Reflecting on the Ebola interventions in West Africa, Fred Martineau et al. emphasise the centrality of local knowledge, of direct and immediate engagement with local actors and their diverse perspectives.55 Livison Chikutu, the current Velemu village head, asserts that the land upon which the refugee camp was built belongs to the Velemu/ Chitsange people, not Chambuta. Chambuta is under chief Masuamele; the Camp is under chief Chilonga. However, when government officials came for consultation, they ended up in Chambuta’s area of jurisdiction. They told people in Chambuta about the plans to set up a refugee camp. The Chambuta village head showed them a piece of land between Chambuta and Velemu villages. Although sparsely populated, the land was inhabited by ten Chitsange Manamere / Making and Unmaking Bonds 47 families, who were not consulted about the refugee camp and were forced out of the designated camp area. “The displaced families lost many of their possessions. They were removed immediately without prior notice so many of them lost livestock, their houses and farming areas”.56 The ten families include Dubula, Bhawa, Manyonga, Mahata, Dumazi, Gara, Ndendereka, Mhame, Chioko and Kunyarara. Families like Kunyarara, Dubula and Bhawa lost farms and had to look for other fields. The government did not compensate them. In contrast to displaced families, those from Chambuta village constituted the majority of the camp workers. 56 Interview: Author with Livison Chikutu, 4 October 2020. 57 Interview: Author with Livison Chikutu, 4 October 2020. 58 Interview: Author with Livison Chikutu, 4 October 2020. 59 Interview: Author with Livison Chikutu, 4 October 2020. 60 Interview: Author with Livison Chikutu, 4 October 2020. 61 Interview: Author with Livison Chikutu, 4 October 2020. 62 Interview: Author with James Chitsange, 1 October 2020. 63 Interview: Author with James Chitsange, 1 October 2020. 64 Interview: Author with James Chitsange, 1 October 2020. 4. UNMAKING BONDS: POOR SANITATION, DISEASE OUTBREAKS AS A CURSE During camp construction, when the organisations that were serving the camp recruited local people for camp construction, they employed from the Chambuta area, Former refugee camp staff, for instance Galela and Madomba, were from Chambuta and gave information to the surveyors and the organisations who came to assist the refugees. They were employed in siting, camp construction, the building of camp infrastructure, toilets, and registration of refugees.57 The Chambuta village head misinformed the government and its partners about that area because he was not the owner of that land.58 The government consulted the wrong people, people who did not only own the land but who were not going to be directly affected by the camp settlement. That is how the camp came to be known as Chambuta.59 Livison Chikutu argues that prior to camp establishment, the Velemu village was inaccessible, while Chambuta village is located along the main road. Therefore, government officials decided to consult the Chambuta instead of moving further to other villages. When they saw how bushy it was, they assumed that it was unoccupied, yet several families were settled there [...] Our forefathers settled there in 1963; that place was sacred; it had shrines. That is why it was left as a bushy area with very few settlements on its outskirts.60 Chikutu claims that the refugees experienced a few challenges when they were setting up the camp but did not know that it was because they had violated the place as a result of misinformation or lack of it. After the camp was set up, people got sick, there were disease outbreaks, and healers and diviners from Mozambique said the diseases and deaths were caused by angry ancestral spirits. Chief Masuamele was consulted, and he held a ritual ceremony, but 56 Interview: Author with Livison Chikutu, 4 October 2020. 57 Interview: Author with Livison Chikutu, 4 October 2020. 58 Interview: Author with Livison Chikutu, 4 October 2020. 59 Interview: Author with Livison Chikutu, 4 October 2020. 60 Interview: Author with Livison Chikutu, 4 October 2020. SJCH 47(2) \| December \| 2022 SJCH 47(2) \| December \| 2022 48 there was no change. The Mozambican traditional leaders said this place does not belong to Masuamele; it belongs to Chilonga. The people who are holding rituals are not the owners of this land, meaning Masuamele, who was not in any way connected to the ancestors. 4. UNMAKING BONDS: POOR SANITATION, DISEASE OUTBREAKS AS A CURSE To end the disease and deaths, the diviners recommended an ancestral appeasement ceremony led by Chilonga. Chief Chilonga came and held the ritual ceremony, and the situation improved. The ancestors were not happy over the name given to the camp. Names are very important in our culture; if you give a family name to a child who does not belong to your lineage, you would have given him spirits that do not belong to him, and things will go wrong for him. He will know no peace.61 Other Velemu villagers, for instance James Chitsange, insist that even the dead should have been repatriated.62 The blame remains long after the refugees returned to Mozambique. He claims that before the refugees arrived, there was enough grazing land, and they even had excess subsistence, but after 1992 there have been continued droughts, and pastures have dried up. Although locals identify the environmental destruction caused by refugees in the area, for instance the burning down of trees which also burnt grass and affected pastures, they argue that the recurrent droughts are not a result of climate change only. According to J Chitsange, Deforestation affected local sacred shrines, which are normally bushy areas. The refugees’ graves, their dead bodies are also a problem. The spirits of the dead refugees cause continued periods of drought because they were people from different places, they are too mixed, and it is difficult to appease them or to repatriate their spirits. We cannot go to their gravesite because we did not know these people. Some of the graves are now open, and skeletons exposed. Those graves are a big problem.63 Moreover, among the refugees were the Barwe, an ethnic group that hosts from both villages believed to be cannibals. The Barwe embalmed their dead, and the locals believe they ate them.64 Embalming a dead body is not acceptable among the Shangani and is associated with the ancestors’ punishment of withholding rain. From the above perspectives, it is clear that the Velemu villagers strongly subscribe to the idea that the disease outbreak was a curse from their ancestors over the loss of land and the presence of cannibals/witches in their village. The plausibility of this perspective is unimportant. 4. UNMAKING BONDS: POOR SANITATION, DISEASE OUTBREAKS AS A CURSE It could as well be possible that the disease outbreak provided the Velemu villagers with the 49 Manamere / Making and Unmaking Bonds opportunity to bring to the fore their simmering grievances on displacement and loss of land and an explanation drawn from their ancestral relationship with the land seemed appealing. Nonetheless, it is important to acknowledge that these alternative explanations of the disease have the power to influence long-term negative attitudes towards refugees. 5. CONCLUSION Drawing from interviews, I examined the diverse attitudes and perspectives of the Chambuta and Velemu villagers on the disease outbreak that killed nearly 200 Mozambicans at Chambuta refugee camp in southeastern Zimbabwe in August 1992. Often, local voices are overshadowed by official perspectives from government officials and non-governmental organisations. In cases where hosts have been included in the literature, they are presented as a homogenous group with similar opinions and attitudes towards refugees. In this article, I emphasised the pertinent need to consider the diverse voices of hosts in humanitarianism studies and policy formulations. Engaging local voices allow us to disaggregate host communities and understand variations in the relationships between hosts and those in need of assistance, as well as formulate context-specific strategies that encourage social cohesion. Governments and organisations involved in refugee settlement adopt several measures to ensure peaceful coexistence between the refugees and their host communities, particularly settling refugees in “uninhabited” or sparsely populated areas as well as ensuring that both populations benefit from the different services available. The provision of shared facilities in healthcare and education, particularly as in the Chambuta case, has encouraged mutual relationships between the refugees and their hosts, who, in most cases, had no access to quality education and healthcare prior to the arrival of refugees. However, the occurrence of epidemic disease in camps often disrupts this peaceful coexistence and leads to “othering”. Stigma, blame and hate take centre stage as host communities perceive refugees as bringing diseases and other misfortunes to them. By paying attention to how the host community understand disease outbreaks in the Chambuta refugee camp, this article shows that although the community appeared undifferentiated and established mutual relations with the refugees before the disease outbreak, this was far from the case. Prior to camp establishment, the host community was differentiated along clan lines, each clan having access and jurisdiction over their own lands which were divided by a stream. The establishment of the refugee camp widened these divisions when the Chambuta village head allocated land that belonged to Velemu village to the camp without consultation. The Velemu village lost a SJCH 47(2) \| December \| 2022 SJCH 47(2) \| December \| 2022 50 piece of their land which they used as grazing land, and ancestral shrines, whilst Chambuta villagers benefited from jobs and recognition. 5. CONCLUSION Consequently, the host community was further divided along socio-economic lines to the extent that when the disease outbreak occurred, those from Velemu village largely blamed refugee presence on their land. This highlights the importance of recognising the politics of host communities. Government authorities treated the local community as one harmonious unit without internal divisions or political cleavages. Receiving governments and local authorities should widely consult local traditional leadership to ensure that refugee camps are not established on disputed land. Whilst it is difficult to find ‘”uninhabited land”, it is important to understand how land is allocated and used at a local level before embarking on large-scale projects leading to internal displacements. The internal displacement of locals, no matter how few the affected people are, encourages discontent among refugee hosts. In the Chambuta case, displaced families argue that they did not have time to communicate with their ancestors, and their link with the ancestors was broken as shrines and ancestral graves were destroyed, and this led to disease outbreaks and low rainfall. Moreover, the spiritual explanations of disease that the Velemu villagers subscribe to point to the need for governments and partnering organisations to identify strategies that deal with alternative explanations of biomedical causes of disease. Such strategies may include public health awareness programmes among host communities before and after refugee settlement. Ensuring that hosts access clinics in refugee camps, as happened at Chambuta, is important but limits health interventions to curative medicine, yet there is a need to educate local communities about the possibilities of communicable disease epidemics among refugees. This may allay several etiological explanations, blame and stigma that threaten peacebuilding between the two communities. Wider consultation with host communities as well as public health and climate change awareness programmes, are important in ensuring a peaceful co-existence between refugees and their host communities.
https://openalex.org/W2781927229	https://europepmc.org/articles/pmc5871646?pdf=render	English	null	Effects of the Positive Allosteric Modulator of Metabotropic Glutamate Receptor 5, VU-29, on Impairment of Novel Object Recognition Induced by Acute Ethanol and Ethanol Withdrawal in Rats	Neurotoxicity research/Neurotoxicity resarch	2,018	cc-by	13,026	* Jolanta H. Kotlinska jolanta.kotlinska@umlub.pl Abstract Glutamate is essential for learning and memory processes, and acute and chronic exposures to ethanol (or protracted abstinence) alter glutamatergic transmission. In the current study, we investigated the effects of VU-29, positive allosteric modulator of metabotropic glutamate 5 (mGlu5) receptor, on the acute ethanol- and ethanol withdrawal-induced impairment of novel object recognition (NOR) task in rats. The influence of VU-29 (30 mg/kg) on memory retrieval was measured (a) at 4-h delay after acute ethanol administration, as well as (b) after acute withdrawal (24 and 48 h) of repeated (2.0 g/kg, once daily for 7 days) ethanol administration. Additionally, the effects of VU-29 on expression of mGlu5 and mGlu2 receptor proteins in the hippocampus, prefrontal cortex, and striatum were determined 48 h after ethanol withdrawal. Our results indicated that VU-29, given before acute ethanol administration, prevented the ethanol-induced impairments in spatial memory retrieval. Furthermore, VU-29 given before the testing session on the first day of abstinence facilitated NOR performance in ethanol-withdrawn rats at 4- and 24-h delay after administration. Our ELISA results show that VU-29 normalized ethanol withdrawal induced increase in expression of mGlu5 receptor protein in the hippocampus, prefrontal cortex, and striatum, as well as expression of mGlu2 receptor protein in the hippocampus. Thus, results from our study indicate that positive modulation of mGlu5 receptor prevented and reversed ethanol-induced memory impairment. Moreover, mGlu5 (hippocampus, prefrontal cortex, and striatum) and mGlu2 (hippocampus) receptors play an important role in the ethanol-induced recognition memory impairment induced by ethanol withdrawal. Keywords Ethanol . VU-29 . mGlu5 receptor . Novel object recognition Effects of the Positive Allosteric Modulator of Metabotropic Glutamate Receptor 5, VU-29, on Impairment of Novel Object Recognition Induced by Acute Ethanol and Ethanol Withdrawal in Rats Marta Marszalek-Grabska1 & Ewa Gibula-Bruzda1 & Anna Bodzon-Kulakowska2 & Piotr Suder2 & Kinga Gawel1,3 & Joanna Filarowska1 & Joanna Listos1 & Wojciech Danysz4 & Jolanta H. Kotlinska1 Marta Marszalek-Grabska1 & Ewa Gibula-Bruzda1 & Anna Bodzon-Kulakowska2 & Piotr Suder2 & Kinga Gawel1,3 & Joanna Filarowska1 & Joanna Listos1 & Wojciech Danysz4 & Jolanta H. Kotlinska1 Received: 8 May 2017 /Revised: 15 December 2017 /Accepted: 18 December 2017 /Published online: 2 January 2018 # The Author(s) 2017. This article is an open access publication Neurotoxicity Research (2018) 33:607–620 https://doi.org/10.1007/s12640-017-9857-z Neurotoxicity Research (2018) 33:607–620 https://doi.org/10.1007/s12640-017-9857-z ORIGINAL ARTICLE 1 Department of Pharmacology and Pharmacodynamics, Medical University, Chodzki 4A, 20-093 Lublin, Poland 2 Department of Biochemistry and Neurobiology, AGH University of Science and Technology, Krakow, Poland 4 Merz Pharmaceuticals GmbH, Frankfurt am Main, Germany jolanta.kotlinska@umlub.pl 1 Department of Pharmacology and Pharmacodynamics, Medical University, Chodzki 4A, 20-093 Lublin, Poland 2 Department of Biochemistry and Neurobiology, AGH University of Science and Technology, Krakow, Poland 3 Department of Experimental and Clinical Pharmacology, Medical University, Lublin, Poland 4 Merz Pharmaceuticals GmbH, Frankfurt am Main, Germany Introduction et al. 2004; Schweizer et al. 2006; Friedman et al. 2011) lead to a broad spectrum of cognitive deficits in humans. In animal studies, ethanol preferentially impairs a variety of hippocampal-dependent spatial memory tasks (Givens et al. 2000; Miki et al. 2000; White and Best 2000; Matthews and Morrow 2000; Chin et al. 2011; Gawel et al. 2016); although, it has also an impairing effect on spatial and non-spatial working memory (Santin et al. 2000; Ryabinin et al. 2002; Silvers et al. 2003; Berry and Matthews 2004) and on object recognition tasks which require non-spatial recognition memory (Brooks et al. 2002; Garcia-Moreno et al. 2002; Garcia-Moreno and Cimadevilla 2012; Yu et al. 2013; Zhao et al. 2013). Acute ethanol intoxication (Ryabinin 1998; White et al. 2000) and long-term ethanol consumption (Meyerhoff Recognition memory is the ability to recognize previously encountered events, objects, or people, and this memory de- pends on the integrity of the medial temporal lobe regions Neurotox Res (2018) 33:607–620 608 (Squire et al. 2007), which include the hippocampus and perirhinal cortex (Squire and Zola-Morgan 1991). As demon- strated before (Crews et al. 2004), these brain structures are very susceptible to ethanol-induced neurodegeneration. The perirhinal cortex is a crucial region for recognition memory performance (Brown et al. 1987; Zhu et al. 1995; Brown and Xiang 1998), but the role of the hippocampus in object recog- nition is still under debate (Mumby et al. 2002; Langston and Wood 2010; Clark et al. 2000; Broadbent et al. 2009; Cohen et al. 2013; Yi et al. 2016). Also, it has been indicated that some forms of recognition memory recruit the medial prefron- tal cortex (Bekinschtein and Weisstaub 2014; Morici et al. 2015) that is associated with successful retrieval of the object recognition task (Bekinschtein and Weisstaub 2014; Morici et al. 2015). retrieval such as the prefrontal cortex (Rugg et al. 2003; Bekinschtein and Weisstaub 2014; Morici et al. 2015), stria- tum (Scimeca and Badre 2012), and hippocampus (Clark et al. 2000; Broadbent et al. 2009; Cohen et al. 2013; Yi et al. 2016). Furthermore, we evaluated the influence of VU-29 pre-treat- ment on ethanol-induced changes in mGlu5 and mGlu2 recep- tor protein expressions in these brain structures after 2 days of withdrawal from repeated ethanol treatment. Also, we per- formed a study to exclude the effects of acute ethanol intoxi- cation (locomotor effects) and ethanol withdrawal (anxiety) on the obtained results. Materials and Methods It has been recently reported that metabotropic glutamate subtype 5 (mGlu5) receptor-dependent signaling, preferential- ly in limbic structures, is involved in cognition (e.g. memory and learning), novelty seeking behavior, and compulsivity. These factors are core personality traits increasing the suscep- tibility to drug addiction (Leurquin-Sterk et al. 2015). Indeed, it has been indicated the mGlu5 receptors are involved in nicotine addiction in humans (Akkus et al. 2012, 2016). Furthermore, preclinical researches suggest the important role of this receptor in ethanol drinking (Cozzoli et al. 2009, 2012; Besheer et al. 2010; Sinclair et al. 2012) and in the memory formation responsible for the chronic relapsing nature of alco- hol abuse (Obara et al. 2009). Therefore, this receptor is a candidate target for the treatment of addictive disorders (Mihov and Hasler 2016). Introduction The data we obtain could, therefore, have therapeutic implications for alcohol addiction. Animals Male naïve Wistar rats (HZL, Warsaw, Poland) weighing 200– 250 g at the initiation of the experimental procedure (age of 8– 9 weeks) were used in our experiments. A total of 136 animals (8–10/group) were used in our study. The rats were housed four per cage with a standard diet (Agropol, Motycz, Poland) and water ad libitum. They were kept under 12-h light/dark cycle and controlled temperature (22 ± 2 °C). The rats were adapted to the laboratory conditions for at least 1 week before the experiments and were handled twice daily for 5 days be- fore the beginning of the tests. All behavioral studies were performed between 9:00 a.m. and 5:00 p.m. The experimental protocols and housing conditions were performed according to the National Institute of Health Guidelines for the Care and Use of Laboratory Animals, the European Community Council Directive of November 2010 for Care and Use of Laboratory Animals (Directive 2010/63/EU), and were ap- proved by the Local Ethics Committee. In the present study, we examined the influence of the mGlu5 receptor-positive allosteric modulator, VU-29, on ethanol-induced impairment of recognition, in rats, using the novel object recognition (NOR) test. Thus, in experiment 1, we examined whether VU-29 given before the test was able to prevent the ethanol-induced impairment of the recognition memory. Thus, we first selected the ethanol dose that induced memory, but not motor impairment. Next, we used this dose in combination with VU-29 to indicate the influence of this com- pound on memory retrieval impaired by acute ethanol administration. Elevated Plus Maze Test The NOR test was based on methods described previously (Reichel et al. 2011; Gomez et al. 2013) with modification of testing time (Davis et al. 2010). Shortly, the animals were handled twice daily for the week preceding testing. The test was carried out in the same Plexiglas square box (63 cm long × 44.5 cm high × 44 cm wide) in a dimly lit (30 ± 5 lx) testing room. All rats were given two 3-min sessions (a train- ing session and a testing session). During the training session (the first session, T1A, familiarization phase), the rats ex- plored two identical objects (A1 and A2). After a 4 h interval, during the testing session (T1B) (short-term memory), rats were allowed to explore one familiar object (A2) from the first session and a novel object (B) that was different enough in shape to be distinguished (Davis et al. 2010; Gomez et al. 2013). The objects were made of glass, plastic, and wood and were chosen after determining, in preliminary experi- ments with other animals, that they were equally preferred. Shapes, colors, and textures were different among these ob- jects. Exploration of the objects was defined as sniffing or touching with the nose towards the object at a distance of less than 1 cm; however, sitting on the object was not considered exploration (Clark et al. 2000). After 24-h delay (long-term memory), the same rats underwent the second memory test without further drug treatment (Davis et al. 2010; Gomez et al. 2013; Reichel et al. 2011). In each test session, the rats were placed back in the box with one familiar and one novel object (C) and allowed to explore freely for a total of 3 min. Object placement was counterbalanced within each group in order to avoid any bias due to a preference that rats may have for a given object or its position in the box. The box and the objects were cleaned with 10% ethanol solution between each trial to remove traces of odor. A discrimination index (DI) was calculated, this being the ability to discriminate the novel from the familiar object: (novel object (s)/novel object (s) + familiar The elevated plus maze test (EPM test) was designed to test general anxiety levels in animal models (Pellow et al. 1985). The EPM apparatus was made of wood and positioned on a height of 50 cm above the floor. Elevated Plus Maze Test Two opposite arms were open (50 × 10 cm), and the other two were walled-off (50 × 10 × 25 cm). The experiment was carried out in a quiet room with constant light of 100 lx. Rats were tested on the EPM 24 h after the last ethanol administration (2.0 g/kg, 20% w/v, i.g., 1 for 7 days). The EPM experiment was performed by placing the rat in the center of the plus maze facing an open arm, after which the number of entries and time spent in the open arms were recorded for a period of 5 min. An Barm entry^ was recorded when the rat entered the arm with all four paws into the arm. The open arms activity was quantified as follows: (A) the number of entries into the open arms as a percentage of the total number of entries into both open and closed arms and (B) the time spent in the open arms as a percent of the total time spent on exploring both the open and closed arms. Locomotor activity of rats (C) was measured as the total number of entries into the closed and open arms of the plus-maze apparatus. The EPM test was conducted 24 h after the last repeated ethanol administration—not only to test for anxiety, but also to test for any possible withdrawal effects of alcohol. Published data suggest that the NOR task conducted immediately after the EPM had no effect on rat performance (Gomez et al. 2013). Locomotor Activity Test To exclude locomotor activity influence on NOR results, the locomotor activity of individual rats was recorded using a photocell apparatus (Porfex, Bialystok, Poland). The animals were individually placed in Plexiglas boxes (square cages, 60 cm each side), in a sound-attenuated experimental room, under moderate illumination (5 lx). Ambulatory activity (dis- tance traveled) was measured for 15 min by two rows of the infrared light-sensitive photocells located along the long axis, 45 and 100 mm above the floor. Locomotor activity test was conducted after acute ethanol (1.5, 1.75, 2.0 g/kg, i.p.) administration. Drugs This compound is a highly selective positive allosteric modulator of mGlu5 over mGlu1 and mGlu2 receptors (de Paulis et al. 2006) with promoting effect on hippocampal synaptic plasticity (Ayala et al. 2009). Drugs For acute injection, ethanol (95%, w/v) was diluted in saline (0.9% NaCl) to a concentration of 10% (w/v) and administered intraperitoneally (i.p.) 30 min prior to testing in three doses (1.5, 1.75, and 2.0 g/kg). This time of ethanol administration was selected (taking into account published data and our pre- liminary experiments) because peak blood ethanol expression following systemic injection of ethanol is obtained 20–45 min post injection, and doses of 1.5 and 2.0 g/kg ethanol corresponded to blood ethanol levels of ~ 125 mg% (Givens and Breese 1990) and of ~ 205 mg% (Devenport et al. 1989), respectively. For repeated administration, ethanol (95%, w/v) was diluted in saline to a concentration of 20% (w/v) and administered intragastrically (i.g.) (preferred route of chronic drug administration) at the dose of 2.0 g/kg (Gomez et al. 2013). Previous study has shown that this ethanol dose In a second experiment, we evaluated the influence of VU- 29 on ethanol-induced memory retrieval impairment during the first 24- and 48-h of ethanol withdrawal. Group II mGlu (mGlu2/3) receptors in combination with the mGlu5 receptor and the N-methyl-D-aspartate (NMDA) receptor are important for recognition memory (Cho et al. 2000), and among the group II mGlu receptors, the mGlu2 receptor particularly plays an essential role in ethanol dependence and relapse (Meinhardt et al. 2013). Thus, we assessed the effect of etha- nol withdrawal on potential changes in mGlu (mGlu5 and mGlu2) receptor protein expression after day 2 and day 7 of ethanol abstinence in brain areas important for memory Neurotox Res (2018) 33:607–620 609 object (s) × 100%). Thus, an index > 50% indicates novel ob- ject preference, < 50%—familiar object preference, and 50%—no preference (Hammond et al. 2004). corresponds to blood ethanol levels of ~ 160 mg% (Gomez et al. 2013). Control rats received saline instead of ethanol. All rats were gavaged with ethanol or saline at the same time every day (8.00). For both routes of ethanol administration (i.p. and i.g.), injection volumes were 1 ml per 200 g body weight. The mGlu5 receptor-positive allosteric modulator N-(1,3-diphenyl-1H-pyrazolo-5-yl)-4-nitrobenzamide (VU- 29) (Chen et al. 2008) (synthesized at Merz Pharmaceuticals GmbH, Frankfurt am Main, Germany) was dissolved in a vehicle consisting of 10% Tween-80 (Sigma) in saline and given at the dose of 30 mg/kg, i.p., in a value of 1 ml/kg, 90 min before test (I. Belozertseva, personal communication, as well as our preliminary study). Brain Dissection and Tissue Preparation A separate group of rats was sacrificed by decapitation on day 2 or 7 of ethanol abstinence, and the brains were removed. The prefrontal cortex, hippocampus, and striatum were immediate- ly dissected, and all samples were stored in a liquid nitrogen tissue bank (LS750, Taylor-Wharton, USA) until analyses. 610 Neurotox Res (2018) 33:607–620 of 1.75 g/kg), and a T1B session was conducted accord- ing to the procedure described above (see Fig. 1a). An observer naive to the experimental conditions manually scored behavior. Next, the samples were mixed with 500 μl of cold phosphate buffered saline and homogenized on ice using a mechanical homogenizer (BioGen PRO200, PRO Scientific, Oxford, USA). Tip speed was set to ca. 15,000 rpm for 5 s. The sam- ples were then frozen/defrozen twice to destroy remaining cell conglomerates and, subsequently, centrifuged (+ 4 °C, 15 min, 4500×g, centrifuge model 5804R, Eppendorf AG, Germany). To estimate protein content in supernatants, all samples were measured colorimetrically with the aid of the Bradford method. Experiment 2. The influence of VU-29 on NOR memory impaired by repeated ethanol administration in NOR test after 4- and 24-h delay: mGlu5 receptor and mGlu2 re- ceptor expressions in the cortex, hippocampus, and striatum The mGlu2 and mGlu5 receptor protein expressions were measured using ELISA kits: for mGlu2 receptor—kit number E02M0093 (receptor’s standard concentration range from 0.5 to 10 ng/ml) and for mGlu5 receptor—kit number E02M0097 (receptor’s standard concentration range from 0.5 to 10 ng/ ml), both from BlueGene Biotech Corp., Shanghai, China. Measurement was done using an ELISA spectrophotometric reader: SpectrostarNano (BMG Labtech, UK), according to the manufacturer’s protocols. After 5 days of habituation to the NOR apparatus, the rats (cohort 2) received ethanol (20% w/v) once daily at the dose of 2.0 g/kg, i.g., for 7 days (Gomez et al. 2013). Next, during the training session (T2A), the rats explored two identical objects for 3 min (day 1 abstinence), and the first memory testing session (T2B) was conducted after a 4-h delay, by allowing the rats to explore an object from the training session, as well as a novel object, for 3 min. After 24-h delay (day 2 absti- nence), the second memory testing session (T2C) was con- ducted (performed 4-h follow the first training session). In this, the rats were given the familiar object and a different novel object. Brain Dissection and Tissue Preparation VU-29 was injected at the dose of 30 mg/kg, i.p., 90 min prior to the first testing session (T2B), on the first day of ethanol abstinence. Subsequently, the groups of rats (N = 8–9) were rapidly decapitated and their brains were re- moved on the second day of abstinence (after T2C session) Experimental Procedure Experiment 1. The influence of acute ethanol administra- tion on NOR memory after 4-h delay and locomotor ac- tivity in rats. The effect of VU-29 on ethanol-induced memory impairment After acclimation to the environment, rats (cohort 1) were habituated to the NOR apparatus for 5 days (Gomez et al. 2013). On day 1, rats were exposed to the testing apparatus for 5 min/session (no object). From days 2–5, once a day for 5 min per session/day, the animals were habituated to the testing apparatus which contained two selected objects (these objects were not used in the following test trials). Next, 24 h after the last session of habituation, the test session for examining object memory was given. This test was based on two 3- min sessions: a training session (T1A) and a testing ses- sion (T1B), separated by 4-h delay. Before the testing session, the rats received either acute ethanol administra- tion (10% w/v) at the dose of 1.5, 1.75, and 2.0 g/kg, i.p. or saline, 30 min prior the trial. Immediately after the NOR test, the influence of ethanol (1.5–2.0 g/kg, i.p.) on locomotor activity (locomotor activity cages) was assessed. This was done to exclude the influence of the ethanol doses on locomotor disturbances so as to exclude nonspecific effects in the memory task. On the basis of this experiment, the dose of ethanol of 1.75 g/kg was selected to determine the effect of mGlu5 receptor- positive allosteric modulator, VU-29, on memory retriev- al impaired by acute ethanol administration. Thus, the rats received VU-29 (30 mg/kg, i.p.), 90 min prior to the testing session (60 min prior to ethanol at the dose NOR II VU-29 NOR I T2C T2A T2A T2B DEC EPM EtOH Administration NOR Habituation D14 D13 D12 D6 D5 Cohort 2 D6 D5 EtOH/SAL VU-29 T1A T1B NOR NOR Habituation Cohort 1 D19 D12 D6 D5 Cohort 3 Acclimation EtOH Administration D1 D1 D1 DEC Fig. 1 Timeline for experiment 1 (cohort 1) and experiment 2 (cohorts 2 and 3). Each tick mark represents 1 day. Longer tick marks represent the beginning or ending of the experiment phase. D day, EtOH ethanol, SAL saline, LOC locomotor activity, EPM elevated plus-maze, NOR novel object recognition, DEC decapitation Cohort 1 NOR Habituation Fig. 1 Timeline for experiment 1 (cohort 1) and experiment 2 (cohorts 2 and 3). Each tick mark represents 1 day. Results In the NOR test during the training session (T1), the subject rats spent a similar period of time exploring two identical objects. Statistical analysis showed no significant difference in the exploration of the identical objects (A1 and A2) during this phase, in any of the treatment groups (data not shown). However, the DI was significantly larger than 50% in saline groups: (Fig. 2: t = 4.954, df = 9, P = 0.0003934856; Fig. 3: t = 7.325, df = 9, P = 2.222113e−05; Fig. 4a: t = 3.094, df = 9, P = 0.006423126; Fig. 4c: t = 2.185 df = 9, P = 0.02835527). This means that all saline groups successfully learned to dis- criminate the novel object. This means that all saline groups successfully learned to dis- criminate the novel object. Statistical Analysis The obtained results were analyzed using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego, California, USA. Statistical significance of the data was assessed by a one-way (one factor with four levels, corre- sponding to four groups shown in Fig. 2: saline and three (1.5, 1.75, and 2 g/kg) concentrations of ethanol) and two- way (two factors in four possible combinations as shown in Figs. 3 and 4: ethanol presence and VU-29 presence) analysis of variance (ANOVA). Post-hoc comparisons were carried out with the Tukey’s (for one-way ANOVA) or with the Bonferroni’s (for two-way ANOVA) test. The Student’s t test was used to analyze EPM data. The value of P < 0.05 was considered statistically significant. Data were reported as mean ± standard error of the mean (SEM) of 8–10 subjects per group. 0 0 5 10 15 20 25 30 Total exploration time [s] b SALINE EtOH (1.5 g/kg) EtOH (1.75 g/kg) EtOH (2 g/kg) 0 5 10 15 20 25 30 * Locomotor activity [m] c Experimental Procedure Longer tick marks represent the beginning or ending of the experiment phase. D day, EtOH ethanol, SAL saline, LOC locomotor activity, EPM elevated plus-maze, NOR novel object recognition, DEC decapitation Fig. 1 Timeline for experiment 1 (cohort 1) and experiment 2 (cohorts 2 and 3). Each tick mark represents 1 day. Longer tick marks represent the beginning or ending of the experiment phase. D day, EtOH ethanol, SAL saline, LOC locomotor activity, EPM elevated plus-maze, NOR novel object recognition, DEC decapitation Neurotox Res (2018) 33:607–620 611 0 10 20 30 40 50 60 70 * * acute EtOH * Discrimination index [%] 0 5 10 15 20 25 30 Total exploration time [s] SALINE EtOH (1.5 g/kg) EtOH (1.75 g/kg) EtOH (2 g/kg) 0 5 10 15 20 25 30 * Locomotor activity [m] a b c Fig. 2 The influence of acute ethanol administration on a memory performance (expressed as a discrimination index) in the NOR test in rats. b Total exploration time displayed by different groups of rats in the NOR test, in T1B. c Locomotor activity. Ethanol was administered at the dose of 1.5, 1.75, and 2.0 g/kg (10%, w/v, i.p.) 30 min before T2B. Each column represents the mean ± SEM (N = 9–10). P < 0.001 vs. vehicle group 611 0 10 20 30 40 50 60 70 * acute EtOH * Discrimination index [%] 0 5 10 15 20 25 30 Total exploration time [s] a b 0 10 20 30 40 50 60 70 * * acute EtOH * Discrimination index [%] a acute EtOH (see Fig. 1b). Furthermore, two additional groups (N = 8) of rats (cohort 3) that received ethanol (20% w/v) once daily at the dose of 2.0 g/kg, i.g., or saline for 7 days were decapitated on the seventh day of abstinence (see Fig. 1c). After this, the expressions of mGlu5 receptor and mGlu2 receptor in the cortex, hippocampus, and striatum were established. a The Influence of VU-29 on the NOR Memory Deficits in Rats Induced by Acute Ethanol Administration, After a 4-h Delay Figure 3 shows the effect of VU-29 on DI impaired by acute ethanol (1.75 g/kg) administration, after a 4-h reten- tion delay. Two-way ANOVA revealed the first factor (VU-29 vs. its absence) as statistically significant: (F(1,34) = 40.38, P < 0.0001; N = 9–10/group) and the second factor (ethanol vs. its absence) (F(1,34) = 55.56, P < 0.0001), also with significant interaction between these factors (F(1,34) = 3.29, P = 0.078). Post-hoc test showed that acute ethanol administration (1.75 g/kg) 30 min before the testing session (T1B) decreased DI, when compared to the saline-treated group (P < 0.001). Notably, VU-29 (30 mg/kg, i.p.) pre-treatment 60 min be- fore ethanol decreased this deficit, as the VU-29/ethanol group showed a significantly increased DI (P < 0.01) when compared to ethanol group, as did the VU-29/saline group (P < 0.01) (Fig. 3a). However, DI in the VU-29/ ethanol group was significantly (P < 0.0001) lower than those of the control (saline) group. Analysis of the total exploration times during T1B did not show a statistically significant interaction between pre-treatment (F(1,34) = 0.68, P = 0.416), treatment (F(1,34) = 6.34, P = 0.017), or pre-treatment × treatment interaction (F(1,34) = 0.60, P = 0.445) (Fig. 3b). In addition, no differences in locomotor activity was observed between treated animals (pre-treat- ment: F(1,34) = 0.07, P = 0.792; treatment: F(1,34) = 1.37, P = 0.250; pretreatment × treatment interaction: F(1,34) = 0.24, P = 0.628) (Fig. 3c). 0 0 5 10 15 20 25 30 Total exploration time [s] b VEHICLE EtOH VU-29 + EtOH VU-29 0 5 10 15 20 25 30 Locomotor activity [m] c The Influence of Acute Ethanol Administration on NOR Memory After a 4-h Delay, in Rats 2c); there- fore, the dose of 1.75 g/kg of ethanol was chosen for future study. The Influence of Acute Ethanol Administration on NOR Memory After a 4-h Delay, in Rats Fig. 2 The influence of acute ethanol administration on a memory performance (expressed as a discrimination index) in the NOR test in rats. b Total exploration time displayed by different groups of rats in the NOR test, in T1B. c Locomotor activity. Ethanol was administered at the dose of 1.5, 1.75, and 2.0 g/kg (10%, w/v, i.p.) 30 min before T2B. Each column represents the mean ± SEM (N = 9–10). *P < 0.001 vs. vehicle group Figure 2 shows the effect of acute ethanol (1.5, 1.75, and 2.0 g/kg) administration before T1B on DI after a 4-h retention delay. One-way ANOVA revealed a significant effect (F(3,35) = 26.95, P < 0.001; N = 9–10/group) (Fig. 2a). Post- hoc test showed that acute ethanol administration at all used doses, 30 min before the testing session (T1B) impaired novel object recognition (P < 0.001), as revealed by significantly lower DI in ethanol groups relative to the saline group (Fig. 2a). Analysis of the total exploration times during T1B session did not show a statistically significant differences between groups (F(3,35) = 2.512, P = 0.074) (Fig. 2b). However, one- way ANOVA revealed significant differences in the locomo- tor activity (F(3,35) = 9.349, P < 0.001) between group of rats. Post-hoc test indicated that acute ethanol administration 0 10 20 30 40 50 60 70 ## * acute EtOH Discrimination index [%] 0 5 10 15 20 25 30 Total exploration time [s] VEHICLE EtOH VU-29 + EtOH VU-29 0 5 10 15 20 25 30 Locomotor activity [m] a b c Fig. 3 The influence of VU-29 on (a) memory performance (expressed as a discrimination index) induced by acute ethanol administration in the NOR test in rats. (b) Total exploration time displayed by different groups of rats in the NOR test, in T2B. (c) Locomotor activity. Ethanol was administered at the dose of 1.75 g/kg (10%, w/v, i.p.) 30 min before T2B and VU-29 was injected at the dose of 30 mg/kg, i.p., 90 min before T2B. Each column represents the mean ± SEM (N = 9–10). P < 0.01, *P < 0.001 vs. vehicle group, ##P < 0.01 vs. ethanol-treated group 612 Neurotox Res (2018) 33:607–620 612 0 10 20 30 40 50 60 70 ## * acute EtOH Discrimination index [%] a indicated only trend towards decreased locomotor activity compared to that of the saline-treated group (Fig. The Influence of VU-29 on the NOR Memory Deficits During Ethanol Abstinence in Rats, After a 4- and a 24-h Delay Ethanol was administered at the dose of 2.0 g/kg (20% w/v, i.g.) for 7 days. VU-29 was injected at the dose of 30 mg/kg, i.p., 90 min before T2B on the first day (24 h) of ethanol withdrawal. Each col- umn represents the mean ± SEM (N = 8–9). P < 0.01, *P < 0.001 vs. vehicle group, ##P < 0.01 vs. ethanol-withdrawal group 48 h EtOH withdrawal (24-h delay) 24 h EtOH withdrawal (4-h delay) VEHICLE VEHICLE VU-29 VU-29 0 5 10 15 20 25 30 Total exploration time [s] VEHICLE VEHICLE VU-29 VU-29 0 5 10 15 20 25 30 Total exploration time [s] b d s. vehicle group, 01 vs. ethanol-withdrawal as observed 48 h after cessation of ethanol treatment (24-h delay) (Fig. 4c). In this set of experiments, analysis of the total exploration times during T2C did not show a statis- tically significant interaction between pre-treatment (F(1,31) = 0.01, P = 0.934), treatment (F(1,31) = 0.00, P = 0.994), or pre-treatment × treatment interaction (F(1,31) = 0.05, P = 0.817) (Fig. 4d). 4a) but only exhibited tendency to increase the DI (P > 0.05) in saline-treated rats. Furthermore, the DI in the VU-29/ethanol group was not significantly different from the saline group (P = 0.0819). Also, analysis of the total exploration times during T2B did not show a statis- tically significant interaction between pre-treatment (F(1,31) = 1.25, P = 0.271), treatment (F(1,31) = 0.01, P = 0.908), or pre-treatment × treatment interaction (F(1,31) = 0.06, P = 0.812) (Fig. 4b). The Influence of VU-29 on the NOR Memory Deficits During Ethanol Abstinence in Rats, After a 4- and a 24-h Delay In this set of experiments, analysis of the total exploration times during T2C did not show a statis- tically significant interaction between pre-treatment (F(1,31) = 0.01, P = 0.934), treatment (F(1,31) = 0.00, P = 0.994), or pre-treatment × treatment interaction (F(1,31) = 0.05, P = 0.817) (Fig. 4d). Influence of Repeated Ethanol Administration on Anxiety-Like Behavior S d ’ i di d h h i ifi 0 10 20 30 40 50 60 70 ## 24 h EtOH withdrawal (4-h delay) * SALINE EtOH Discrimination index [%] 0 10 20 30 40 50 60 70 48 h EtOH withdrawal (24-h delay) ## SALINE EtOH Discrimination index [%] VEHICLE VEHICLE VU-29 VU-29 0 5 10 15 20 25 30 Total exploration time [s] VEHICLE VEHICLE VU-29 VU-29 0 5 10 15 20 25 30 Total exploration time [s] a c b d Fig. 4 The influence of VU-29 on memory performance (expressed as a discrimination index) in- duced by repeated ethanol ad- ministration at (a) 24 and (c) 48 h post-treatment in the NOR test in rats. Total exploration time displayed by different groups of rats in the NOR test (b, d). Ethanol was administered at the dose of 2.0 g/kg (20% w/v, i.g.) for 7 days. VU-29 was injected at the dose of 30 mg/kg, i.p., 90 min before T2B on the first day (24 h) of ethanol withdrawal. Each col- umn represents the mean ± SEM (N = 8–9). P < 0.01, *P < 0.001 vs. vehicle group, ##P < 0.01 vs. ethanol-withdrawal group Neurotox Res (2018) 33:607–620 613 Neurotox Res (2018) 33:607–620 613 0 10 20 30 40 50 60 70 ## 24 h EtOH withdrawal (4-h delay) * SALINE EtOH Discrimination index [%] 0 10 20 30 40 50 60 70 48 h EtOH withdrawal (24-h delay) ## SALINE EtOH Discrimination index [%] VEHICLE VEHICLE VU-29 VU-29 0 5 10 15 20 25 30 Total exploration time [s] VEHICLE VEHICLE VU-29 VU-29 0 5 10 15 20 25 30 Total exploration time [s] a c b d Fig. 4 The influence of VU-29 on memory performance (expressed as a discrimination index) in- duced by repeated ethanol ad- ministration at (a) 24 and (c) 48 h post-treatment in the NOR test in rats. Total exploration time displayed by different groups of rats in the NOR test (b, d). The Influence of VU-29 on the NOR Memory Deficits During Ethanol Abstinence in Rats, After a 4- and a 24-h Delay Ethanol was administered at the dose of 2.0 g/kg (20% w/v, i.g.) for 7 days. VU-29 was injected at the dose of 30 mg/kg, i.p., 90 min before T2B on the first day (24 h) of ethanol withdrawal. Each col- umn represents the mean ± SEM (N = 8–9). P < 0.01, *P < 0.001 vs. vehicle group, ##P < 0.01 vs. ethanol-withdrawal group 0 10 20 30 40 50 60 70 ## 24 h EtOH withdrawal (4-h delay) * SALINE EtOH Discrimination index [%] 0 10 20 30 40 50 60 70 48 h EtOH withdrawal (24-h delay) ## SALINE EtOH Discrimination index [%] VEHICLE VEHICLE VU-29 VU-29 0 5 10 15 20 25 30 Total exploration time [s] VEHICLE VEHICLE VU-29 VU-29 0 5 10 15 20 25 30 Total exploration time [s] a c b d Fig. 4 The influence of VU-29 on memory performance (expressed as a discrimination index) in- duced by repeated ethanol ad- ministration at (a) 24 and (c) 48 h post-treatment in the NOR test in rats. Total exploration time displayed by different groups of rats in the NOR test (b, d). Ethanol was administered at the dose of 2.0 g/kg (20% w/v, i.g.) for 7 days. VU-29 was injected at the dose of 30 mg/kg, i.p., 90 min before T2B on the first day (24 h) of ethanol withdrawal. Each col- umn represents the mean ± SEM (N = 8–9). P < 0.01, *P < 0.001 vs. vehicle group, ##P < 0.01 vs. ethanol-withdrawal group 0 10 20 30 40 50 60 70 ## 24 h EtOH withdrawal (4-h delay) * SALINE EtOH Discrimination index [%] 0 10 20 30 40 50 60 70 48 h EtOH withdrawal (24-h delay) ## SALINE EtOH Discrimination index [%] a c 0 10 20 30 40 50 60 70 48 h EtOH withdrawal (24-h delay) ## SALINE EtOH Discrimination index [%] c 0 10 20 30 40 50 60 70 ## 24 h EtOH withdrawal (4-h delay) * SALINE EtOH Discrimination index [%] a Fig. 4 The influence of VU-29 on memory performance (expressed as a discrimination index) in- duced by repeated ethanol ad- ministration at (a) 24 and (c) 48 h post-treatment in the NOR test in rats. Total exploration time displayed by different groups of rats in the NOR test (b, d). The Influence of VU-29 on the NOR Memory Deficits During Ethanol Abstinence in Rats, After a 4- and a 24-h Delay After 24 h of ethanol abstinence, during the testing ses- sion (T2B) (after a 4-h retention delay), two-way ANOVA indicated statistically significant differences between pre- treatment (ethanol effect) (F(1,31) = 25.25, P < 0.0001; N = 8–9/group), treatment (VU-29 effect) (F(1,31) = 8.38, P = 0.007), and pre-treatment × treatment interaction (F(1,31) = 4.75, P = 0.037). Post-hoc test showed that the ethanol withdrawal rats discriminated worse the novel ob- ject than did the saline-treated group (P < 0.001). Moreover, a single injection of VU-29 (30 mg/kg, i.p.) 90 min before the testing session (T2B) significantly in- creased the DI in ethanol-treated animals (P < 0.01) (Fig. Fig. 3 The influence of VU-29 on (a) memory performance (expressed as a discrimination index) induced by acute ethanol administration in the NOR test in rats. (b) Total exploration time displayed by different groups of rats in the NOR test, in T2B. (c) Locomotor activity. Ethanol was administered at the dose of 1.75 g/kg (10%, w/v, i.p.) 30 min before T2B and VU-29 was injected at the dose of 30 mg/kg, i.p., 90 min before T2B. Each column represents the mean ± SEM (N = 9–10). P < 0.01, *P < 0.001 vs. vehicle group, ##P < 0.01 vs. ethanol-treated group significantly decreased at the dose of 2.0 g/kg (P < 0.001), but at the doses of 1.75 and 1.5 g/kg (30 min before the test), it 4a) but only exhibited tendency to increase the DI (P > 0.05) in saline-treated rats. Furthermore, the DI in the VU-29/ethanol group was not significantly different from the saline group (P = 0.0819). Also, analysis of the total exploration times during T2B did not show a statis- tically significant interaction between pre-treatment (F(1,31) = 1.25, P = 0.271), treatment (F(1,31) = 0.01, P = 0.908), or pre-treatment × treatment interaction (F(1,31) = 0.06, P = 0.812) (Fig. 4b). After 48 h of ethanol abstinence, during the testing session (T2C) (after a 24-h retention delay), two-way ANOVA indicated statistically significant differences be- ( h l ff ) ( (1 31) 10 83 as observed 48 h after cessation of ethanol treatment (24-h delay) (Fig. 4c). Influence of Repeated Ethanol Administration on Anxiety-Like Behavior Furthermore, two-way ANOVA indicated statistically signifi- cant differences in the hippocampus between treatment (saline and ethanol) (F(1,32) = 80.94; P < 0.0001), time (2 and 7 days of ethanol withdrawal) (F(1,32) = 29.37; P < 0.0001), and ex- posing also significant interactions (F(1,32 = 38.63; P < 0.0001). Repeated ethanol administration induced a statis- tically significant increase in mGlu5 receptor protein expres- sion in the hippocampus at 2 days of ethanol withdrawal (P < 0.001), when compared to the saline-treated rats (Fig. 5b). Moreover, two-way ANOVA indicated statistically sig- nificant differences in the striatum time (2 and 7 days of eth- anol withdrawal) (F(1,32) = 20.55; P < 0.0001). Repeated eth- anol administration induced statistically significant increases in mGlu5 protein expression in the striatum after 2 days of ethanol withdrawal (P < 0.001), when compared to the saline- treated rats (Fig. 5c). 7 days withdrawal 7 days withdrawal 2 days withdrawal 0.0 0.1 0.2 0.3 0.4 0.5 2 days withdrawal 7 days withdrawal mGlu5 Striatum ### mGlu5 expression [mg/ml] c 7 days withdrawal Fig. 5 The effect of ethanol and VU-29 on mGlu5 levels in (a) the cortex, (b) hippocampus, and (c) striatum 2 and 7 days after last ethanol injection. Ethanol was administered at the dose of 2.0 g/kg (20% w/v, i.g.) for 7 days. VU-29 was injected at the dose of 30 mg/kg, i.p., 90 min before T2B on the first day (24 h) of ethanol withdrawal. Each column represents the mean ± SEM (N = 8–9). P < 0.01, P < 0.001 vs. vehicle group, ##P < 0.01, ###P < 0.001 vs. ethanol-withdrawal group Fig. 5 The effect of ethanol and VU-29 on mGlu5 levels in (a) the cortex, (b) hippocampus, and (c) striatum 2 and 7 days after last ethanol injection. Ethanol was administered at the dose of 2.0 g/kg (20% w/v, i.g.) for 7 days. VU-29 was injected at the dose of 30 mg/kg, i.p., 90 min before T2B on the first day (24 h) of ethanol withdrawal. Each column represents the mean ± SEM (N = 8–9). P < 0.01, P < 0.001 vs. vehicle group, ##P < 0.01, ###P < 0.001 vs. ethanol-withdrawal group Furthermore, one-way ANOVA indicated statistically sig- nificant effects of single VU-29 injection on mGlu5 receptor expression in the cortex (F(2,25) = 7.55; P = 0.0027) (Fig. 5a), hippocampus (F(2,25) = 49.08; P < 0.0001) (Fig. Influence of Repeated Ethanol Administration on Anxiety-Like Behavior vehicle group, ##P < 0.01, ###P < 0.001 vs. ethanol-withdrawal group epeated ethanol administration (2.0 g/kg, i.g., 1 ke behavior in the EPM test on the first day of ercent of entries into the open arms; (B) percent arms; (C) locomotor activity measured as a total pen arms entries. Each column represents the Vehicle EtOH 35.33 ± 9.255 40.75 ± 6.123 36.74 ± 6.006 42.85 ± 3.575 9.75 ± 0.796 9.50 ± 0.654 0.0 0.1 0.2 0.3 0.4 0.5 VEHICLE ETOH mGlu5 Cortex ETOH + VU-29 ## 2 days withdrawal 7 days withdrawal mGlu5 expression [mg/ml] a a Table 1 The effect of repeated ethanol administration (2.0 g/kg, i.g., 1 for 7 days) on anxiety-like behavior in the EPM test on the first day of ethanol withdrawal: (A) percent of entries into the open arms; (B) percent of time spent in the open arms; (C) locomotor activity measured as a total number of closed and open arms entries. Each column represents the mean ± SEM (N = 8) Vehicle EtOH % Time in open arms 35.33 ± 9.255 40.75 ± 6.123 % Entries in open arms 36.74 ± 6.006 42.85 ± 3.575 Number of total entries 9.75 ± 0.796 9.50 ± 0.654 Influence of Repeated Ethanol Administration on mGlu5 Receptor Protein Expression in the Cortex, Hippocampus, and Striatum After 2 and 7 days of Withdrawal. Effect of VU-29 on mGlu5 Protein Expression at Day 2 of Ethanol Withdrawal 0.0 0.1 0.2 0.3 0.4 0.5 * mGlu5 Hippocampus ### 7 days withdrawal 2 days withdrawal mGlu5 expression [mg/ml] b Two-way ANOVA indicated statistically insignificant differ- ences in the cortex between treatment factor (saline and etha- nol) (F(1,32) = 0.05; P > 0.05), time factor (2 and 7 days of ethanol withdrawal) (F(1,32) = 8.64; P = 0.0064), and without significant interactions between these two factors (F(1,32) = 0.52; P > 0.05). Post-hoc test showed that repeated ethanol administration (2.0 g/kg, 20% w/v × 7 days) induced a statis- tically significant increase in mGlu5 receptor protein expres- sion in the cortex after 2 days of ethanol withdrawal (P < 0.01), compared to the saline-treated rats (Fig. 5a). Influence of Repeated Ethanol Administration on Anxiety-Like Behavior After 48 h of ethanol abstinence, during the testing session (T2C) (after a 24-h retention delay), two-way ANOVA indicated statistically significant differences be- tween pre-treatment (ethanol effect) (F(1,31) = 10.83, P = 0.003; N = 8–9/group), treatment (VU-29 effect) (F(1,31) = 10.01, P = 0.004), and pre-treatment × treat- ment interaction (F(1,31) = 6.54, P = 0.016). Post-hoc test showed that animals during withdrawal from repeated eth- anol discriminated worse the novel object than did the saline-treated group (P < 0.01). Furthermore, VU-29, giv- en on the first day of ethanol abstinence (90 min before T2B), prevented (P < 0.01) this ethanol withdrawal- induced deficit in recognition memory (the DI was not significantly different from the saline group, P = 0.9298) Student’s t test indicated that there were no significant differences between repeated ethanol (2.0 g/kg, for 7 days) and saline administration on the number of open arm en- tries (t = 0.875, P = 0.396; N = 8) and the percent of time spent by rats in the open arm compartment of the maze (t = 0.487, P = 0.633) (Table 1). Ethanol withdrawal also did not produce any changes in locomotion of rats mea- sured as the total number of entries into both the open and closed arms of the apparatus (t = 0.242, P = 0.812). Thus, treatment with ethanol had no effect on anxiety-like be- haviors as compared to the control group (Table 1). Neurotox Res (2018) 33:607–620 614 0.0 0.1 0.2 0.3 0.4 0.5 VEHICLE ETOH mGlu5 Cortex ETOH + VU-29 ## 2 days withdrawal 7 days withdrawal mGlu5 expression [mg/ml] 0.0 0.1 0.2 0.3 0.4 0.5 * mGlu5 Hippocampus ### 7 days withdrawal 2 days withdrawal mGlu5 expression [mg/ml] 0.0 0.1 0.2 0.3 0.4 0.5 2 days withdrawal 7 days withdrawal mGlu5 Striatum ### mGlu5 expression [mg/ml] b c a Fig. 5 The effect of ethanol and VU-29 on mGlu5 levels in (a) the cortex, (b) hippocampus, and (c) striatum 2 and 7 days after last ethanol injection. Ethanol was administered at the dose of 2.0 g/kg (20% w/v, i.g.) for 7 days. VU-29 was injected at the dose of 30 mg/kg, i.p., 90 min before T2B on the first day (24 h) of ethanol withdrawal. Each column represents the mean ± SEM (N = 8–9). P < 0.01, P < 0.001 vs. vehicle group, ##P < 0.01, ###P < 0.001 vs. Influence of Repeated Ethanol Administration on Anxiety-Like Behavior ethanol-withdrawal group Neurotox Res (2018) 33:607–620 ted Ethanol Administration r Protein Expression in the Cortex, Striatum After 2 and 7 days ect of VU-29 on mGlu5 Protein 2 of Ethanol Withdrawal ndicated statistically insignificant differ- etween treatment factor (saline and etha- P > 0.05), time factor (2 and 7 days of F(1,32) = 8.64; P = 0.0064), and without ns between these two factors (F(1,32) = -hoc test showed that repeated ethanol kg, 20% w/v × 7 days) induced a statis- rease in mGlu5 receptor protein expres- after 2 days of ethanol withdrawal d to the saline-treated rats (Fig. 5a). y ANOVA indicated statistically signifi- hippocampus between treatment (saline = 80.94; P < 0.0001), time (2 and 7 days l) (F(1,32) = 29.37; P < 0.0001), and ex- icant interactions (F(1,32 = 38.63; d ethanol administration induced a statis- rease in mGlu5 receptor protein expres- mpus at 2 days of ethanol withdrawal ompared to the saline-treated rats (Fig. way ANOVA indicated statistically sig- n the striatum time (2 and 7 days of eth- ,32) = 20.55; P < 0.0001). Repeated eth- nduced statistically significant increases pression in the striatum after 2 days of P < 0.001), when compared to the saline- way ANOVA indicated statistically sig- gle VU-29 injection on mGlu5 receptor ex (F(2,25) = 7.55; P = 0.0027) (Fig. 5a), ) = 49.08; P < 0.0001) (Fig. 5b), and stri- VU-29 to the ethanol group (VU-29/ethanol group) significant- ly decreased mGlu5 protein expression vs. ethanol group (P = 0.0 0.1 0.2 0.3 0.4 0.5 VEHICLE ETOH mGlu5 Cortex * ETOH + VU-29 ## 2 days withdrawal 7 days withdrawal mGlu5 expression [mg/ml] 0.0 0.1 0.2 0.3 0.4 0.5 * mGlu5 Hippocampus ### 7 days withdrawal 2 days withdrawal mGlu5 expression [mg/ml] 0.0 0.1 0.2 0.3 0.4 0.5 2 days withdrawal 7 days withdrawal mGlu5 Striatum ### mGlu5 expression [mg/ml] b c a Fig. 5 The effect of ethanol and VU-29 on mGlu5 levels in (a) the cortex, (b) hippocampus, and (c) striatum 2 and 7 days after last ethanol injection. Ethanol was administered at the dose of 2.0 g/kg (20% w/v, i.g.) for 7 days. VU-29 was injected at the dose of 30 mg/kg, i.p., 90 min before T2B on the first day (24 h) of ethanol withdrawal. Each column represents the mean ± SEM (N = 8–9). P < 0.01, P < 0.001 vs. The Effect of Ethanol and VU-29 on mGlu2 Receptor Protein Expression in the Cortex, Hippocampus, and Striatum at 2 and 7 days of Ethanol Withdrawal The Effect of Ethanol and VU-29 on mGlu2 Receptor Protein Expression in the Cortex, Hippocampus, and Striatum at 2 and 7 days of Ethanol Withdrawal 2 days withdrawal 7 days withdrawal 0.0 0.1 0.2 0.3 0.4 0.5 ETOH + VU-29 mGlu2 Hippocampus ### 2 days withdrawal 7 days withdrawal mGlu2 expression [mg/ml] b Two-way ANOVA did not indicate statistically significant dif- ferences in mGlu2 receptor protein expression in the cortex between treatment (saline and ethanol) (F(1,32) = 4.01; P > 0.05), time (2 and 7 days of ethanol withdrawal) (F(1,32) = 0.11; P > 0.05), and treatment × time interaction (F(1,32) = 0.05; P > 0.05) (Fig. 6a). However, two-way ANOVA did indicate statistically significant differences in the hippocampus between treatment (saline and ethanol) (F(1,32) = 0.71; P > 0.05), time (2 and 7 days of ethanol with- drawal) (F(1,32) = 62.80; P < 0.0001), and treatment × time interaction (F(1,32) = 21.35; P < 0.0001) (Fig. 6b). Moreover, post-hoc test showed that repeated ethanol administration (2.0 g/kg, 20% w/v × 7 days) brought a statistically significant increase in mGluR2 receptor protein expression in the hippo- campus 2 days of ethanol withdrawal (P < 0.001), compared to saline-treated rats (Fig. 6b). Still, two-way ANOVA did not indicate statistically significant differences in the striatum be- tween treatment (saline and ethanol) (F(1,32) = 3.22; P > 0.05), time (2 and 7 days of ethanol withdrawal) (F(1,32) = 0.80; P > 0.05), and treatment × time interaction (F(1,32) = 0.69; P > 0.05) (Fig. 6c). ETOH + VU-29 7 days withdrawal One-way ANOVA indicated statistically significant differ- ences between mGlu2 receptor protein expression in the hip- pocampus, in the ethanol withdrawal, and in control (saline) groups, on the second day (day 2) after single injection of VU- 29 (F(2,24) = 50.34; P < 0.0001) (Fig. 6b). What is more, post-hoc test showed that acute injection of VU-29 (30 mg/ kg, i.p.) on the first day of ethanol withdrawal, 90 min prior to the testing trial, decreased mGluR2 receptor protein expres- sion in the hippocampus 24 h later (P < 0.001) (Fig. 6b) but the results did not differ significantly from the saline group (P = 0.0980). 2 days withdrawal 7 days withdrawal Fig. 6 The effect of ethanol (a, b, c) and VU-29 (b) on mGlu2 levels in the cortex, hippocampus and striatum 2 and 7 days after last ethanol injection. The Effect of Ethanol and VU-29 on mGlu2 Receptor Protein Expression in the Cortex, Hippocampus, and Striatum at 2 and 7 days of Ethanol Withdrawal Ethanol was administered at the dose of 2.0 g/kg (20% w/v, i.g.) for 7 days. VU-29 was injected at the dose of 30 mg/kg, i.p., 90 min before T2B on the first day (24 h) of ethanol withdrawal. Each column represents the mean ± SEM (N = 8–9). P < 0.001 vs. vehicle group, ###P < 0.001 vs. ethanol-withdrawal group failed to recognized a novel object during the test phase. Moreover, pretreatment with VU-29, a positive allosteric modulator of mGlu5 receptor, before ethanol administration, significantly decreased ethanol-induced memory impairment. Furthermore, repeated (1× for 7 days) ethanol administration also caused NOR memory deficits in the acute (24–48 h) with- drawal phase. These cognitive deficits were accompanied by Influence of Repeated Ethanol Administration on Anxiety-Like Behavior mGlu2 Cortex Influence of Repeated Ethanol Administration on Anxiety-Like Behavior 5b), and stri- atum (F(2,25) = 23.97; P < 0.0001) (Fig. 5c) after 2 days of ethanol withdrawal. The post-hoc test showed that VU-29 (30 mg/kg, i.p.) given 90 min prior the testing trial on the first day of ethanol abstinence, decreased mGlu5 receptor protein expression in the cortex (P < 0.01) (Fig. 5a). Administration of VU-29 to the ethanol group (VU-29/ethanol group) significant- ly decreased mGlu5 protein expression vs. ethanol group (P = 0.0045). Herein, the protein expression was not significantly different from the saline group (P = 0.1360). The post-hoc test showed that VU-29 (30 mg/kg, i.p.), given 90 min prior the testing trial on the first day of ethanol abstinence, decreased mGlu5 receptor protein expression in the hippocampus 615 Neurotox Res (2018) 33:607–620 f il d t i d l bj t d i th t t h 0.0 0.1 0.2 0.3 0.4 0.5 VEHICLE ETOH mGlu2 Cortex 2 days withdrawal 7 days withdrawal mGlu2 expression [mg/ml] 0.0 0.1 0.2 0.3 0.4 0.5 ETOH + VU-29 mGlu2 Hippocampus ### 2 days withdrawal 7 days withdrawal mGlu2 expression [mg/ml] 0.0 0.1 0.2 0.3 0.4 0.5 mGlu2 Striatum 2 days withdrawal 7 days withdrawal mGlu2 expression [mg/ml] b c a Fig. 6 The effect of ethanol (a, b, c) and VU-29 (b) on mGlu2 levels in the cortex, hippocampus and striatum 2 and 7 days after last ethanol injection. Ethanol was administered at the dose of 2.0 g/kg (20% w/v, i.g.) for 7 days. VU-29 was injected at the dose of 30 mg/kg, i.p., 90 min before T2B on the first day (24 h) of ethanol withdrawal. Each column represents the mean ± SEM (N = 8–9). *P < 0.001 vs. vehicle group, ###P < 0.001 vs. ethanol-withdrawal group 615 0.0 0.1 0.2 0.3 0.4 0.5 VEHICLE ETOH mGlu2 Cortex 2 days withdrawal 7 days withdrawal mGlu2 expression [mg/ml] a a (P < 0.001) (Fig. 5b). Protein expression in the VU-29/ethanol group did not differ significantly from the control (saline) group (P = 0.3790). The post-hoc test showed that VU-29 (30 mg/kg, i.p.), given 90 min prior the testing trial on the first day of ethanol abstinence, decreased mGlu5 receptor protein expres- sion in the striatum (P < 0.001) (Fig. 5c) on the second day, when compared to ethanol-withdrawal rats. The mGlu5 protein expression in the VU-29/ethanol group differed significantly from the control (saline) group (P = 0.0009). Discussion Our ELISA assay show that such VU-29 admin- istration returned to control values the elevated mGlu5 recep- tor protein expressions in the hippocampus and prefrontal cor- tex, as well as mGlu2 receptor protein expressions in the hip- pocampus at 48 h of ethanol abstinence. Discussion Our study shows that acute ethanol administration, prior to the testing session, impaired retrieval of the recognition memory in the NOR task in rats. Animals treated with acute ethanol Neurotox Res (2018) 33:607–620 616 2005). Systemic administration of mGlu2/3 receptor, as well as mGlu5 receptor antagonists, before novel objects training and before testing, impaired long- (Barker et al. 2006a, b) and short-term recognition memories or retrieval of such memo- ries (Christoffersen et al. 2008). In turn, stimulation of mGlu5 receptor via positive allosteric modulators (CDPPB or ADX47273) enhanced recognition memory in normal rats and mice (Liu et al. 2008; Uslaner et al. 2009; Fowler et al. 2013). Published data indicated that VU-29 (Ayala et al. 2009) potentiated long-term potentiation (LTP) or long-term depres- sion (LTD) in hippocampal slices in vitro, both widely accept- ed processes of memory formation (Neyman and Manahan- Vaughan 2008). In our study, VU-29, a mGlu5 receptor- positive allosteric modulator, given before the test enhanced recognition memory in control rats. Furthermore, a single pre- treatment with VU-29 decreased NOR deficit induced by acute ethanol administration. It should be noted that mGlu5 receptor is physically linked to NMDA receptors via several anchoring proteins, including Homer proteins (Fagni et al. 2004), and mGlu5 receptor activation can overcome or pre- vent the consequences of inhibition of NMDA receptors. As previously stated, acute ethanol administration inhibits, or an- tagonizes, the action of the NMDA receptors in several brain regions (Dildy and Leslie 1989; Hoffman et al. 1989; Lovinger et al. 1989; Simson et al. 1991). Thus, in our study, the protective effect of VU-29, a mGlu5 receptor-positive al- losteric modulator on ethanol-induced memory deficit, might occur via the ability of this agent to decrease the consequences of ethanol-induced inhibition of the NMDA receptor. alterations in hippocampal expression of mGlu5 and mGlu2 receptors proteins, and to a lesser extent, in expression of cortical and striatal mGlu5 receptor protein during acute eth- anol withdrawal (day 2 of abstinence). The casual relationship between these events remains to be demonstrated. Single ad- ministration of VU-29 prior to the testing session on the first abstinence day recovered the impaired recognition memory measured at both 4-h delay (short-term memory) and at 24 h-delay (long-term memory) intervals (the second day of abstinence). Effect of VU-29, a mGlu5 Receptor-Positive Allosteric Modulator, on the Acute Effect of Ethanol on Retrieval of Recognition Memory Preclinical studies suggest that acute ethanol administration has deleterious effects on recognition memory (Brooks et al. 2002; Ryabinin et al. 2002; Garcia-Moreno and Cimadevilla 2012; Yu et al. 2013), especially when it is administered be- fore the training session of the test. Our experiments in rats extended these findings and indicated that acute ethanol ad- ministration induced a deficit in memory retrieval in the NOR test. Analysis of animal behavior showed that rats injected with ethanol at the doses tested (1.5, 1.75, and 2 g/kg) before the testing session demonstrated decreased preference for a novel object. Thus, ethanol produced retrograde amnesia for previously acquired information, including information about familiarity of objects previously encountered. Our experi- ments also indicated that ethanol at the dose of 2 g/kg signif- icantly decreased locomotor activity, and, at lower doses (1.5 and 1.75 g/kg), showed a trend towards decreasing it. However, the total exploration time of the objects (sniffing or touching with the nose towards the object) for the rats did not differ significantly between the groups. Thus, our data support other research that reveals that there is clear dissocia- tion between locomotor activity and exploration, and no cor- relation exist between these two measures. Such results un- derline the distinction for the two behaviors (Leyland 1976). Because exploration time generates information about the ob- ject via direct (animal) contact with it and this type of move- ment is important for the novel object recognition, but loco- motor activity concerns movement from one location to other and could also be important for our study, we choose such dose of ethanol (1.75 g/kg) for our study that did not change significantly the locomotor activity. Effect of VU-29, a mGlu5 Receptor-Positive Allosteric Modulator, on Retrieval of Recognition Memory Impaired by Withdrawal from Repeated Ethanol Administration After the first 24–48 h of withdrawal from repeated etha- nol treatment (2.0 g/kg, i.g. for 7 days, once daily), the rats showed significant NOR memory deficits. Unlike the controls, rats treated with ethanol failed to recognize a novel object during the testing sessions. With the proce- dure used in this experiment, it is difficult to determine if the decreased novel subject exploration on the test day was due to a deficit in initial learning or in retrieval dur- ing the test. This effect was observed with no symptoms of withdrawal such as anxiety. However, published data (using other procedures of object recognition tests) have shown that withdrawal from other abuse drugs such as cocaine or methamphetamine also produce deficits in rec- ognition (Briand et al. 2008; Rogers et al. 2008; Reichel et al. 2011) and CDPPB, a mGlu5 receptor-positive allo- steric modulator, and reversed deficits in recognition memory during methamphetamine abstinence in rats (Reichel et al. 2011). In our study, VU-29, given 90 min Published data indicated that mGlu5 receptors are found in brain regions important for cognition, including the prefrontal cortex, striatum, and hippocampus (Packard et al. 2001; Melendez et al. 2004; Manahan-Vaughan and Braunewell Neurotox Res (2018) 33:607–620 617 before the testing session on the first day of ethanol ab- stinence, facilitated retrieval of NOR performance after a 4-h delay. This effect was long-lasting since it was also observed after a 24-h delay (Fig. 4). In summary, our experiments indicated that acute ethanol administration or withdrawal from repeated ethanol administra- tion induced deficits in recognition memory that were associ- ated with an increase in expressions of mGlu5 (hippocampus, prefrontal cortex, and striatum) and mGlu2 (hippocampus) re- ceptor proteins in brain structures important for this type of memory. VU-29, a mGlu5 receptor-positive allosteric modula- tor, reversed/decreased ethanol-induced recognition memory deficits and normalized the expression of mGlu5 and mGlu2 receptors in the hippocampus and cortex. Thus, our data show the pivotal role of mGlu5 receptor in recognition memory im- pairment induced by acute and repeated ethanol administration. It also suggests the possible prospect for compounds acting as mGlu5 receptor-positive allosteric modulators in countering the effects of alcoholism. before the testing session on the first day of ethanol ab- stinence, facilitated retrieval of NOR performance after a 4-h delay. This effect was long-lasting since it was also observed after a 24-h delay (Fig. 4). Effect of VU-29, a mGlu5 Receptor-Positive Allosteric Modulator, on Retrieval of Recognition Memory Impaired by Withdrawal from Repeated Ethanol Administration Herein, ethanol withdrawal-induced deficits in the NOR test were associated with increased mGlu5 receptor protein expression at 48 h of abstinence, in brain structures important for retrieval of recognition memory, such as the hippocampus, prefrontal cortex, and striatum. VU-29 administration re- versed this ethanol effect in the hippocampus and prefrontal cortex, but only decreased it in the striatum. Thus, we hypoth- esize that the positive allosteric modulator, VU-29 (given on the first day of ethanol withdrawal), potentiated mGlu5 recep- tor function by acting at allosteric sites and shifting the con- centration response for glutamate to the left, without the risk of inducing excitotoxicity (Gass and Olive 2009). Still, a mGlu5 receptor-positive allosteric modulator can stabilize a conformation of the receptor (Homayoun and Moghaddam 2010) that would facilitate activation of neuroprotective path- ways against neurotoxicity induced by glutamate release dur- ing withdrawal. A preventive effect of mGlu5 receptor- positive allosteric modulators against glutamate overstimula- tion of NMDA receptors was earlier described (Ribeiro et al. 2010; Doria et al. 2013). We put forward that such stabiliza- tion of neuronal function by VU-29 could have normalized the elevated expression of mGlu5 receptor in the hippocampus and prefrontal cortex in our study. Funding information This work was supported by the Statutory Funds of the Medical University of Lublin (DS 22/16). Compliance with Ethical Standard The experimental protocols and hous- ing conditions were performed according to the National Institute of Health Guidelines for the Care and Use of Laboratory Animals, the European Community Council Directive of November 2010 for Care and Use of Laboratory Animals (Directive 2010/63/EU), and were ap- proved by the Local Ethics Committee. All applicable international, na- tional, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in the studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted. This article does not contain any stud- ies with human participants performed by any of the authors. In addition, given the finding that combined mGlu5 recep- tor and mGlu2/3 receptor activity in the perirhinal cortex is important for recognition memory (Barker et al. 2006a, b), we also examined changes in expression of mGlu2 receptor in brain structures important for retrieval of recognition memory, such as the prefrontal cortex, the hippocampus, and the stria- tum. Effect of VU-29, a mGlu5 Receptor-Positive Allosteric Modulator, on Retrieval of Recognition Memory Impaired by Withdrawal from Repeated Ethanol Administration Although the magnitude of increase was similar between mGlu5 receptor and mGlu2 receptor in the hippocampus on abstinence day 2, the changes in mGlu2 receptor were not observed in the prefrontal cortex and striatum. Taken together, these data suggest that the mGlu5 receptor is more engaged in the effects of repeated ethanol exposure than the mGlu2 re- ceptor. Moreover, the impact of ethanol on mGlu2 receptor is dependent on brain structure. Generally, in our experiments, changes in expression of mGlu5 receptor and mGlu2 receptor were the most evident in the hippocampus. This implies that the hippocampus plays an important role in ethanol-induced deficits in the retrieval of recognition memory during acute ethanol abstinence. Furthermore, VU-29 returned expression of mGlu2 receptor to the control in the hippocampus, as it did with mGlu5 receptor. Although, published data indicated that both mGlu5 and mGlu2 receptors in the hippocampus are independently involved in different forms of synaptic plastic- ity (Manahan-Vaughan 1997), we cannot rule out the possi- bility that functional linkage between these receptors exists in the hippocampus. Conflict of Interest The authors declare that they have no conflict of interest. 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https://openalex.org/W3213548330	https://figshare.com/articles/thesis/Design_and_development_of_the_Hyperloop_Deployable_wheel_system/14645238/1/files/28125228.pdf	English	null	Design and development of the Hyperloop Deployable wheel system	null	2,023	cc-by	129,602	by Toronto, Ontario, Canada, 2018 © Graeme P.A Klim 2018 AUTHOR'S DECLARATION FOR ELECTRONIC SUBMISSION OF A TH I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I authorize Ryerson University to lend this thesis to other institutions or individuals for the purpose of scholarly research. I further authorize Ryerson University to reproduce this thesis by photocopying or by other means, in total or in part, at the request of other institutions or individuals for the purpose of scholarly research. I understand that my thesis may be made electronically available to the public. ii ii DESIGN AND DEVELOPMENT OF THE HYPERLOOP DEPLOYABLE WHEEL SYSTEM Graeme P.A Klim Master of Applied Science, Aerospace Engineering, Ryerson University, Toronto (2018) Abstract In 2013 Elon Musk inspired engineers and entrepreneurs with his idea for a 5th mode of transportation: the Hyperloop. Using large near-vacuum tubes as a medium, Musk envisioned sending humans and cargo in levitating pods from Los Angeles to San Francisco California in 35 minutes or less. Consisting of multiple subsystems, these pods would use magnetic or air-bearing technology for primary levitation to accommodate speeds approaching 700 mph. To address Musk’s call for a traditional deployable wheel system to provide added safety and low-speed mobility for the pods, a patent-pending Hyperloop Deployable Wheel System (HDWS) was developed. This report details the author’s contribution to the design and development of the award-winning HDWS and examines the constraints and limitations imposed by the Hyperloop concept: small operational space, near-vacuum low-pressure conditions, high-speed use and smooth ride requirements. iii iii Acknowledgements I would like to sincerely thank Dr. Seyed M. Hashemi for supporting and guiding me throughout the entirety of my university education. Your attentive attitude towards my educational and personal ambitions has been greatly appreciated. I would like to give a huge “shout-out” to the student team members of Ryerson’s International Hyperloop Team – Tayo Shonibare, Whintta Ghebreiyesus, Jesse Brito, Min Adhikari and Moeid Elahi – for their contributions to the present work. I am indebted to the project’s academic and industrial advisors: Steve Amberg, Clement Rodrigues, Primoz Cresnik, Faizan Rehematullah and Seyed M. Hashemi who spent a great deal of their personal time supporting the development of the Hyperloop Deployable Wheel System. I would like to thank the large “team” of supporters at Safran Landing Systems Canada Inc. who provided me with access to development funding, engineering expertise, test facilities and even provided me a desk onsite for the duration of the project. By the same token, the assistance of Ryerson University, from the president’s office to the lab technicians, was unwavering and greatly appreciated. The additional financial support from the Natural Sciences and Engineering Research Council of Canada (NSERC) through an ENGAGE grant is also acknowledged. I would like to extend a deep sense of gratitude to Mr. Pete Cooper who spent a great deal of his 2016 calendar year manufacturing the structural components for the Hyperloop Deployable Wheel System and its test fixtures. Pete’s commitment to manufacturing excellence ensured that the final product not only functioned well, but looked great too! Finally, I would like to thank my friends and family, whose love and encouragement has enabled me to complete this work. Thank you to my partner, Kelly Firth, for always encouraging and supporting me throughout this project. Thank you to my Mom and Dad, and my grandparents, Alex (2010) and Blanche (2015) Klim, Clarence and Walterina Mountain, for all your love and support throughout the years. Without the support of all these people and organizations, the Hyperloop Deployable Wheel System would have remained a great idea yet to be realized. iv Table of Contents Abstract .......................................................................................................................................... iii Acknowledgements ........................................................................................................................ iv List of Figures ................................................................................................................................ ix List of Tables ............................................................................................................................... xiv List of Symbols, Constants and Abbreviations ............................................................................. xv Symbols ..................................................................................................................................... xv Constants .................................................................................................................................. xvi Abbreviations ........................................................................................................................... xvi 1 Introduction ............................................................................................................................. 1 1.1 Preamble ........................................................................................................................... 1 1.2 The SpaceX Hyperloop Competition ............................................................................... 1 1.3 Ryerson’s International Hyperloop Team ........................................................................ 2 1.4 Research Motivation ........................................................................................................ 2 1.5 Key Objectives and Contributions ................................................................................... 3 1.6 Limitation of Current Work ............................................................................................. 4 1.7 Thesis Outline .................................................................................................................. 5 2 Literature Review .................................................................................................................... 6 2.1 The Hyperloop.................................................................................................................. 6 2.1.1 Pod Overview............................................................................................................ 6 2.1.2 Major Subsystems Breakdown ................................................................................. 7 2.1.3 The Tube System ...................................................................................................... 9 2.1.4 SpaceX Hyperloop Tube System ............................................................................ 10 2.2 Hyperloop Pod Operation Environment ......................................................................... 11 v v 2.2.1 Low-pressure Environment ..................................................................................... 11 2.2.2 High-speed Operation ............................................................................................. 12 2.2.3 Thermal Management ............................................................................................. 12 2.3 Aircraft Landing Gear .................................................................................................... 13 2.3.1 A Brief History ....................................................................................................... 13 2.3.2 Key Components and Configurations ..................................................................... 14 2.3.3 Common Components ............................................................................................ 15 2.4 Existing Patents .............................................................................................................. 17 3 Product Definition and Preliminary Design .......................................................................... 23 3.1 Precondition: Why Wheels? ........................................................................................... 23 3.1.1 Air Levitation Pod................................................................................................... 23 3.1.2 Magnetic Levitation Pod ......................................................................................... 24 3.2 Former University of Toronto Partnership ..................................................................... 25 3.3 Design Objectives .......................................................................................................... 25 3.4 Product Development Guidelines and Constraints......................................................... 26 3.4.1 Design Constraints .................................................................................................. 27 3.4.2 Performance Constraints and Guidelines ................................................................ 28 3.4.3 Material and Lubrication Guidelines ...................................................................... 29 3.5 HDWS Architecture and Kinematics ............................................................................. 30 3.5.1 HDWS Architecture Definition .............................................................................. 31 3.5.2 HDWS Kinematic Layouts ..................................................................................... 34 3.6 Component Definition, Product Structure and Process Scope ....................................... 38 3.6.1 Component Definition ............................................................................................ 38 3.6.2 Product Structure Breakdown ................................................................................. 39 3.6.3 Design and Development Process Scope ................................................................ 40 vi 4 Design and Development of the HDWS ................................................................................ 42 4.1 HDWS Dressed Assembly (P/N 10-1000-00) ................................................................ 42 4.1.1 HDWS Undressed Assembly (P/N 20-1000-00) .................................................... 43 4.1.2 Electric Motor Assembly (P/N 20-2000-00)........................................................... 44 4.1.3 Rolling Stock .......................................................................................................... 45 4.1.4 Electrical Dressings and Hardware ......................................................................... 48 4.2 HDWS Undressed Assembly Components .................................................................... 50 4.2.1 Trailing Arm Sub-assembly (P/N 30-1100-00) ...................................................... 50 4.2.2 Shock Strut Sub-assembly (P/N 30-1200-00) ......................................................... 53 4.2.3 Retraction Sub-assembly (P/N 30-1300-00) ........................................................... 56 4.2.4 Torque Link Sub-assembly (P/N 30-1400-00) ....................................................... 57 4.2.5 Axle (P/N 40-1501-00) ........................................................................................... 58 4.3 Structural Analyses ........................................................................................................ 59 4.3.1 Classical Analyses Overview .................................................................................. 60 4.3.2 Finite Element Analyses and Applications ............................................................. 65 4.4 Part and Assembly Engineering Drawings..................................................................... 68 4.5 Manufacturing and Treatment Processes ....................................................................... Table of Contents 70 4.5.1 Subtractive Manufacturing...................................................................................... 71 4.5.2 Protective Finishes and Processes ........................................................................... 72 4.5.3 Additive Manufacturing .......................................................................................... 74 4.6 Chapter Summary ........................................................................................................... 75 5 The Deployable and Retractable Shock Strut ........................................................................ 76 5.1 System Configuration ..................................................................................................... 76 5.2 System Operation ........................................................................................................... 78 5.3 HDWS Shock Absorber Design ..................................................................................... 79 vii 5.3.1 Oleo-Pneumatic Shock Absorber Introduction ....................................................... 79 5.3.2 Shock Absorber Gas Spring Preliminary Design .................................................... 81 5.3.3 Internal Geometry Definition .................................................................................. 90 5.3.4 Detailed Gas Spring Design .................................................................................... 94 5.3.5 Shock Absorber Detail Design................................................................................ 98 5.4 HDWS Actuation Mechanism Design ......................................................................... 103 5.5 Chapter Summary ......................................................................................................... 107 6 Experimental Results ........................................................................................................... 108 6.1 Polyurethane Wheel Performance Testing ................................................................... 108 6.2 Gas Spring Testing ....................................................................................................... 115 6.3 Full Single-System Testing .......................................................................................... 118 6.4 Dummy Pod Test (4 Units) .......................................................................................... 123 7 Concluding Remarks ........................................................................................................... 124 7.1 Conclusion .................................................................................................................... 124 7.2 Future Work ................................................................................................................. 125 Appendix A: HDWS Component Drawings ............................................................................... 126 Appendix B: HDWS Tooling Drawings ..................................................................................... 167 Appendix C: HDWS Test Fixture Drawings .............................................................................. 172 Appendix D: Gas Spring Test Result Data ................................................................................. 183 Appendix E: Retractable Shock Strut Concept Art..................................................................... 189 Appendix F: Galvanic Chart ....................................................................................................... 192 References ................................................................................................................................... 193 viii List of Figures g Figure 2.1: Example of a Hyperloop passenger pod, subsystem notional locations (not to scale) [1] ......................................................................................................................................................... 7 Figure 2.2: Hyperloop One’s test “sled” is shown to include (1) magnets for levitating and (2) a rudimentary wheel system in tow [14]............................................................................................ 8 Figure 2.3: Rotor and stator 3D diagram [1]................................................................................... 9 Figure 2.4: Artist rendering of a Hyperloop tube cutaway equipped with solar arrays [1] .......... 10 Figure 2.5: Image of the SpaceX Hyperloop sub-scale test track, image taken January 2017 ..... 11 Figure 2.6: Tail wheel configuration landing gear on a STOL Maule MX-7-235 Super Rocket . 14 Figure 2.7: General arrangement of a retractable MLG Trailing arm configuration [23] ............ 15 Figure 2.8: Sharples kinematic retraction configuration (left) with a cutaway of the contractive shock strut (right), shown as items 11 and 25 in the kinematic configuration (left) [25] ............. 18 Figure 2.9: An illustration provided in Veaux et al. illustrating a motor and gearbox assembly embedded into the lower half of a piston to lengthen the strut [26] ............................................. 19 Figure 2.10: An illustration of the design proposed by Currey and Renshaw [27] ...................... 20 Figure 2.11: An image (left) illustrating key components and with the advantageous kinematic architecture displayed (right), proposed by Waide [8] ................................................................. 21 Figure 2.12: Two possible configurations of a retractable shock strut, proposed by Waide [8] .. 21 Figure 3.1: Normalized force of the EDS Maglev plotted against translational velocity [29] ..... 25 Figure 3.2: A 2D side view of a Hyperloop pod with the HDWS positioned, not to scale .......... 27 Figure 3.3: Partial cross-section view of the SpaceX Hyperloop sub-track interface, dimensions shown in inches, with reference to the specification [4] ............................................................... 28 Figure 3.4: The HDWS preliminary configuration, stick diagram, not to final scale ................... 32 Figure 3.5: The HDWS Hybrid actuator concept illustration ....................................................... 33 Figure 3.6: The HDWS Hybrid actuator concept #2 illustration .................................................. 34 Figure 3.7: An example of a preliminary HDWS kinematic layout, retracted, shown in CATIA® V5 .................................................................................................................................................. 35 Figure 3.8: U of T HDWS kinematic layout, retracted position (left) and WOW position (right)37 Figure 3.9: U of T HDWS kinematic layout, sample static stroke (left) and sample static stroke + pod height adjustment of 0.5 in (12.7 mm) from the reference position (right) .......................... 37 ix x Figure 3.10: The HDWS product structure tree ............................................................................ List of Figures 39 Figure 3.11: The HDWS design and development process scope overview ................................ 40 Figure 4.1: The HDWS Dressed assembly, CATIA® V5 rendering ............................................. 42 Figure 4.2: The HDWS Undressed assembly, CATIA® V5 rendering ......................................... 43 Figure 4.3: The HDWS Electric Motor assembly, installed on the Retract sub-assembly ........... 44 Figure 4.4: CATIA® V5 renderings of the final HDWS Wheel Assembly (left), and alternate wheel concept as designed with input from the author (right) ................................................................ 45 Figure 4.5: A Wheel and Tire assembly, 3D cross-sectional view, as drawn by Khan [45] ........ 47 Figure 4.6: The rolling stock as installed on the HDWS Dressed ................................................ 47 Figure 4.7: Sensors, brackets, electrical and hardware mount (orange) shown on the HDWS Dressed .......................................................................................................................................... 48 Figure 4.8: Sensors, brackets, electrical and hardware labeled (some features hidden for clarity) ....................................................................................................................................................... 49 Figure 4.9: The Trailing Arm sub-assembly, parts labeled .......................................................... 51 Figure 4.10: The Trailing Arm sub-assembly cross-sectional view ............................................. 52 Figure 4.11: The Trailing Arm axle installation pin ..................................................................... 53 Figure 4.12: The HDWS Shock Strut sub-assembly, CATIA® V5 rendering .............................. 53 Figure 4.13: Full cross-section view of the Shock Strut sub-assembly, Find Numbers shown .... 54 Figure 4.14: Partial cross-section view of the shock strut sub-assembly, Find Numbers shown . 54 Figure 4.15: Retract sub-assembly 3D view (left) and cross section (right), Find Numbers shown ....................................................................................................................................................... 57 Figure 4.16: Torque Link sub-assembly (left) and exploded view (right), Find Numbers shown 58 Figure 4.17: The axle component as equipped to the trailing arm sub-assembly ......................... 59 Figure 4.18: Section cut, illustrating a typical double shear loading on the HDWS .................... 61 Figure 4.19: Section cut, illustrating a shear stress around the circumference of the bolt head ... 63 Figure 4.20: Flanged bush (left), illustrating a projected bearing area (right) used for the preliminary calculation of the bearing stress ................................................................................ 64 Figure 4.21: Flanged bush illustrating the features involved in a bearing length refinement ...... 64 Figure 4.22: Sample FEA result from ANSYS®, trailing arm lug analysis [40] .......................... 65 Figure 4.23: HDWS motor mount bracket, machine from solid variant (maroon colored) [17] .. 66 Figure 4.24: Final bracket design, produced from an FEA based optization [17] ........................ 67 x xi Figure 4.25: Example of a bracket displacement analysis, gravity load applied [17] .................. 67 Figure 4.26: Sample of a typical HDWS engineering drawing, key elements encircled.............. List of Figures 68 Figure 4.27: Sample of title block layout used for the HDWS ..................................................... 69 Figure 4.28: Typical design notes used for the HDWS aluminum parts ...................................... 70 Figure 4.29: Images depicting from (1) through (3) the stages in the turning process performed for the manufacturing of the shock absorber outercylinder ACME 3G thread .................................. 71 Figure 4.30: Images (1) through (3) depict the milling process starting from a solid metal block, and image (4) depicts the internal thread screw cut performed on a lathe .................................. 72 Figure 4.31: Trailing arm pictured after undergoing blue anodizing............................................ 73 Figure 4.32: A shock strut outer cylinder shown with black oxide surface treatment .................. 74 Figure 4.33: Images (1) through (2) depict the DMLS process used to create the final (3) AMM part [17] ......................................................................................................................................... 75 Figure 5.1: Deployable and retractabe shock strut system configuration, partial cross-section ... 77 Figure 5.2: HDWS extension and retraction process, some parts hidden for clarity .................... 78 Figure 5.3: An example of an oleo-pneumatic shock strut with oil and gas free to mix [5] ........ 80 Figure 5.4: Example of a hypothetical pod layout, loads applied to the HDWS in a static position ....................................................................................................................................................... 82 Figure 5.5: The HDWS preliminary configuration, stick diagram ............................................... 83 Figure 5.6: Illustration of oleo-pneumatic shock strut key operational states .............................. 86 Figure 5.7: Graphic representation of the isothermal oleo-pneumatic gas spring curve produced using the results in Table 5.2 ........................................................................................................ 89 Figure 5.8: A simplified oleo-pneumatic strut, key preliminary design components labeled for clarity ............................................................................................................................................ 91 Figure 5.9: An example of an oleo-pneumatic shock strut highlighting preliminary geometric features .......................................................................................................................................... 92 Figure 5.10: Piston rod gland design, key dimensions labeled, as defined by Greene Tweed [59] ....................................................................................................................................................... 94 Figure 5.11: Dynatool© HDWS gas spring curve (static and dynamic), load vs. stroke .............. 96 Figure 5.12: Dynatool© HDWS gas spring curve (static and dynamic), pressure vs. stroke ....... 96 Figure 5.13: Cross-section view showing the shock strut while fully compressed at servicing, total oil volume (red) 6.426 in3 ............................................................................................................. 97 xi Figure 5.14: Cross-section view showing the total volume when extended (purple) 9.174 in3, gas+oil ........................................................................................................................................... 97 Figure 5.15: Cross-section view of the shock strut trapped air volume (white) 0.106 in3, max piston compression .................................................................................................................................. 97 Figure 5.16: Cross-section, lower bearing, key design details labeled ......................................... 98 Figure 5.17: 3D cross-section of the filler tube, key design details labeled ............................... List of Figures 100 Figure 5.18: 3D cross-section view of the filler tube with trapped gas illustrated ..................... 101 Figure 5.19: 3D view of the piston, spherical bearing installed, key features labeled ............... 101 Figure 5.20: 3D cross-sectional view of the shock strut outer cylinder, key features labeled .... 102 Figure 5.21: 3D cross-sectional view of the retract assembly, trunnion pins labeled ................ 103 Figure 5.22: Retract housing (bushed), key design features labeled .......................................... 104 Figure 5.23: 3D cross-section view, ACME retract nut, key design features labeled ................ 105 Figure 5.24: Retract assembly 3D exploaded view, key design features labeled ....................... 106 Figure 6.1: Wheel test configuration .......................................................................................... 109 Figure 6.2: Reflector tape positioning and wheel temperature measurement region shown ...... 110 Figure 6.3: Plot illustrating Test #1, the pre-spin of the spin-up machine, engaging the stationary pod mounted wheel at 180 mph, and braking to stop at a decceleration rate of 0.6g ................. 111 Figure 6.4: Localized wear marking, followed by a trail-off effect, observed after Test #1 ...... 112 Figure 6.5: Thin polyurethane deposited on the aluminum fly-wheel observed after Test #2, wheel deterioration increased under same loading and speeds ............................................................. 112 Figure 6.6: Shifting of the reflective tape caused by the expanding and contracting outer diameter of the wheel after disengagement from the spin-up machine at 180 mph .................................. 113 Figure 6.7: Grease splatter and roller rotation evident as traced by the orange anti-rotation paint ..................................................................................................................................................... 114 Figure 6.8: Graph displaying the average load measured by the load cell as applied by the spin machine, load spikes at initial contact encircled in red .............................................................. 114 Figure 6.9: Shock absorber gas spring verification initial, initial setup ..................................... 115 Figure 6.10: Shock absorber gas spring verification test, final setup ......................................... 116 Figure 6.11: HDWS full system test configuration (temperature sensors out of view) .............. 118 Figure 6.12: Example of torque paint (orange) applied, used to track loosening of the hardware ..................................................................................................................................................... 119 xii Figure 6.13: General pod mission profile inidcating key regions/events of interest (not to scale) ..................................................................................................................................................... 120 Figure 6.14: Graph of the data recorded during Test Case #4, with the HDWS subjected to 2.4g acceleration and decceleration, key events labeled for clarity .................................................... 121 Figure 6.15: An example of typical wheel wear as shown after full systems testing ................. List of Figures 122 Figure 6.16: Dummy pod test bench, control system testing fixture with 200 lbm mobile dummy mass to simulate shifts in CG...................................................................................................... 123 xiii List of Tables Table 3.1: Representative SpaceX pusher acceleration values (2016) [4] .................................... 28 Table 3.2: Levitation ride height comparison ............................................................................... 36 Table 4.1: Electric Motor assembly breakdown summary ........................................................... 44 Table 4.2: Electric Motor assembly breakdown summary ........................................................... 50 Table 4.3: Trailing Arm sub-assembly breakdown summary ....................................................... 51 Table 4.4: Shock Strut sub-assembly breakdown summary ......................................................... 55 Table 4.5: Retraction sub-assembly component breakdown summary ........................................ 56 Table 4.6: Torque Link sub-assembly component breakdown summary ..................................... 58 Table 5.1: Shock strut design standards and specifications referenced ........................................ 79 Table 5.2: Shock strut design tool developed by the author using equations derived in Currey [5] ....................................................................................................................................................... 85 Table 5.3: Key shock strut stroke position outputs (ideal gas law, isothermal assumption) ........ 85 Table 5.4: Shock strut gas spring design features and definitions per Currey [5] ........................ 87 Table 5.5: Shock strut mechanical design features and definitions .............................................. 91 Table 5.6: Piston detail design template, as defined in the author’s shock strut design tool ........ 93 Table 5.7: Piston rod dynamic seal gland dimensions, per Greene Tweed [59], based on SAE AS4716B ....................................................................................................................................... 93 Table 5.8: Sample set of the Dynatool© program interface inputs required from the user ........... 95 Table 6.1: Gas spring test verification results vs. Dynatool© theoretical data ............................ 117 xiv Symbols 𝐴𝐵 Bearing area (projected) 𝐴𝑇 Tensile area 𝐴𝑝 Piston area 𝐴𝑠𝑐 Shear area 𝐹𝐴 Force applied or Axial force 𝐹𝑇 Tensile Force 𝐹𝑠 Static force (at static piston stroke) 𝐿𝐵 Bearing length 𝑃𝐵 Bearing pressure 𝑃𝑠 Static pressure (at static piston stroke) 𝜎𝑇 Tensile stress 𝜏𝑠 Shear stress 𝐴 Area 𝐶𝐻 Chamfer length 𝐹 Force 𝐼𝐷 Inner Diameter 𝑀 Moment 𝑂𝐷 Outer Diameter 𝑃 Pressure 𝑉 Volume 𝑊 Weight 𝑔 Gravity Force 𝑚 Mass 𝑟 Radius 𝑡 Thickness xv xv 𝑥 Cartesian coordinate 𝑦 Cartesian coordinate Constants 𝜋 3.141592654 𝑔 9.81 m/s2 or 32.2 ft/s2 Abbreviations 2D Two Dimensional 3D Three Dimensional AFT Aft (rear) of an aircraft AIA Aerospace Industries Association Al-Br Aluminum Bronze AlSi10Mg Aluminum Silicon 10 Magnesium AM Additive Manufacturing AMM Additive Metal Manufacturing ANSI American National Standards Institute ASME American Society of Mechanical Engineers BLDC Brushless Direct Current CAD Computer Aided Design CG Center of Gravity CRES Corrosion Resistant (Steel) DC Direct Current DMLS Direct Metal Laser Sintering EDS Electrodynamic Suspension EMS Electromagnetic Suspension FEA Finite Element Analysis FWD Forward 1.1 Preamble In the summer of 2013, Elon Musk (Tesla, SpaceX) released a white paper proposing a 5th mode of transportation: the Hyperloop [1]. He imagined levitating pods traveling through a low-pressure, near-vacuum tube capable of achieving speeds more than 700 mph (1126 kph) while free of significant air resistance. Consisting of multiple subsystems, Hyperloop pods are expected to include either magnetic or air-bearing levitation as a primary suspension system during high-speed travel, with the magnetic method leading most proposed designs [2]. For added safety and low- speed travel, 100mph (160 kph), the Hyperloop pods should include a traditional deployable wheel system, similar to an aircraft landing gear [1]. When realized, the Hyperloop is expected to be the fastest form of ground transportation on Earth. Constants xvi GD&T Geometric Design and Tolerancing HDWS Hyperloop Deployable Wheel System ID Inner Diameter INBD Inboard ISO International Standards Organization LOI Letter of Intent LTL Lower Torque Link MLG Main Landing Gear MMPDS Metallic Materials Properties Development and Standardization NAS National Aerospace Standards NLG Nose Landing Gear OD Outer diameter OML Outer Mold Line P/N Part Number QTY Quantity RIHT Ryerson’s International Hyperloop Team RPM Rotations per minute SAE Society of Automotive Engineers SS Stainless Steel STOL Short Takeoff and Landing TPIS Tire Pressure Indication System U of T University of Toronto UTL Upper Torque Link VDC Voltage-Direct Current WOW Weight on Wheels GD&T Geometric Design and Tolerancing HDWS Hyperloop Deployable Wheel System ID Inner Diameter INBD Inboard ISO International Standards Organization LOI Letter of Intent LTL Lower Torque Link MLG Main Landing Gear MMPDS Metallic Materials Properties Development and Standardization NAS National Aerospace Standards NLG Nose Landing Gear OD Outer diameter OML Outer Mold Line P/N Part Number QTY Quantity RIHT Ryerson’s International Hyperloop Team RPM Rotations per minute SAE Society of Automotive Engineers SS Stainless Steel STOL Short Takeoff and Landing TPIS Tire Pressure Indication System U of T University of Toronto UTL Upper Torque Link VDC Voltage-Direct Current WOW Weight on Wheels xvii 1.2 The SpaceX Hyperloop Competition On June 15th 2015, Musk and company announced the SpaceX Hyperloop Competition I. The first of its kind, the competition was created to accelerate the development of the Hyperloop but also succeeded in generating public interest. The competition was divided into three main categories: Design and Build (Pod Team), Conceptual Design Only (Pod Team) and Subsystem (Subsystem Team). In support of the competition, SpaceX organized a design weekend (January 2016, Texas A&M) and hosted the final pod race at their headquarters (January 2017, Hawthorne California). To facilitate the races and testing of key technologies, SpaceX built the world’s first Hyperloop test track outside of their headquarters, a near vacuum tube running approximately 1.5 km (1 mile) in length [3], [4]. Out of over 1000 student teams from all over the world, 124 were invited to the Design Weekend at Texas A&M. One year later, in January 2017, 34 teams, comprised of competitors and exhibitors, descended on SpaceX headquarters for the final pod races and product demonstrations. 1 1 1.3 Ryerson’s International Hyperloop Team In September 2015, Ryerson’s International Hyperloop Team (RIHT), led by the author, submitted a letter of intent (LOI) to compete in the SpaceX Hyperloop Competition I. By October 2015, our team comprised of three graduate students and three advisors (industrial and academic), committed to compete in the Subsystem category of the competition. In January 2016, RIHT joined 124 student teams from over 20 countries at the Texas A&M design weekend where we presented our concept for a Hyperloop Deployable Wheel System (HDWS). Selected by the judges for the “Subsystem Innovation Award”, the team spent the entirety of 2016 building a network of 20 sponsors, enabling the development, manufacturing, marketing and testing of our design. To facilitate the increase in workload and to address the need for additional expertise, the team expanded to 6 students and 5 advisors throughout 2016. In January 2017, our team presented 5 completed prototypes at the final phase of the SpaceX Hyperloop Competition in Hawthorne California. 1.4 Research Motivation The author’s motivation to develop a deployable wheel system spun out of text in Musk’s Alpha Document that reads the Hyperloop pod “may also include traditional deployable wheels similar to aircraft landing gear for ease of movement at speeds under 100 mph (160 kph) and as a component of the overall safety system” [1]. Considering the operational requirements, environment, power sources available, and hypothetical geometrical constraints of the Hyperloop transportation system, it became apparent that an-off-the-shelf landing gear or the like would not be sufficient for this unique application. To use aspects of existing technologies and lessons learned for a new design, a detailed literature and patent review of landing gear systems was conducted. The works of Currey [5] and Conway [6], experts in the field of landing gear design, provided many starting points for a new Hyperloop-suited design, particularly in the area of oleo- pneumatic shock absorber design. However, these reviews also left many questions unanswered (e.g. performance impacts of a system in near-vacuum conditions, active control stability, compact design for small operations spaces). These items are not typically addressed in landing gear design (although the author acknowledges the existence of the Space Shuttle landing gears). 2 One key challenge, as it relates to the Hyperloop, is associated with the kinematics required by the design while ensuring performance requirements (e.g. smooth ride operations) are met. It is well understood that some aircraft, helicopters and Hyperloop pods, have very limited space for housing a deployable wheel system and/or landing gear. Prior patent art includes systems that attempt to solve the problem by compressing the shock strut element of a landing gear prior to retraction, and, thus, for the duration of stowage, such as presented in Churchill et al. [7]. These are commonly referred to as shrink or contracting struts [5]. The closest viable solution came from Waide [8], who proposed an electrically contracted shock strut, using a coaxial motor. However, in the configuration proposed by Waide, the shock absorption portion of the system is impacted and does not allow for desired energy dissipation (the purpose of such). Additionally, operational space required by both Waide and Churchill et al.’s designs is likely too large to accomplish the space saving goals imposed by the pod geometry. 1.4 Research Motivation To address the gap in available solutions, the Hyperloop Deployable Wheel System (HDWS) was designed for emergency and low-speed travel, a necessary addition to future Hyperloop transportation systems. Using an innovative, patent-pending deployable and retractable shock strut [9], the system is operable within small spaces and employs advanced technologies and software that enables the system to provide active height stabilization control and can be fitted with wheel motors as well as regenerative braking capabilities [9]. 1.5 Key Objectives and Contributions The objective of the present thesis was to design and develop a deployable wheel system proposed for Hyperloop pods. Using the functional requirements defined within Musk’s Hyperloop Alpha document as a baseline, alongside historical and modern landing gear developments, the HDWS was designed and developed. This process demonstrates how the many disciplines of engineering, such as: strength of materials; statics; dynamics; compressible adiabatic & isothermal gas theory; stress analysis; galvanic corrosion & material compatibility; tribology; cost versus weight; computer aided optimization and program management, interact in a highly iterative design process to develop the HDWS. The key objectives and contributions are to: 1. Investigate what requirements must be considered to develop a Hyperloop subsystem 3 2. Investigate the guidelines, requirements and constraints necessary to develop a deployable wheel system for the Hyperloop and apply those to the creation of the HDWS 2. Investigate the guidelines, requirements and constraints necessary to develop a deployable wheel system for the Hyperloop and apply those to the creation of the HDWS 3. Develop a baseline system architecture of a deployable wheel system for the Hyperloop 4. Address and discuss technical design challenges imposed by the environment of the Hyperloop as they pertain to the developed HDWS system architecture 4. Address and discuss technical design challenges imposed by the environment of the Hyperloop as they pertain to the developed HDWS system architecture 5. Provide a detail design overview of the developed HDWS system architecture for the SpaceX Hyperloop Competition I 5. Provide a detail design overview of the developed HDWS system architecture for the SpaceX Hyperloop Competition I 6. Present key tests performed on prototypes developed under the HDWS system architecture and discuss future challenges and opportunities for future improvements The author’s contribution through the present research and development has led to a patent- pending application, for the design of a “Deployable and Retractable Shock Strut,” filed with the US Patent Office on April 26th, 2017. The application was sponsored by Safran Landing Systems Canada Inc., the assignee. 1.6 Limitation of Current Work The current work is bound by the following limitations: 1. The five prototype Hyperloop Deployable Wheel Systems (HDWS) were not tested in a near vacuum low-pressure environment due to logistical reasons. 1. The five prototype Hyperloop Deployable Wheel Systems (HDWS) were not tested in a near vacuum low-pressure environment due to logistical reasons. 2. Time constraints within the framework of the SpaceX Hyperloop Competition often forced prototype development shortcuts. Whenever present, short comings due to the time constraints are addressed within this report. 3. Budget constraints typically drove a design-to-cost methodology. Consequently, components were not optimized for weight savings, although savings were often realized because of a system performance requirement or a key technology demonstration, such as additive metal manufacturing. 4. For prototyping purposes of proving the basic operational concepts, production quality materials and hardware were not used unless required to meet key performance drivers. 4. For prototyping purposes of proving the basic operational concepts, production quality materials and hardware were not used unless required to meet key performance drivers. 5. The interface architecture was limited by available information (Fall 2015) on pod geometry, layout etc. 4 2 Literature Review This chapter is intended to familiarize the reader with the Hyperloop concept, its major components and the medium of pod transportation. The operational environment and constraints imposed on the system are discussed. A brief section is dedicated to the fundamentals of aircraft landing gear, providing necessary background terminology. Finally, background information on the field of advanced aircraft landing gear system design is presented. Relevant information discussed in this chapter, required to design the Hyperloop Deployable Wheel System (HDWS), is then utilized in Chapter 3. 2.1 The Hyperloop There currently exists only four fundamental forms of transportation: planes, trains, ships and automobiles. These modes of transport use air, rail, water and roads as a medium for operation. Proposed as the 5th mode of transport in 2013, the Hyperloop is conceptualized as a new, very high speed, intercity transportation system [1], [2], [10]. At its core, the Hyperloop transportation system consists of: a set of near-vacuum tubes running between cities, pods traveling through the tubes transporting cargo and humans, and stations at the beginning and end of each route. As a result of sending pods through a self-contained low-pressure tube, the Hyperloop transportation system is immune to wind, rain, ice and fog – weather elements that cause delays or cancelations for every other form of transportation [1], [2], [10]. Acknowledging the potential benefits of quiet supersonic air travel for longer distances, the proposal is intended for corridors in the 900-mile (1500 km) range, such as the now infamous Los Angeles to San Francisco route [1]. As an open-sourced idea first proposed in 2013, various companies such as Hyperloop One [11], Hyperloop Transportation Technologies [12] and Transpod [13] are working to bring the Hyperloop to life in parallel to SpaceX’s Hyperloop Competition. 1.7 Thesis Outline This thesis report is divided into seven chapters, outlined as follows: This thesis report is divided into seven chapters, outlined as follows: Chapter 1 provides the backstory and motivation of this thesis. The contributions that are discussed throughout the thesis are summarized and the limitations of the current work are stated. Chapter 2 contains a literature review of the relevant background information and theory that was used to develop the present HDWS. Background information on the Hyperloop is presented to lay the ground work for the interfacing systems and operational environment challenges. Research on potential solutions is discussed, followed by a brief history of aircraft landing gears. Following the literature review, Chapter 3 provides the product definition for the HDWS by highlighting key functional requirements and performance goals, and defining the prototype’s development scope within the SpaceX Hyperloop Competition framework. Chapter 4 develops the HDWS solution and breaks it down to the subsystem and component level subject to the system architecture defined and highlighted in Chapter 3. Following the detailed system overview, the detailed design of the deployable and retractable shock strut, a complex interdisciplinary concept, is presented in Chapter 5. Chapter 6 outlines the experimental tests performed on the HDWS prototypes. Qualitative and quantitative results are provided where available. Finally, Chapter 7 concludes with a summary of the current work and proposed future work to be considered. The author’s contributions, including co-authored conference papers and presentations, are highlighted in list form. The appendices provide readers interested in design details access to the associated part and assembly drawings, selected concept art, and a sample of raw test data. 5 2.1.1 Pod Overview Key to the Hyperloop architecture is the use of pod structures to transport humans and cargo through the near-vacuum low-pressure environment. Current configurations, as proposed by Musk 6 and developers, expect to transport up to 28 passengers per pod [1]. Because of the environment, the pods are imagined by most, particularly in the aerospace industry, to have an aircraft fuselage frame structure best suited for the cyclic loading induced by traveling between standard (station) and low-pressure environments (tube). A typical pod shall use either passive magnets or air- bearings for the primary suspension (or levitation) system. Both suspension systems have been demonstrated. Hyperloop One has conducted successful tests on the passive magnetic system [14], and student teams have recently demonstrated air-bearing levitation in the SpaceX Hyperloop Competition II [3]. As originally proposed by Musk, current pod designs also use wheels as a secondary mobility system for loading and unloading at stations as well as low-speed travel [14]. 2.1.2 Major Subsystems Breakdown Figure 2.1: Example of a Hyperloop passenger pod, subsystem notional locations (not to scale) [1] Figure 2.1: Example of a Hyperloop passenger pod, subsystem notional locations (not to scale) [1] A typical Hyperloop pod architecture, as illustrated in Figure 2.1, is considered to include, but is not limited to, the following subsystems: A typical Hyperloop pod architecture, as illustrated in Figure 2.1, is considered to include, but is not limited to, the following subsystems: 1) Life Support System: Pressurized air, collected and/or stored in onboard tanks, is required for pressurized cargo and passengers. The design of pressurized section of the pod will be similar to that of aircraft fuselage and or spacecraft [1]. A life support system should also be capable of circulating air and provide a method of regulating temperature. 1) Life Support System: Pressurized air, collected and/or stored in onboard tanks, is required for pressurized cargo and passengers. The design of pressurized section of the pod will be similar to that of aircraft fuselage and or spacecraft [1]. A life support system should also be capable of circulating air and provide a method of regulating temperature. 2) Onboard Power: The pods are proposed to run exclusively on electrical power. The various capsule configurations are to include batteries, like the type used onboard the Tesla Model S, for energy storage, which can be quickly swapped out and charged at each station [1]. 7 7 3) High-speed Levitation System: To achieve the high speeds proposed within the Hyperloop architecture, the pod shall traverse the track using a low-friction interface to reduce the energy required to sustain the speeds and provide a smooth ride [1]. The initial proposal called for air- bearing (or air-cushion) based levitation, like an air-hockey table. However, more recent concepts, like the one shown in Figure 2.2, have opted to use a more familiar magnetic levitation source with the added penalty of larger drag forces at low speeds [15], [16]. 3) High-speed Levitation System: To achieve the high speeds proposed within the Hyperloop architecture, the pod shall traverse the track using a low-friction interface to reduce the energy required to sustain the speeds and provide a smooth ride [1]. The initial proposal called for air- bearing (or air-cushion) based levitation, like an air-hockey table. 2.1.2 Major Subsystems Breakdown Hyperloop One has created a similar “proprietary electric propulsion system” that was first successfully demonstrated in May 2016 [2]. Figure 2.3: Rotor and stator 3D diagram [1] Figure 2.3: Rotor and stator 3D diagram [1] 6) Braking System: Current pod designs propose the use of permanent magnets actuated by electrical mechanical actuators, as a primary braking source. No clear secondary braking system has been proposed, although this requirement has been recognized by those in the industry for safety and future certification activities [2]. The author proposes that a secondary, wheel-mounted braking system, for lower speeds, should be investigated. To design a subsystem for integration to a pod, it is important to understand the interdisciplinary couplings inherent to the Hyperloop System [18]. Thus, a non-exhaustive list of key subsystems has been presented. Information gathered from the present summary, such as the pod’s proposed reliance on electrical power, begs one to consider the available resources at hand when proposing a Hyperloop wheel system design. 2.1.2 Major Subsystems Breakdown However, more recent concepts, like the one shown in Figure 2.2, have opted to use a more familiar magnetic levitation source with the added penalty of larger drag forces at low speeds [15], [16]. Figure 2.2: Hyperloop One’s test “sled” is shown to include (1) magnets for levitating and (2) a rudimentary wheel system in tow [14] Figure 2.2: Hyperloop One’s test “sled” is shown to include (1) magnets for levitating and (2) a rudimentary wheel system in tow [14] 4) Low-speed Levitation System: For low-speed travel less than 100 mph (160 kph), loading passengers and cargo at stations, and as a component of the overall safety system, a deployable wheel system, similar to an aircraft landing gear is included [1], [17]. As recently shown in July 2017, Hyperloop One’s test sled (Figure 2.2) is shown to incorporate a rudimentary retractable wheel system. 5) Propulsion System: The Hyperloop will use an advanced linear motor, suitable for operation within a vacuum and capable of dissipating generated heat. The linear motor proposed by Musk [1] is being developed to accelerate the pod at speeds approaching 760 mph (1220 kph) at a maximum of 1g acceleration to ensure passenger comfort. Musk proposes to equip the rotor (or moving element) on the pod, and the stator (stationary motor element) to the tube structure. 5) Propulsion System: The Hyperloop will use an advanced linear motor, suitable for operation within a vacuum and capable of dissipating generated heat. The linear motor proposed by Musk [1] is being developed to accelerate the pod at speeds approaching 760 mph (1220 kph) at a maximum of 1g acceleration to ensure passenger comfort. Musk proposes to equip the rotor (or moving element) on the pod, and the stator (stationary motor element) to the tube structure. 5) Propulsion System: The Hyperloop will use an advanced linear motor, suitable for operation within a vacuum and capable of dissipating generated heat. The linear motor proposed by Musk [1] is being developed to accelerate the pod at speeds approaching 760 mph (1220 kph) at a maximum of 1g acceleration to ensure passenger comfort. Musk proposes to equip the rotor (or moving element) on the pod, and the stator (stationary motor element) to the tube structure. 8 Hyperloop One has created a similar “proprietary electric propulsion system” that was first successfully demonstrated in May 2016 [2]. 2.1.3 The Tube System The tube system, shown in Figure 2.4, is comprised of large, partially evacuated (0.015 psi or 100 Pa) steel tubes mounted on support pillars. Steel has been the common choice among Hyperloop tube developers for its low cost and weldability. Although two Hyperloop tube systems are currently in operation, no “final size” for the tube has been selected. A recent study in 2015, 9 conducted by a group of researchers at NASA’s Glen Research Center, indicated that the required tube size is highly dependent on the pod geometry required and the intended speeds to travel [18]. Thus, this may be a route-specific decision. Figure 2.4: Artist rendering of a Hyperloop tube cutaway equipped with solar arrays [1] Figure 2.4: Artist rendering of a Hyperloop tube cutaway equipped with solar arrays [1] The tube support columns, to be made of reinforced concrete, will be equipped with specially designed slip joints to combat thermal expansion [1]. Hyperloop One recently announced that they will be incorporating “isolators and dampers” to limit the effects of earthquakes [2]. The tube support columns, to be made of reinforced concrete, will be equipped with specially designed slip joints to combat thermal expansion [1]. Hyperloop One recently announced that they will be incorporating “isolators and dampers” to limit the effects of earthquakes [2]. 2.1.4 SpaceX Hyperloop Tube System In support of the SpaceX Hyperloop Competition, SpaceX built a one-mile long, six-foot in diameter Hyperloop tube system (see Figure 2.5), located at SpaceX headquarters in Hawthorne, California. The tube is lined with concrete base on which aluminium (Al 6101) track plates and an aluminium I-beam are fixed [3]. SpaceX claims that the Hyperloop system developed is approximately half scale, when compared to the eventual production size [19]. Since the present work was originally designed at scale for the SpaceX Competition, the specifications of the tube/track, referenced for the author’s design in Chapter 3, correspond to the SpaceX Hyperloop tube system. 10 Figure 2.5: Image of the SpaceX Hyperloop sub-scale test track, image taken January 2017 Figure 2.5: Image of the SpaceX Hyperloop sub-scale test track, image taken January 2017 2.2 Hyperloop Pod Operation Environment In addition to understanding the interdisciplinary interactions between subsystems within the pod architecture while developing a deployable wheel system, an understanding of the external operational environment must be understood and quantified when necessary. 2.2.2 High-speed Operation The Hyperloop pod will theoretically travel at speeds of up to 760 mph (1,220 kph, Mach 0.99 at 68 ºF or 20 ºC) [1]. These speeds, not yet achieved during Hyperloop testing [14], are capable of being reached in part due to the passive magnetic or air bearing levitation systems. That is, at high speeds, conventional wheel and axle systems, like on a car or landing gear, become impractical due to high frictional losses and dynamic instability [1]. A reader familiar with aircraft landing gear design can appreciate that the instability of a wheeled system at high speeds can result in shimmy loading [5]. Liu et al. [21], published authors in the art of Maglev train design, have also indicated the extreme wear and tear from the frictional loading between wheels on rails at high speeds. This may also lead to accelerated equipment deterioration. Since a wheel system designed for the Hyperloop may be required in high-speed emergency situations, a system shall be designed with consideration of all the above aspects. Particularly, one can now appreciate another reason for the retractability of a wheeled system. That is, a fixed system, constantly cycling through touch-and-go loading due to changes in magnetic and air levitation distances, may cause premature wear, especially at high speeds. 2.2.1 Low-pressure Environment The pod will be subjected to cyclic pressure loading as it transitions from the pressurized stations (14.7psi or 101.3kPa) to the low-pressure tube (0.015 psi, or 100 Pa). In emergency situations, the rate of depressurisation, due to a tube breach or leak, would be uncontrolled. Regardless, it will be necessary to design the pod frame to withstand this pressure loading. If a deployable wheel system is to use a typical pneumatic tire/wheel configuration, the pressure changes must be accounted for or an alternate “tire” material, perhaps unpressurized, must be selected. However, the loading due to pressure is only one consequence of a low-pressure tube. Another critical factor imposed on the design of the pod and its non-pressurized subsystems, like a deployable wheel system, is the effects of outgassing. Outgassing is the release or evaporation of gases that were trapped, dissolved or absorbed in a material [20]. Thus, the design engineer 11 must be cautious of selected lubricants, adhesives and even materials that can be prone to outgassing. Gases omitted from materials within the vacuum have the potential to contaminate and sometimes cloud optical sensor equipment. The tendency of a material to evaporate is defined by its volatility [20]. This becomes a critical design element when considering the lubrication of joints and the seizing thereof after a long period of outgassing. The design of the HDWS proposes methods to reduce and eliminate the possibility of seizure at all rotational wear surfaces, as discussed further in Chapter 3. 2.2.3 Thermal Management As noted by Musk [1] and Chin et al. [18], each pod passing through the tube adds energy to the surrounding low-pressure air in the form of heat. When the frequency of pod launches is considered, the heating of the overall environment becomes non-trivial. Thus, in the absence of normal atmospheric pressure, and the circulation thereof (convective cooling), a significant 12 medium for heat dissipation is lost [1]. Consequently, when designing subsystems, such as a deployable wheel system, the thermal management must be considered and addressed. medium for heat dissipation is lost [1]. Consequently, when designing subsystems, such as a deployable wheel system, the thermal management must be considered and addressed. 2.3.1 A Brief History Landing gears support the entire weight of the aircraft during takeoff, landing and ground operations [22]. They are designed to endure the harsh loading conditions at touchdown and ensure a soft and safe landing. Because of this, one author refers to landing gears as “the essential intermediary between the aeroplane and catastrophe” [6]. According to Currey [5], the first aircraft to use wheeled landing gears appeared shortly after the Wright Brothers’ first flight in 1903. However, it wasn’t until World War II that aircraft began to adopt a more recognizable architecture. The addition of shock absorbers, a key component of modern landing gear, became necessary as aircraft weight and rate of descent (sink) speeds increased while runways surfaces became harder and wartimes demanded performance on uneven terrain. These shock absorption designs included modern oleo-pneumatic (oil and gas) struts in addition to less common rubber blocks, compression springs, and liquid springs, to name a few [5]. In parallel, as aircraft technologies advanced and enabled higher flight speeds, the retractable landing gear became a popular method to improve aerodynamic efficiency. Nowadays, modern aircraft utilize state-of-the-art landing gears equipped with numerous and typically complex systems such as steering, electrically operated composite brakes, hydraulic extension and retraction actuators in addition to complex electrical sensing and control instrumentation. It is important to note that not all landing gears use wheels – some use skis, while others that land on water use pontoons. However, the subject of this thesis is closely related to retractable wheeled landing gears so other concepts are omitted for brevity. For more detailed information regarding the history of landing gear, wheeled and otherwise, reference [5], [6] and [22] provide a great starting point. 13 2.3.2 Key Components and Configurations This thesis presents a novel solution to retain the benefits of a trailing arm configuration, shown in Figure 2.7, while at the same time achieving better retraction architecture for the Hyperloop pod. To appreciate the new approach, common landing gear components are briefly defined next to equip the reader with basic landing gear terminology. 2.3.2 Key Components and Configurations Landing gear architecture is constantly evolving to meet the needs of each new aircraft’s unique performance requirements. Early configurations, such as those seen in World War I, made use of a tail gear arrangement (Figure 2.6) with the main landing gear (MLG), fixed, or, in later years, retractable, located forwards of the aircrafts center of gravity (CG). Consequently, this required support in the tail from a third wheel [22]. Later, during World War II, as aircraft achieved higher speeds, the retractable landing gear in a tricycle configuration became a popular method to reduce parasite drag and improve aerodynamic efficiency at high speeds. These configurations typically consisted of a single nose landing gear (NLG) at the front and at least two MLGs slightly aft of the aircraft CG [5]. In all cases, landing gears are attached to rigid primary structural elements – typically wing spars, and the fuselage structure (nose and mid) are primary candidates. The most relevant designs to the current subject are wheeled configurations. Figure 2.6: Tail wheel configuration landing gear on a STOL Maule MX-7-235 Super Rocket Figure 2.6: Tail wheel configuration landing gear on a STOL Maule MX-7-235 Super Rocket The development of landing gear kinematics and part geometries were discovered to be interdependent during the design process. Since landing gear designs are heavily centered on their retraction and extension capabilities, landing gear was used as a baseline for the HDWS conceptualization, rather than attempting to adapt fixed wheel/tire configurations on cars and trains. After reviewing existing architectures, the author chose to closely mimic that of an aircraft trailing arm MLG, an example of which is shown in Figure 2.7. However, a distinct feature of this concept is the requirement of an inboard swinging action for retraction – an action that would be logistically difficult within a Hyperloop pod design space, if only because of its proximity to the ground (sub-track). 14 14 Figure 2.7: General arrangement of a retractable MLG Trailing arm configuration [23] Figure 2.7: General arrangement of a retractable MLG Trailing arm configuration [23] This thesis presents a novel solution to retain the benefits of a trailing arm configuration, shown in Figure 2.7, while at the same time achieving better retraction architecture for the Hyperloop pod. To appreciate the new approach, common landing gear components are briefly defined next to equip the reader with basic landing gear terminology. 2.3.3 Common Components It is fair to say that most landing gears, at least within similar classes of aircraft, use very similar design features and components. Landing gears typically consist of the following key components [22]: Shock Absorber – Referred to by Currey as “usually the most complex part of the landing gear,” the shock absorber (or shock strut) is designed to dissipate energy during taxi, landing and takeoff [5]. The most common modern configuration is oleo-pneumatic with damping to limit the piston’s rate of motion and avoid spring back effects [24]. The shock absorber is typically used as a main structural member, interfacing with the aircraft frame in NLG configurations, subject to bending 15 loads, or as an axially loaded member of the MLG trailing arm configuration, shown in Figure 2.7. The design of the shock absorber for the present work is covered in Chapter 5. Main Fitting – In MLG configurations, the main fitting or “trunnion leg” as shown in Figure 2.7 is the primary interface between the landing gear and the aircraft. A main fitting is typically rotationally mounted via oppositely disposed trunnion pins as can be seen at the top left section of Figure 2.7. Trailing Arm – In MLG configurations, the trailing arm acts as a lever arm, connected to both the shock strut assembly and main fitting through pinned sockets or lugs. Typically, trailing arms are subject to tension loading and are designed to resist torsion induced at landing, during taxi and otherwise. Generally, the trailing arm is designed to ensure only axial loading is transferred to the attached shock strut. It is further noted, as added value to the author’s present work, that the trailing arm configuration is highly stable at landing and taxiing due to its lever arm configuration. Geometrically, referring to Figure 2.7, the vertical wheel axle travel (VAT) and the travel (stroke) of the shock absorber piston are not the same. This allows for energy absorption through relatively large displacements at the axle, compared to the direct 1:1 relationship in cantilever shock struts like the NLG configuration previously discussed. Drag and/or Side Brace Arm – Drag and/or side brace arms are often required as additional load bearing members. Some larger aircraft with higher loading and longer main fitting/shock strut members tend to incorporate both drag and side braces (see Figure 2.7) in their design for added stability and load carrying capacity. 2.3.3 Common Components Steering Systems – Used almost exclusively on NLG architectures, landing gears can be equipped with either rack and pinion or tandem actuator steering subsystems. As a steering system was not considered in the present work interested readers are referred to Currey [5] who provides an excellent detailed overview into steering system designs and their operation. Wheels and Brakes – Landing gear are typically equipped with specially designed pneumatic aircraft tires. Most gears, typically the MLG, contain carbon or steel brakes, actuated either by hydraulic or electrical means. Retraction Mechanism– The retraction of a landing gear is generally accomplished, in part or wholly, by a retraction actuator disposed between the landing gear structure and the airframe. 16 These systems typically operate using hydraulic fluid supplied throughout the aircraft. As can be found in various patents the design of electric or hybrid retraction actuators is an ongoing subject of interest. Feedback Sensor Equipment – Weight on Wheel (WOW) sensors are common main stay alongside tire pressure indication systems (TPIS) and steering feed-back transducers. As landing gears become more and more electric, there is likely to be a demand for increased sensor presence, including health monitoring. 2.4 Existing Patents A patent search was conducted to find state-of-the-art landing gear retraction systems, shock strut designs, and kinematic layouts suited for tight spaces. The review revealed that the subject of “contracting” the shock absorber prior to retraction, but retaining a separate traditional retract actuator, is frequently addressed. These “shortening” or “contracting” mechanisms frequently provided excellent solutions for cantilever type landing gears, such as an NLG and canted MLG configuration. In some cases, as presented by Sharples [25], designs enable a kneeling function that is useful for helicopters and military transports. Mostly, however, the goal is to ensure the landing gear would fit inside the allotted stowage volume in the aircraft. Churchill et al. [7] presented a configuration that is capable of retracting and extending the shock strut piston into the outer cylinder by transferring pressurized hydraulic fluid into a shrink chamber, resulting in an overall lengthening and shortening of the shock strut. Although this provides a potential means for saving space when stowed, it relies on external pressurized hydraulic fluid sources, which would not be available on Hyperloop pods. Sharples [25] presents a tempting solution (Figure 2.8) to not only shrink the effective length of the shock strut, but also to retract it into the aircraft using a ball screw and ball nut guide, both externally mounted. The design uses both electric power (for retraction) and hydraulic fluid pressure (for contraction) ported from the aircraft. The adaptation and detail design of the proposed architecture would require the addition of a hydraulic reservoir and a significant portion of vertical space in a pod frame design. The use of a screw and nut for retraction will eventually be adopted, in another form, by the author. 17 Figure 2.8: Sharples kinematic retraction configuration (left) with a cutaway of the contractive shock strut (right), shown as items 11 and 25 in the kinematic configuration (left) [25] Figure 2.8: Sharples kinematic retraction configuration (left) with a cutaway of the contractive shock strut (right), shown as items 11 and 25 in the kinematic configuration (left) [25] In another patent, Veaux et al. [26] provided a different approach, internalizing the contraction mechanism of the shock strut. In one embodiment, shown in Figure 2.9, the design integrates an electric motor into the bottom of a modified shock strut piston. 2.4 Existing Patents Using a rotating internal ball screw shaft and a fixed internal ball nut, the total length of the landing gear strut can be adjusted to modify the attitude of the airplane. This procedure is to be done when stationary or at low speeds without affecting internal gas spring properties. In its current embodiment, this concept does not aid in the act of retraction, nor in any appreciable shortening and may in fact add to the overall length of such strut by design. Furthermore, a large internal diameter is required to house the motor, the size of which is typically a function of the required torque output. In another patent, Veaux et al. [26] provided a different approach, internalizing the contraction mechanism of the shock strut. In one embodiment, shown in Figure 2.9, the design integrates an electric motor into the bottom of a modified shock strut piston. Using a rotating internal ball screw shaft and a fixed internal ball nut, the total length of the landing gear strut can be adjusted to modify the attitude of the airplane. This procedure is to be done when stationary or at low speeds without affecting internal gas spring properties. In its current embodiment, this concept does not aid in the act of retraction, nor in any appreciable shortening and may in fact add to the overall length of such strut by design. Furthermore, a large internal diameter is required to house the motor, the size of which is typically a function of the required torque output. 18 Figure 2.9: An illustration provided in Veaux et al. illustrating a motor and gearbox assembly embedded into the lower half of a piston to lengthen the strut [26] Figure 2.9: An illustration provided in Veaux et al. illustrating a motor and gearbox assembly embedded into the lower half of a piston to lengthen the strut [26] Another variant on an internally integrated screw shaft, nut and motor system is proposed by Currey and Renshaw [27]. This configuration, proposed for short takeoff and landing (STOL) vehicles, allows for a considerable amount of lengthening and shortening and thus provides a means for vehicle attitude adjustment. As with the design of Veaux et al. [26], Currey and Renshaw’s design is not capable of providing a decreased storage length. 2.4 Existing Patents It does however include a clever variance in the design – a rotating ball nut and a fixed lead screw by means of an adjacent torque rod, shown in Figure 2.10. 19 Figure 2.10: An illustration of the design proposed by Currey and Renshaw [27] Figure 2.10: An illustration of the design proposed by Currey and Renshaw [27] Waide [8] presents a landing gear, developed for use in helicopters, that enables small space operation, planner motion (advantageous for the Hyperloop design), electrical operation and even active stabilization capabilities. Although the overall kinematic system of the design by Waide, shown in Figure 2.11 is far more advantageous than the traditional inward swinging MLG retraction, the gear still occupies a considerable vertical stowage space within the aircraft body. One familiar with the art of shock strut design will note that the system exhibits a questionable interference with the internal shock strut geometry, due to the space allocated for the lead screw rod, shown in Figure 2.12, that would prohibit adequate gas spring properties (mainly pressure over area), drive a large internal shock strut diameter, and limit the integration of dampening features. 3 Product Definition and Preliminary Design In Chapter 2 the reader was introduced to the Hyperloop transportation system concept and received a breakdown of its major components and subsystems. The author’s review and understanding of the operational environment along with its challenges were addressed and will be frequently referenced throughout the remainder of this thesis. Further, a brief overview of aircraft landing gear and a patent search regarding innovative retraction and shock absorbing configurations were presented. As will be discovered in the present chapter, these subjects together provide a necessary background to appreciate the author’s Hyperloop Deployable Wheel System (HDWS) design. This chapter outlines the product definition for the HDWS, including an overview of the system architecture and kinematic arrangement. This will provide the basis of knowledge required to appreciate the detailed component discussions in Chapter 4 and Chapter 5. A component definition scheme is presented and partially illustrated using a product structure tree – an essential tool in organization of a design project. Lastly, the design process followed for the present development is summarized. 3.1 Precondition: Why Wheels? As discussed in Chapter 2, the Hyperloop pod may utilize magnetic or air-bearing levitation to enable high speed travel without frictional losses. The necessity of an additional system, in the form of a wheeled system, will now be affirmed. 2.4 Existing Patents 20 Figure 2.11: An image (left) illustrating key components and with the advantageous kinematic architecture displayed (right), proposed by Waide [8] Figure 2.11: An image (left) illustrating key components and with the advantageous kinematic architecture displayed (right), proposed by Waide [8] Figure 2.11: An image (left) illustrating key components and with the advantageous kinematic architecture displayed (right), proposed by Waide [8] Figure 2.12: Two possible configurations of a retractable shock strut, proposed by Waide [8] Figure 2.12: Two possible configurations of a retractable shock strut, proposed by Waide [8] 21 Generally, the design proposed by Waide [8] introduces undesirable load paths highlighted by: • A long, slender, loaded lead screw rod, that when subject to axial loading, could tend to buckle • A configuration that allows direct axial load transfer through the motor gear shaft • A long, slender, loaded lead screw rod, that when subject to axial loading, could tend to buckle • A configuration that allows direct axial load transfer through the motor gear shaft • Anti-rotation using an internal sliding rod linked directly to the shock strut piston, fixed to a trailing arm with a spherical bearing (this is not a good design, spherical bearings cannot react torque) The designs’ clear optimization of shock strut dead length (the limiting of unused volume), kinematic simplicity and reduction in part count (no side brace, no separate retraction actuator) are acknowledged. The designs’ clear optimization of shock strut dead length (the limiting of unused volume), kinematic simplicity and reduction in part count (no side brace, no separate retraction actuator) are acknowledged. The proposed shock strut configuration for the design of the HDWS, to be discussed in subsequent chapters, addresses the specific volumetric constraints and retraction/extension requirements imposed by Hyperloop pod and tube geometry and thus provides a clear benefit over the configurations mentioned here. 22 22 3.1.1 Air Levitation Pod A Hyperloop pod relying on air-bearing levitation, as defined by Musk [1], uses compressed air ejected through pod-mounted skis to enable the pod levitation. During a typical journey, the pod replenishes onboard air storage tanks with compressed air received through the intake compressor mounted at the front of the pod. However, when at rest, such as in a station, it is not reasonable for a pod to use the stored air to maintain levitation as it awaits passengers and cargo. Further, if air- levitation is to be used because of the efficiencies at high speed (no rolling friction or excessive 23 wear), then why not utilize a wheeled system for low-speed and tight turns, as proposed by Musk. Further, if a pod is to rely on compressed air for levitation, what happens if there is a failure in the air supply system? As proposed by Musk [1] in the Alpha document, the Hyperloop shall utilize a retractable wheel system to increase the safety of the pod. 3.2 Former University of Toronto Partnership In Spring 2016 the author established a partnership with the University of Toronto (U of T) Hyperloop pod team to install four deployable wheel systems. Therefore, the original design, submitted to SpaceX in January 2016, was scaled down in size to better suit the U of T configuration. Thus, a large portion of the work presented in the following sections has been completed utilizing information provided by the U of T team, which influenced the design of the system’s kinematics (Section 3.5.2) and shock strut gas spring design (Chapter 5). In the summer of 2016, the U of T withdrew from the competition but the author’s present embodiment of the HDWS remains developed with information exchanged throughout that time. 3.1.2 Magnetic Levitation Pod Hyperloop pods using passive magnetic levitation have been successfully demonstrated in the SpaceX Hyperloop Competition [3] and by industry leader Hyperloop One [14]. This arrangement uses permanent magnets fixed to the Hyperloop pod, typically on externally mounted pod skis, which when translated along a conductive sheet at high speeds (aluminum sub-track) induces a current in the sub-track and generates an opposing magnetic field. The magnetic field generated by the relative motion between the pod and the conductive sub-track causes a repulsive force, lifting the vehicle. This method, however, is not novel and falls under a specific category of Maglev known as electrodynamic suspension (EDS) which has been demonstrated by engineers in Japan [28]. The permanent magnetic EDS system is passive and thus does not require a constant power source like the most common Maglev technology, electromagnetic suspension system (EMS). Using a Maglev based EDS system would ensure that a failure in the pod’s propulsion system would not cause the pod to fall onto the track instantly. This contrasts with EMS that requires a constant power to maintain levitation and would require a back-up power source to ensure levitation is maintained in case of a power failure [21]. A drawback to the EDS passive magnetic approach is that the induced current on the conductive sub-track results in a magnetic drag force. The drag force has been recorded in literature to increase with speed until it reaches a peak value and is then slowly decreased as the speed increases [29]. A plot of the normalized force from the EDS Maglev is shown in Figure 3.1 with respect to translational velocity. As an added result, the passive EDS system cannot levitate the Hyperloop pod at a station and can only levitate the pod at sufficiently high speeds, about 94 mph (150 kph) [21]. Consequently, a Hyperloop pod equipped with passive magnetic levitation should use wheels at rest and when accelerated, like an aircraft, as suggested for EDS Maglev configurations by He et al. [28] and Paudel [29]. 24 Figure 3.1: Normalized force of the EDS Maglev plotted against translational velocity [29] Figure 3.1: Normalized force of the EDS Maglev plotted against translational velocity [2 3.3 Design Objectives The main objective of this work was to design and develop a wheeled suspension system that operates in tandem with the proposed air bearing or passive magnetic levitation systems. The system shall be primarily designed to operate at low speeds within the tube environment defined in Chapter 2, be retractable to minimize drag, and act as a component of the overall safety system. 25 A goal was to consider as many key design drivers as possible throughout development to ensure a scalable and complete solution is presented. Using the functional requirements defined within Musk’s Hyperloop Alpha document as a baseline, alongside historical and current landing gear development literature, the HDWS was designed and developed. The specific objectives targeted throughout the design and development of the HDWS are as follows: The specific objectives targeted throughout the design and development of the HDWS are as follows: 1) Develop a prototype wheeled system capable of operating nominally at 180 mph (290 kph) to ensure that it can operate at predicted max speeds, in case of emergency, within the SpaceX pod competition framework (lower max speeds) 2) Ensure that the deployment of the wheel system limits the direct transfer of load and vibrations throughout the vehicle using a shock absorber (+ damper if required) 3) Develop a system with advantageous kinematics that is scalable and operable in the least amount of volume possible 4) Develop a system that is electrically powered (no requirement for the porting of pressurized fluids) and thus compatible with known pod power supplies (e.g. lithium-ion batteries) 5) Develop a system that is simple to integrate, preferably two mounting points 6) Ensure that a wheel system is capable of retracting and extending quickly, a key feature in an emergency scenario 7) Address the issues presented by near-vacuum operation (e.g. outgassing) 8) Design a common wheel system that can be duplicated and distributed throughout the pod with minimal modifications required for left-hand, right-hand, front and back installations 9) Identify and investigate key weight saving opportunities since a wheeled system is “dead weight” during most of regular vehicle operation 9) Identify and investigate key weight saving opportunities since a wheeled system is “dead weight” during most of regular vehicle operation 3.4 Product Development Guidelines and Constraints At present time, no regulatory body has been established to govern the certification requirements for the Hyperloop and its various subsystems. Consequently, the author’s present work has used a combination of aircraft landing gear industry design standards, examples of performance 26 guidelines found in literature, and the information contained within the SpaceX competition tube specifications [4] and competition rules manual [19]. guidelines found in literature, and the information contained within the SpaceX competition tube specifications [4] and competition rules manual [19]. 5) All high-pressure gas systems shall have vent and relief valves [19]. 3.4.1 Design Constraints The HDWS was subject to extreme space constraints imposed by the U of T pod configuration. That is, the HDWS was to operate within a 20-in by 20-in box, as imposed on a side view of a pod layout, shown in Figure 3.2. Furthermore, the wheel system was to be fully compatible with the SpaceX test track as outlined in the SpaceX Tube Specification [4] and partially illustrated in Figure 3.3. Based on the width of the aluminum sub-track, shown Figure 3.3, the HDWS was designed to fit within the maximum sub-track engagement width of 12-in. Figure 3.2: A 2D side view of a Hyperloop pod with the HDWS positioned, not to scale Figure 3.2: A 2D side view of a Hyperloop pod with the HDWS positioned, not to scale Figure 3.2: A 2D side view of a Hyperloop pod with the HDWS positioned, not to scale The following design constraints and guidelines are presented as follows: The following design constraints and guidelines are presented as follows: 1) The HDWS shall be operable within the volume provided by the mating pod, using approximated values selected by the author in consultation with the U of T Pod team (L=20- in, W=12-in, H=20-in). 2) Designs shall be compatible with the SpaceX tube (sub-track) structural tolerances, defined by the SpaceX Hyperloop tube specification [19]. 3) All components shall maintain structural safety factors (SF). SpaceX specifies a SF of 2.0 for reasonable load cases (e.g. acceleration, deceleration, pressure) [19]. 4) All compressed gas shall be limited to pressures of 4,000 psi (27.6 MPa) [19]. 5) All high-pressure gas systems shall have vent and relief valves [19]. 27 6) Flow directions through vent and relief valves shall be oriented for the safest possible outcome in the event of a pressure burst [19]. 6) Flow directions through vent and relief valves shall be oriented for the safest possible outcome in the event of a pressure burst [19]. Figure 3.3: Partial cross-section view of the SpaceX Hyperloop sub-track interface, dimensions shown in inches, with reference to the specification [4] Figure 3.3: Partial cross-section view of the SpaceX Hyperloop sub-track interface, dimensions shown in inches, with reference to the specification [4] 3.4.2 Performance Constraints and Guidelines 1) The HDWS shall be designed to operate under the accelerations highlighted in Table 3.1, provided by SpaceX, for the purposes of the Hyperloop competition. The HDWS shall be tested under these conditions, where possible, prior to initial full pod testing and integration. Table 3.1: Representative SpaceX pusher acceleration values (2016) [4] Pod Mass (kg) Pod Mass (lbm) Pod Acceleration (g) 250 550 2.4 500 1100 2.0 750 1650 1.7 1000 2200 1.5 1500 3300 1.2 2000 4400 1.0 2500 5500 0.9 3000 6600 0.8 4000 8800 0.6 5000 11000 0.5 28 2) A wheeled system shall perform nominally when subject to the Hyperloop system operating pressures ranging from 14.7 psi (101.3 kPa) to 0.15 psi (100 Pa) as per the alpha document [1] and tube specifications [4]. 2) A wheeled system shall perform nominally when subject to the Hyperloop system operating pressures ranging from 14.7 psi (101.3 kPa) to 0.15 psi (100 Pa) as per the alpha document [1] and tube specifications [4]. 3) A wheeled system shall be nominally operable, at a minimum, at sustained speeds between 0- 100 mph (0-160 kph) as specified per the alpha document [1]. 3) A wheeled system shall be nominally operable, at a minimum, at sustained speeds between 0- 100 mph (0-160 kph) as specified per the alpha document [1]. 4) While deployed, a wheeled system’s operation shall ensure that the pod and any externally mounted features (e.g. air-bearing skis) do not contact the SpaceX tube sub-track 5) A wheel system should deploy to an agreed upon position (agreed between the supplier and pod manufacturer) within a “reasonable time” to assist in a “levitation loss” emergency scenario. The retraction speeds shall be determined in the same manor and the following discussion points are now offered. a. A “reasonable time” shall be defined based on future pod emergency scenario analysis b. As a reference, Currey [5] notes that U.S. Navy aircraft require that the gear “shall be retractable in no more than 10s” and “extensible in less than 15s” 3.4.3 Material and Lubrication Guidelines passivation shall be performed on all corrosion resistant steel (CRES) components [37] e. other methods of surface protection, as available, shall be used to satisfy material compatibility as per the galvanic tables [32] and to protect from natural corrosion 4) Material selection shall be selected based on the desired end-form and function (e.g. a bronze based material shall be used in a rotating and wear surface due to its low friction interaction with aluminum and steel) [5]. 4) Material selection shall be selected based on the desired end-form and function (e.g. a bronze based material shall be used in a rotating and wear surface due to its low friction interaction with aluminum and steel) [5]. 5) Wherever possible, corrosion resistant steel, passivated, shall be the preferred choice of steel, especially for pins and sealing surfaces [5]. 6) Bushings shall be procured and designed using CRES or bronze based materials only, as is the typical standard in the industry [5]. 7) Lubrication used shall be certified for use in a vacuum, ensuring lubricative properties are retained. Consideration shall be given to lubrication forms such as: a. Dow Corning® high-vacuum silicone grease [36] b. Self-lubricating liners, such as KamaticsRWG’s KAron® [31] As a final note, it is important to recognize that above guidelines should be satisfied for the development of a production grade HDWS product. However, the present work, due to budgetary reasons, could not always use the optimal materials to meet the above guidelines. 3.4.3 Material and Lubrication Guidelines The material and lubrication used for the HDWS were based on standard material and tribology practices, specifically those defined in the aircraft design industry. Based on this information, and the information presented in Chapter 2 regarding the effects of outgassing, the following requirements and guidelines were established: 1) Material strength properties, specified by part suppliers and the Metallic Materials Properties Development and Standardization (MMPDS-07) handbook [30], shall be observed, including: a. static yield pressures 𝜎𝑌𝑆 1) Material strength properties, specified by part suppliers and the Metallic Materials Properties Development and Standardization (MMPDS-07) handbook [30], shall be observed, including: a. static yield pressures 𝜎𝑌𝑆 1) Material strength properties, specified by part suppliers and the Metallic Materials Properties Development and Standardization (MMPDS-07) handbook [30], shall be observed, including: Development and Standardization (MMPDS-07) handbook [30], shall be observed, including: a. static yield pressures 𝜎𝑌𝑆 a. static yield pressures 𝜎𝑌𝑆 b. metallic bearing allowable pressures 𝜎𝑌𝐵 b. metallic bearing allowable pressures 𝜎𝑌𝐵 c. self-lubricating bearing liner allowable pressures, dynamic and static [31] 2) Material compatibility, utilizing the galvanic tables found in industry literature [32] shall be enforced to ensure compatible materials were selected for joints, or: 3) The materials shall be treated for corrosion protection, considering: a. anodization, all over exposed aluminum components for corrosion resistance (anodization is also used in spacecraft design with a low-risk of outgassing [33], [34]) 29 b. cadmium plating, applied to outer diameter of off-the-shelf bushings (Note: best avoided if possible because of its susceptibility to outgassing) [35] b. cadmium plating, applied to outer diameter of off-the-shelf bushings (Note: best avoided if possible because of its susceptibility to outgassing) [35] b. cadmium plating, applied to outer diameter of off-the-shelf bushings (Note: best avoided if possible because of its susceptibility to outgassing) [35] b. cadmium plating, applied to outer diameter of off-the-shelf bushings (Note: best avoided if possible because of its susceptibility to outgassing) [35] b. cadmium plating, applied to outer diameter of off-the-shelf bushings (Note: best avoided if possible because of its susceptibility to outgassing) [35] c. installation and assembly of matting components at joints shall be lubricated [5] using grease (preferably vacuum certified, such as Dow Corning® [36] high-vacuum silicone grease) c. installation and assembly of matting components at joints shall be lubricated [5] using grease (preferably vacuum certified, such as Dow Corning® [36] high-vacuum silicone grease) d. 3.5 HDWS Architecture and Kinematics A key design consideration for the development of a deployable wheel system for the Hyperloop pod is that its wheel system be deployable and retractable. This creates minimal impact to the aerodynamic efficiency of the pod outer mold line (OML) and insures that drag, produced at high speeds within the enclosed near-vacuum tube diameter, is kept to an absolute minimum [5], [18]. Of initial importance was ensuring that the HDWS’s geometry allowed for operation within the small space 30 provided by the typical Hyperloop competition pod. Thus, a key element in the design of the deployable wheel system, like landing gear, was the development of a product architecture and kinematic layout. Since the end goals – retractability, extensibility and operation at high speeds – are all common elements in aircraft landing gear design, this work focused on evolving these designs to fit the needs of the Hyperloop. 3.5.1 HDWS Architecture Definition Canted landing gears are found to require a large forward sweep area for retraction and extension, without any shortening mechanism [5]. A stowed landing gear with the shock strut in an initial extended position (no load applied), as is common during flight, requires an unrestricted sweep volume to be successfully swung into “landing” position from a FWD, AFT or INBD direction. After successfully deploying, the landing gear would touch the ground with an unloaded shock strut, and compress to a static shock absorber stroke thereafter. In the context of the Hyperloop pod, the pod would “land” on the tube sub-track and begin to compress the stroke using the extension actuator. For an optimized shock strut to reach a typical static stroke, as discussed by Currey [5], it would require at least 70% of the piston stroke, meaning that the pod will have surely “touched down” on the tube surface through its magnetic or air-bearing skis prior to the strut reaching a statically loaded position capable of maintaining a steady pod’s weight. Thus, a canted gear without shortening prior to touchdown or without lengthening capabilities at touchdown (fully extended piston stroke) is not considered feasible for the present application. Further, should a canted gear be sized and shrinkable to an appreciable stowage size, the design still requires the addition of a drag brace member and retraction mechanism, with retraction classically accomplished using a hydraulic actuator. Considering the canted gear’s stated deficiencies for the current application, an MLG trailing arm architecture was adopted to the benefit of stability and the possibility of kinematic alterations. Specifically, the elimination of the inboard swinging element, requiring a retraction actuator and typically a side brace member, was proposed in favor of an “all-in-one” shock strut and electric retraction solution. Of interest for preliminary 2D kinematic analysis are the stowage compartment dimensions imposing constraints on the space in which the HDWS must operate. It was decided, based on 31 these constraints, that a fully planar 2D HDWS operation best suited the deployment characteristics desired. However, this posed a problem with space requirements due to the nature of gear storage in the retracted position. The hybrid actuator concept was proposed to address this issue, as will be described in the present section. An initial proposal for a preliminary system architecture is shown in Figure 3.4. 3.5.1 HDWS Architecture Definition This diagram depicts a stowage frame (approximated by a 2D box as drawn), the tube surface, of which the HDWS will be deployed onto, and a familiar trailing arm architecture. Figure 3.4: The HDWS preliminary configuration, stick diagram, not to final scale Figure 3.4: The HDWS preliminary configuration, stick diagram, not to final scale For the initial configuration, five key points were proposed to define the geometry of the system. In this early configuration, referring to Figure 3.4, a trailing arm, comprised of points (1) – (4) is attached to a pod-fixed point (1) consisting of a revolute joint, and thus allowing the trailing arm 32 to rotate. The trailing arm is equipped with a wheel at an axle (2). As is typical in an landing gear trailing arm configuration, a shock strut, comprised of points (4) – (5) is proposed and fixed at point (4) to the trailing arm by a 2D revolute joint (later a spherical joint in 3D) and by a revolute joint, fixed to the pod frame at point (5). Thus, in the current description, a force applied at (2) in the vertical y-direction would cause the trailing arm to pivot counterclockwise about point (1) and the shock strut member disposed between points (4) and (5), consisting of a piston and cylinder, to contract in length. This is a desirable result; however, the present description (Figure 3.4) does not yet propose a method for extending and retracting the system from the present state. Now, consider a member (4) – (5), shown in Figure 3.5, further comprised of a piston, shock strut cylinder and lengthening device of unknown design, co-axially arranged, mounted between points (4) and (5) in Figure 3.4 . In such a configuration, it becomes conceivable to develop kinematics that allow for the new member (4) – (5) to provide both extension, retraction, shock absorbing and possibility active height adjustment during operation to adjust the pod height above the tube surface as required. Figure 3.5: The HDWS Hybrid actuator concept illustration Figure 3.5: The HDWS Hybrid actuator concept illustration Such a system could be designed and developed using hydraulic fluid, supplied by a reservoir, to pressurize the area within the “lengthening device” and cause the configuration shown in Figure 3.4 to extend and contract. 3.5.1 HDWS Architecture Definition A solution like this, however, poses two main problems: the 33 requirement for an externally supplied hydraulic line (hydraulic systems are not in the current pod architectures) and the mounting point (5) would need to be placed relatively far vertically inside the pod structure to accommodate a mechanism long enough to provide adequate lengthening at static stroke (pod attitude adjustment) and extension/retraction. If one was to utilize this configuration with an electrical deployment and retraction, a design such as that proposed by Waide [8] and in Conway and Renshaw [27] could be explored to similar space and operational limitations. A new configuration is shown in Figure 3.6. This configuration includes the same key elements of Figure 3.5, arranged so that the point (5), which the lengthening device is mounted and the point at which the member (4) – (5) pivots, is movable during the design process. Such a configuration could be accomplished at point (5) by utilizing oppositely disposed trunnion pins, on each side of the lengthening device. The author therefore proposes a design in the spirit of Figure 3.6 and will provide kinematic layouts based on this concept in the following section. Figure 3.6: The HDWS Hybrid actuator concept #2 illustration Figure 3.6: The HDWS Hybrid actuator concept #2 illustration 3.5.2 HDWS Kinematic Layouts This section illustrates the kinematic arrangement of the HDWS using this work’s deployable and retractable shock absorber. Its configuration will be shown in Chapter 4 and discussed in greater detail within Chapter 5. Based on the basic architecture defined in Figure 3.4, a kinematic mock- 34 up was drawn using CATIA® V5 [38]. As a preliminary example, the mock-up is shown in Figure 3.7 with the HDWS in a stowed (retracted) position and bound within boxed dimensions: L= 20- in and W = 14-in. The pod frame is shown to be 3.75-in above the surface of the sub-track. Figure 3.7: An example of a preliminary HDWS kinematic layout, retracted, shown in CATIA® V5 Figure 3.7: An example of a preliminary HDWS kinematic layout, retracted, shown in CATIA® V5 During deployment, the wheels reach a point where they touch the bottom of the tube, typically referred to as weight on wheels (WOW). At this point, the HDWS system must use its retractable and deployable shock strut to further deploy the wheels until the static load is reached. The static load is the piston stroke at which the total forces in each of the shock struts, produced by the internal pressure times the piston area, are equal to the total weight of the Hyperloop pod distributed proportionally across the wheel systems. At this point, it is suggested that the Hyperloop pod should be raised further above the tube surface using the same actuation mechanism. The adjustment of the pod ride height (“ride height maintenance”) is believed to be especially important since the air-bearings or magnetic skis which the vehicle rides on throughout the journey, are known to have extremely small operational clearances, as detailed in Table 3.2. Per Janzen [39], the air-bearing levitation system is particularly sensitive to small irregularities in the Hyperloop tube sub-track. Janzen suggests that a momentary loss in levitation can “cause the [air] 35 bearing to slam down on the [sub-track] and risk self-damage.” Based on the above discussion, and tabulated data in Table 3.2, the decision was made to enforce a minimum height adjustment of 0.5 in (12.7 mm) after static stroke is achieved in order to combat the risk of damaging a pod’s levitation feature that evidently requires a low operational clearance. 3.5.2 HDWS Kinematic Layouts Table 3.2: Levitation ride height comparison Levitation Source Levitation Gap [in] [mm] Air bearing [1] 0.020 – 0.050 0.5 – 1.3 Magnetic (Passive) [21] 0.31 – 0.39 8 – 10 Table 3.2: Levitation ride height comparison Table 3.2: Levitation ride height comparison Ideally, the HDWS will lift the vehicle, relative to the bottom of the air or magnetic levitation skis, by a distance that enables the shock strut, when deployed, to compress and extend freely (i.e. absorb and react to disturbances during operation) without the pod touching the tube sub-track surface. The minimum height adjustment value selected was directly related to the stiffness of the shock absorber gas spring. That is, a stiffer gas spring, as will be discussed in Chapter 5, requires less height maintenance versus a softer gas spring that will allow the pod to compress greater distances under loads. Based on the anticipated loading during braking and acceleration that was analyzed by Ryerson’s International Hyperloop Team [40], the gas spring model for the HDWS required a gap of at least 0.5 in (12.7 mm) to ensure the system remains clear of sub-track in all cases. In addition to the pre-emptive raising of the pod to allow for load oscillations, a desired gap between the pod and the sub-track is monitored and controlled by the HDWS’s active control and leveling capabilities based on a program written and designed by Shonibare [16]. The pod kinematic attachment points, (1) and (5), as selected for the pod originally proposed by U of T, are shown in Figure 3.8 and Figure 3.9. These kinematic illustrations, designed in CATIA® V5, use dimensional characteristics that were selected based on an iterative trial and error method cycling between the required kinematic ranges of motion, the anticipated part geometry (e.g. lug sizing, member lengths, etc.), and clearance with the pod based on the anticipated part geometry. The final number of HDWS’s integrated to a pod, and their distribution throughout the pod’s length, is a function of the available interface geometry and the distribution of the pod weight – like aircraft landing gear distribution methodologies [30]. 3.6.1 Component Definition A part numbering system was developed for the HDWS to manage the various parts, assemblies and configurations. The present scheme was developed alongside the product structure configuration tree presented in Section 3.6.2. The scheme operates on five levels, with 10- representing the complete assembly at level one, and 40-, level five, representing the numbering for individual parts and thus encompassing the HDWS product from the top-level assembly down to each standard nut and bolt. As is standard practice in the industry, custom components designed by the author were assigned individual part numbers and standard hardware retained their corresponding part numbers. Standard components sourced from other industry partners such as the Greene Tweed [41] seals, SKF [42] roller bearings, and KamaticsRWG [43] bushings also retained their respective supplier part numbers. 3.5.2 HDWS Kinematic Layouts 36 Figure 3.8: U of T HDWS kinematic layout, retracted position (left) and WOW position (right) Figure 3.8: U of T HDWS kinematic layout, retracted position (left) and WOW position (right) Figure 3.9: U of T HDWS kinematic layout, sample static stroke (left) and sample static stroke + pod height adjustment of 0.5 in (12.7 mm) from the reference position (right) Figure 3.9: U of T HDWS kinematic layout, sample static stroke (left) and sample static stroke + pod height adjustment of 0.5 in (12.7 mm) from the reference position (right) 37 Example: 40-1101-01 – Corresponds to a level 5 part (40, lowest), will assemble into the undressed assembly (1), falls under the sub-assembly “Trailing Arm” (1), carries the first part number in that sub- assembly (01), and is at revision 1 (01). 38 3.6.2 Product Structure Breakdown For organizational purposes, a product structure tree was developed (Figure 3.10) to establish the HDWS component layout. The top-level model on the product structure tree, “HDWS Dressed”, encompasses the entire structure, motor, mounted electrical equipment (sensors, wires) and rolling stock. Chapter 4 provides a detailed pictorial view of the components described here in Figure 3.10. Figure 3.10: The HDWS product structure tree Figure 3.10: The HDWS product structure tree 39 39 3 Design and Development Process Scope Figure 3.11: The HDWS design and development process scope overview 3.6.3 Design and Development Process Scope 4 Design and Development of the HDWS This chapter provides an overview of the main assemblies that make up the Hyperloop Deployable Wheel System (HDWS). The system Undressed (structural) assembly is subsequently broken into its components and key functions and features are described. Following the system description, the various design activities that were carried out (including a discussion on the drawing arrangement, common structural analysis exercises, and component optimization) are outlined. This chapter concludes with a brief discussion of manufacturing. 3.6.3 Design and Development Process Scope Figure 3.11: The HDWS design and development process scope overview 40 The scope of the HDWS’s design and development, as illustrated in Figure 3.11, was highly iterative in nature and generally followed a typical engineering development project architecture. The early stages of the project consisted of the background literature survey and problem definition. Here, the need for a wheeled system was investigated and key design drivers (e.g. it must be sufficiently compact) were defined. After conducting this review, the constraints, guidelines and possible requirements that would apply to a wheeled subsystem for the Hyperloop were defined. Using the information collected in the early stages, a preliminary design phase commenced. During this time, the general architecture (e.g. shock absorber + wheel(s)) was chosen and the initial sizing of a shock strut gas spring (see Chapter 5) was defined. The goal at this phase was to develop a system baseline concept that could be demonstrated as manufacturable and scalable. The development of the product architecture and kinematics (Chapter 3) and the design of a functioning shock strut become interdependent at this stage in the development. Trade studies were performed on the fly, varying key dimensions to retain the desired kinematic motion yet allow for the required geometry to design a functioning 3D product within a 2D kinematic wireframe. After a baseline model was developed and 3D printed using plastics for demonstration purposes, the detailed design phase commenced. At this point, the structural design was scaled to meet the needs of the prospective Hyperloop pod partner, the U of T. Although the adherence to the baseline design remained firm, during the detail design stage, the scaling of shock absorber internal geometries required adjustments to the gas spring design while manufacturability constraints drove additional changes to the product geometry and kinematic definition. Once the baseline was fully adapted to the needs of the prospective pod partner, the 2D drawings generated using CATIA® V5 were submitted for manufacturing. Although not discussed in the present thesis, the development of the electronic control system architecture began during the detailed design phase of the present work. After being defined by the author, the features of the control system were developed by dedicated electronic and control system actors. Once integrated with the structural elements of the HDWS developed by the author, the electronics and control system became the driving force behind the kinematic operation capabilities of the HDWS. 41 4.1 HDWS Dressed Assembly (P/N 10-1000-00) The HDWS Dressed assembly (Figure 4.1) is the top-level assembly containing the primary structure (HDWS Undressed), electric motor, assembly hardware, sensors and the rolling stock. Since the HDWS design consists of many sub-assemblies and parts, as shown in Figure 4.1, defining a top-level assembly was important to realise a smooth integration of all subassemblies that ultimately made up the complete system. Figure 4.1: The HDWS Dressed assembly, CATIA® V5 rendering Figure 4.1: The HDWS Dressed assembly, CATIA® V5 rendering 42 At the HDWS Dressed level, the system is fully connected to its kinematic skeleton defined in Chapter 3. Using the kinematic operations within CATIA® V5, the HDWS could be cycled through all operational positions and checked for any clashing of components on the HDWS and any external interference with a mating pod structure. At the HDWS Dressed level, the system is fully connected to its kinematic skeleton defined in Chapter 3. Using the kinematic operations within CATIA® V5, the HDWS could be cycled through all operational positions and checked for any clashing of components on the HDWS and any external interference with a mating pod structure. 4.1.1 HDWS Undressed Assembly (P/N 20-1000-00) The HDWS Undressed Figure 4.2 is the first major sub-assembly of the HDWS Dressed and contains five key sub-assemblies and most of the major HDWS structural components. The Undressed assembly includes the axle, the Trailing arm sub-assembly, Retraction sub-assembly, Torque link sub-assembly and Shock absorber sub-assembly. Thus, main structure “dressings” such as the electric motor, including its sensors and wiring as well as the rolling stock (wheels) are covered as standalone sub-assemblies that are integrated to the Undressed assembly to create the final Dressed assembly. Figure 4.2: The HDWS Undressed assembly, CATIA® V5 rendering Figure 4.2: The HDWS Undressed assembly, CATIA® V5 rendering 43 4.1.2 Electric Motor Assembly (P/N 20-2000-00) The HDWS Electric Motor assembly, shown in Figure 4.3, is equipped to the HDWS Dressed assembly and is the driving force that enables the extension, retraction and active leveling capabilities of the HDWS. This assembly is comprised of the motor (1) with integrated electrical wiring (2), attached to a motor mounting bracket (3) that is fixed to the Retract sub-assembly using standard NAS bolts. The motor shaft is equipped with a pinion spur gear (4) which interfaces with the retract assembly leading to the actuation of the system further discussed in Chapter 5. Figure 4.3: The HDWS Electric Motor assembly, installed on the Retract sub-assembly Figure 4.3: The HDWS Electric Motor assembly, installed on the Retract sub-assembly Table 4.1: Electric Motor assembly breakdown summary Find # P/N Part Description Material QTY 1 40-2101-00 BLDC Motor & Gearbox (48 VDC, 3200 RPM) Various 1 2 --- Electrical wiring (power, signal transfer) Various --- 3 40-2102-00 Motor mount bracket (AMM) AlSi10Mg 1 4 40-2103-00 Custom designed Spur Gear pinion 303 SS 1 5 M6T25L M6 Screws CRES 316 4 44 4.1.3 Rolling Stock The rolling stock are the last components integrated to the HDWS dressed assembly. In the current embodiment, the rolling stock is comprised of two wheel assemblies, identical for the left and right mounting on the axle. The wheel assembly shown in the left image of Figure 4.4 was designed and produced to meet the needs of the U of T Hyperloop Pod. To enable a quick turnaround time, a wheel configuration was produced from a solid polyurethane 95A durometer disk that was balanced and equipped with roller bearings at the axle interface. Per the manufacturer, Sunray Inc. [44], polyurethane is proposed to outperform a standard rubber tire in terms of wear-resistance and abrasion. However, the manufacturer notes that at temperatures above 90°C (195°F), rubber becomes a preferred option. Figure 4.4: CATIA® V5 renderings of the final HDWS Wheel Assembly (left), and alternate wheel concept as designed with input from the author (right) Figure 4.4: CATIA® V5 renderings of the final HDWS Wheel Assembly (left), and alternate wheel concept as designed with input from the author (right) In another embodiment, a more traditional framed wheel concept was conceived, consisting of a solid polyurethane or rubber lining trapped between an aluminum frame, shown in Figure 4.4. The advantage of this design over the one currently used for the HDWS is the light-weight aluminum 45 frame, which also provides added structural stability. A significant challenge however comes in designing a reliable retention for the externally mounted polyurethane (or rubber) liner, shown in black, on the outer diameter of the wheel frame (right image, Figure 4.4). In the proposed concept, the external liner could tend to rotate within the aluminum frame under high speeds and loads (even with a pre-squeeze) and the liner could tend to lift from the outer aluminum frame under high rotational speeds. To solve this, adhesives could be used in conjunction with a mechanical lock as drawn. However, after consulting with design advisors in support of the HDWS project, it was deemed too high-risk and complex to develop this design within the project timelines. As will be discussed in the testing results, Chapter 6, such a design will need to be investigated to best address the bearing dislodgment due to radial wheel growth at high speeds that was identified during test. In a first iteration, a wheel and tire configuration as used on aircraft and cars was conceived (Figure 4.5). 4.1.3 Rolling Stock Originally sized for a first HDWS iteration circa January 2016, a wheel was designed and developed by Khan [45] and fit with a standard aircraft tire capable of withstanding the high speeds and loads. Although a typical pneumatic tire was investigated in the early stages, there were uncertainties associated with using such a system in a low-pressure environment. It was hypothesized that the application of such a design would require an active pressurization control unit to ensure the tire was adequately inflated during all external conditions (e.g. low-pressure, sudden tube de-pressurization and waiting in a pressurized station). After a review of wheel and tire architecture it was found that most pneumatic tire configurations were developed to absorb irregular operating surface conditions, such as pot holes, and in the case of landing gear, the high loads (due to the rate of decent on impact) and speeds at landing [5]. Since the Hyperloop will operate inside a highly-controlled environment without large sub-track misalignments but it is instead subjected to the varying environmental pressure discussed in Chapter 2, the present solid polyurethane design was explored, as shown in Figure 4.4. This design draws inspiration from roller coasters, which also operate at relatively high speeds on a highly-controlled track surface. The design proposed by Khan [45] was presented as part of the SpaceX Hyperloop Competition design package but was ultimately not used for the final HDWS configuration which is shown in Figure 4.6. 46 Figure 4.5: A Wheel and Tire assembly, 3D cross-sectional view, as drawn by Khan [45] Figure 4.5: A Wheel and Tire assembly, 3D cross-sectional view, as drawn by Khan [45 Figure 4.6: The rolling stock as installed on the HDWS Dressed Figure 4.6: The rolling stock as installed on the HDWS Dressed 47 4.1.4 Electrical Dressings and Hardware The HDWS is not only capable of extending and retracting into a Hyperloop Pod, but can also operate as an active suspension system. Using externally mounted sensors shown in Figure 4.7, the HDWS uses real time data to control the position of the Hyperloop pod frame relative to the tube sub-track. The sensor data is filtered through a closed-loop control system, developed by Shonibare [16]. The sensors provide the signal and the motor performs the active adjustments, retraction and extension using the sensor feedback and commands from the external controller. Figure 4.7: Sensors, brackets, electrical and hardware mount (orange) shown on the HDWS Dressed Figure 4.7: Sensors, brackets, electrical and hardware mount (orange) shown on the HDWS Dressed 48 To accomplish the preceding tasks, the HDWS Dressed Assembly is equipped (Figure 4.8) with two externally mounted cable potentiometers (1) and (2) that monitor the translation of the shock strut cylinder and shock strut piston stroke respectively. The potentiometers are fixed to a sensor bracket (3) via four cap screws (4), per potentiometer, and locked using spring back washers. The bracket (3) is itself fixed to the outer frame of the Retraction sub-assembly via four NAS cap screws (5), and locked using lock-wire at install (not shown here). The shock strut cylinder potentiometer cable is attached to an eyelet receiver bracket (6) and locked on installation using a cotter pin. The piston stroke potentiometer cable is routed via a transfer bracket (7) and attached at the piston rod-end eyelet receiver bracket (8). 4.1.4 Electrical Dressings and Hardware Figure 4.8: Sensors, brackets, electrical and hardware labeled (some features hidden for clarity) Figure 4.8: Sensors, brackets, electrical and hardware labeled (some features hidden for clarity) 49 Table 4.2: Electric Motor assembly breakdown summary Table 4.2: Electric Motor assembly breakdown summary Find # P/N Part Description Material QTY 1 SM2-12.5 Miniature String Potentiometer (12.5-in range) Various 1 2 SM2-25 Miniature String Potentiometer (25-in range) Various 1 3 40-1310-01 Bracket, Cable Potentiometers ABS 1 4 NAS1352C06-6 NAS Cap Screw CRES 8 5 NAS1351C3H8 Cap Screw, Drilled Head CRES 4 6 40-1211-00 Eyelet bracket, ACME Potentiometer ABS 1 7 40-1212-00 Eyelet transfer bracket, Piston Potentiometer ABS 1 8 40-1404-00 Eyelet bracket, Oleo Potentiometer ABS 1 9 NGCMB10AX01B Limit Switch Various 1 10 NAS1352C08-8 Cap Screw CRES 2 11 40-1311-00 Bracket, Limit Switch ABS 1 12 NAS1352C06-8 Cap Screw CRES 2 Once reaching a fully retracted state, a limit switch (9), attached via two NAS cap screws (10) to a mounting bracket (11) is triggered by a bumper on the upper torque link indicating a “fully retracted” system state and subsequently shuts down the motor. The bracket (11) is mounted to the Retraction sub-assembly via two NAS cap screws (12). 4.2.1 Trailing Arm Sub-assembly (P/N 30-1100-00) The Trailing Arm sub-assembly (Figure 4.9) was designed as a primary load-carrying member of the HDWS. The design of the trailing arm was sized principally on anticipated side loads and dynamic braking loads (external source braking) that the system could face during operation within the SpaceX Hyperloop Competition. Table 4.3 details the individual components that make up the trailing arm sub-assembly that is itself part of the HDWS Undressed assembly. 50 Figure 4.9: The Trailing Arm sub-assembly, parts labeled Figure 4.9: The Trailing Arm sub-assembly, parts labeled In Figure 4.9, Find Numbers (used to identify key parts of the system), corresponding to the information in Table 4.3, can be found. Find Number (1) consists of the machined from solid trailing arm component. This part, made from aluminum (Al) 6061-T6, was anodized blue for aesthetic appearance and proactively controls and initiates the natural oxidization for increased corrosion protection. The flanged bushings at the trailing arm pivot socket (2) are machined from aluminum bronze (Al-Br) and installed using an interference press fit method. The bushings (2) material choice and design protect the aluminum trailing arm structure by providing a stronger bearing surface, resisting wear and acting as a sacrificial repair component that enable the protection of the main structure. Table 4.3: Trailing Arm sub-assembly breakdown summary Find # P/N Part Description Material QTY 1 40-1101-66 Trailing Arm Al 6061-T6 1 2 40-1105-00 Bush, Pivot Pin, Trailing Arm Al-Br 2 3 40-1106-00 Bush, Axle, Trailing Arm Al-Br 2 4 KRJ5-UDSVC-008 KAron® Bush, Piston Clevis CRES 17-4PH 4 Table 4.3: Trailing Arm sub-assembly breakdown summary 51 The flanged bushings (3) for the axle interface socket are designed specifically to ensure contact with the inner-race of the wheel mounted bearings. In contrast, if the bushings at (2) were used for the axle interface socket, the wheel bearing’s inner and outer race would contact the bush flange face causing undesirable friction and wear to the roller bearings and trailing arm bushings. The bushings at (2) and (3) are typically greased prior to pin installation [5], especially when the joint is subject to rotation. In this application, the bushings at (2) and (3) will contain a pin and axle respectively, locked in rotation and axial translation by a standard NAS bolt, washer and nut set. The locations of the two bores housing the bolt, washer and nut sets are shown in Figure 4.11. 4.2.1 Trailing Arm Sub-assembly (P/N 30-1100-00) Figure 4.10: The Trailing Arm sub-assembly cross-sectional view Figure 4.10: The Trailing Arm sub-assembly cross-sectional view As discussed in Chapter 2, since the HDWS operates in a near vacuum, typical greasing methods are unacceptable due to outgassing and the evaporation of oil based lubricants within a vacuum. To combat the issue, self-lubricated bushings were sourced from Kamatics Speciality Bearings (subsidy of KAMAN Aerospace [43]), suppliers of the patented KAron® dry-lubricated bearings. These bearings are specially designed to operate within a vacuum environment and are used on various spacecraft and satellites. These self-lubricated bushings (4) are installed on the trailing arm at the piston lug interfaces and lower torque-link interfaces. The KAron® lubrication was particularly required on both the flange and inner diameter of the bushing to provide lubrication for the rotating parts and NAS bolt fitted through the ID of the part. The bushings (4) were installed using a press fit method at laboratory temperature, 68 °F (20°C). In an earlier concept of the trailing arm, illustrated in Figure 4.11, the trailing arm had two small lugs machined on the end. These small lugs enabled the easy locking of a thread-less eye-bolt, using a dowel pin, shown in Figure 4.11. This feature did not end up on the final design due to the 52 added manufacturing cost associated with these small features. However, for a future production variant, such a system should be considered for the quick removal and attachment of the axle. Figure 4.11: The Trailing Arm axle installation pin Figure 4.11: The Trailing Arm axle installation pin 4.2.2 Shock Strut Sub-assembly (P/N 30-1200-00) The Shock Strut sub-assembly (Figure 4.12) is one of the most important sub-assemblies of the HDWS and was included to ensure a smooth ride for the Hyperloop pod and its payload. The shock absorber was designed as an oleo-pneumatic type, a typical choice for aircraft landing gear due to its high efficiency and relatively low mass [5]. The detailed design and performance characteristics, principally characterized by the sizing of the gas spring, is one of the most complex features of the HDWS and will be presented in Chapter 5. Figure 4.12: The HDWS Shock Strut sub-assembly, CATIA® V5 rendering Figure 4.12: The HDWS Shock Strut sub-assembly, CATIA® V5 rendering 53 53 Figure 4.13: Full cross-section view of the Shock Strut sub-assembly, Find Numbers shown Figure 4.13: Full cross-section view of the Shock Strut sub-assembly, Find Numbers shown Figure 4.13: Full cross-section view of the Shock Strut sub-assembly, Find Numbers shown Figure 4.14: Partial cross-section view of the shock strut sub-assembly, Find Numbers shown Figure 4.14: Partial cross-section view of the shock strut sub-assembly, Find Numbers shown Figure 4.14: Partial cross-section view of the shock strut sub-assembly, Find Numbers shown 54 Table 4.4: Shock Strut sub-assembly breakdown summary Table 4.4: Shock Strut sub-assembly breakdown summary Find # P/N Part Description Material QTY 1 40-1201-00 Piston 303 SS 1 2 40-1202-00 Cylinder AISI 12L14 1 3 40-1205-00 Piston Upper Bearing Al-Br 1 4 40-1207-00 Gland Nut (Lower Bearing) Al-Br 1 5 40-1209-00 Support Tube Al 6061-T6 1 6 NAS1423-14 Jam Nut (Hex, Drilled Head) 4340 1 7 MS28889-2 Charge Valve (Gas/Oil) CRES 1 8 KRJ4-UDSVC-008 KAron® Bush, UTL Lug CRES 17-4PH 2 9 KR5-CNGBP Spherical Bearing CRES 17-4PH 1 10 711D6MTE-161-P15 AGT Ring (Static Seal) --- 1 11 40-1208-00 Lock tab AISI 12L14 1 12 R2361-215E344 MSE Scraper --- 1 13 7125MT-964-P15 AGT Ring (Static Seal) --- 1 14 7215FT-964-P17 AGT Ring (Dynamic Seal) --- 1 15 NAS1352C08H8 Cap Screw (Drilled Head) CRES 2 As illustrated in Figure 4.13 and Figure 4.14 and described in Table 4.4, the assembly consists of a piston (1) and outer cylinder (2). The outer cylinder (2) is finished on the exterior with 3G ACME threads to interface with the retraction assembly. A set of bushed lugs protruding at the base of the cylinder mates with the upper torque link (UTL) as seen in Figure 4.2. 4.2.2 Shock Strut Sub-assembly (P/N 30-1200-00) 303 SS (stainless steel) was used to manufacture the piston (1) with its outer diameter (OD) ground to a surface roughness specified by the seal manufacturer to ensure excellent sealing characteristics. The piston is locked inside the outer cylinder by a gland nut (4), retained by a lock tab (11), and standard NAS drilled head cap screws (15) locked in place using lock-wire (not shown). The piston is attached to the trailing arm lugs using a KAron® lined spherical bearing (9) and NAS bolt (not shown, equipped at Undressed level). The piston itself is equipped with an upper bearing (3) which maintains the contact (acts as an axial bearing) of the piston with the outer cylinder wall during stroke operation and functions as a restrictive flow path for the oil. The shock absorber is charged with nitrogen and filled with oil using the filler support tube (5), fastened using a jam nut (6) and fitted with a standard MS charge valve (7). All pathways to the outside environment are sealed using static and dynamic seals. A 55 Green Tweed AGT static seal (13) (with 2 back-up rings) is used to seal the gland nut to the cylinder inner diameter (ID). A Green Tweed AGT® dynamic seal (14) is equipped in the ID of the gland nut (4) and is used to seal the piston OD. Lastly, a static seal (10) is fit between the support tube (5) and the shock strut outer cylinder ID, completing the sealing of the inner chamber. 4.2.3 Retraction Sub-assembly (P/N 30-1300-00) The Retraction sub-assembly, as shown in Figure 4.15, plays a primary role in the successful operation of the HDWS. The sub-assembly converts the externally mounted BLDC motor’s rotational motion (torque) into translational motion (at the shock strut), enabling the retraction and extension of the HDWS. By the same token, this sub-assembly, whose components are defined in Table 4.5, provides the translational motion for the active height control of the pod using feedback from various sensors mounted to the retraction sub-assembly at the dressed level. The retraction housing (1) is connected to the pod using two oppositely disposed pins (not shown) at the bearings (5) and locked in translation and rotation using NAS shear bolts. A large flange at the back of the housing (1) is used to mount the BLDC electric motor, as seen in Figure 4.3. The internally mounted lead screw retract nut (2), which is lined on its OD with standard spur gear teeth, is mounted inside the housing (1) and held in place by two deep groove ball bearings (6). The nut and thrust bearings are packed into the housing via a large castellated packing nut (3) and locked from rotation via a lock tab (4), fastened to the housing (1) via two standard NAS cap screws (7). The two cap screws (7) are lock-wired on assembly, lock-wire not shown here. Table 4.5: Retraction sub-assembly component breakdown summary Find # P/N Part Description Material QTY 1 40-1301-02 Retract Housing Al 6061-T6 1 2 40-1302-02 Retraction Nut Al-Br 1 3 40-1303-01 Packing Nut Al 6061-T6 1 4 40-1304-00 Lock Tab, Retract Housing AISI 12L14 1 5 40-1308-00 Trunnion Bush, Trunnion Pin Al-Br 2 6 61813-2RZ Deep Groove Ball Bearing CRES 2 7 NAS1352C08H4 Cap Screw, Drilled Head CRES 2 Table 4.5: Retraction sub-assembly component breakdown summary 56 Figure 4.15: Retract sub-assembly 3D view (left) and cross section (right), Find Numbers shown Figure 4.15: Retract sub-assembly 3D view (left) and cross section (right), Find Numbers shown 4.2.4 Torque Link Sub-assembly (P/N 30-1400-00) As shown in Figure 4.16, the Torque Link sub-assembly is composed of two major components, the upper (1) and lower (2) torque links. The torque links are required to react the loads induced by the friction between the ACME thread retract nut, as shown in Figure 4.15, and the shock strut cylinder, as shown in Figure 4.12. The reaction of these loads enables the Shock Strut sub- assembly to translate axially and cause the HDWS to extend, retract, and operate in an actively controlled manner. Since the torque links will be under rotation and under load (as the shock absorber strokes), the joints are equipped with KAron® lined CRES bushings (4, 5 and 6) providing lubrication in the low-pressure environment and a tough wear surface protecting the Al 6061-T6 torque link bores. The upper torque link (UTL) and the lower torque link (LTL) are connected at the apex joint using a standard NAS bolt (7). The LTL is attached to the trailing arm piston lug and the UTL is attached to the shock strut cylinder lug (see Figure 4.2). Lastly, a limit switch bumper (3) is equipped to the UTL using two standard NAS Cap screws (8). Upon reaching a fully retracted position, the UTL bumper triggers a limit switch sensor, signaling that the HDWS is now retracted. 57 Figure 4.16: Torque Link sub-assembly (left) and exploded view (right), Find Numbers shown Figure 4.16: Torque Link sub-assembly (left) and exploded view (right), Find Numbers shown Figure 4.16: Torque Link sub-assembly (left) and exploded view (right), Find Numbers shown Table 4.6: Torque Link sub-assembly component breakdown summary Find # P/N Part Description Material QTY 1 40-1401-00 Upper Torque Link Al 6061-T6 1 2 40-1402-02 Lower Torque Link Al 6061-T6 1 3 40-1403-00 Limit Switch Bumper ABS (AM) 1 4 KRJ5-UDSVC-008 KAron® Bush, LTL Lug CRES 17-4PH 2 5 KRJ4-UDSVC-008 KAron ® Bush, LTL Apex Lug CRES 17-4PH 2 6 KRJ4-USVC-008 KAron® Bush, UTL CRES 17-4PH 4 7 NAS6204-22D NAS Bolt 4340 1 8 NAS1352C06-06 NAS Cap Screw CRES 2 Table 4.6: Torque Link sub-assembly component breakdown summary 4.2.5 Axle (P/N 40-1501-00) The axle component (Figure 4.17) is an integral part of the Undressed assembly. The axle was manufactured from 303 SS and designed with a 1-in (25.4 mm) shaft OD. It is equipped, as shown in Figure 4.17, to the Trailing Arm sub-assembly through the axle bushings located at the back of 58 the arm. The axle is further constrained from rotation and translation using a standard NAS shear bolt equipped at the Undressed level of the assembly and features externally threaded ends with locking features for the axle nut, used to retain the wheels at installation (see Figure 4.6). Figure 4.17: The axle component as equipped to the trailing arm sub-assembly Figure 4.17: The axle component as equipped to the trailing arm sub-assembly 4.3 Structural Analyses The structural analysis of the HDWS components was principally split into two broadly defined areas: classical hand calculation analysis and finite element analysis (FEA). The common use cases for each broad method of analyses are discussed here using applicable examples to illustrate. It shall be stated that the HDWS project had the benefit of a dedicated structural analyst, Moeid Elahi, hereby referred to as the “actor”, who was the primary source of the project’s official FEA and classical hand calculations. Wherever the work of the actor is shown or discussed, a differentiation will be made between the actor’s contribution and the author’s. In all cases, the minimum SF requirement (SF=2), defined by SpaceX [19], was achieved by comparing the stresses in the component to its relevant material allowable. The static yield and bearing allowable stresses for all materials utilized on the HDWS can be found in the MMPDS-07 handbook [30] or obtained from the part and material suppliers. 59 4.3.1 Classical Analyses Overview Classical hand calculations rely on the fundamental strength of materials and statics equations that can be found in standard engineering textbooks, such as those authored by Hibbeler [46]. Classical hand calculations were performed whenever possible on simple features and cross sections. During the design process these fundamentals were frequently used to perform initial sizing calculations, two common samples of which are discussed in this section. Bolts Analysis: Depending on the application, bolts can be subjected to a variety of loading including shear, tension, bending or a combination. As an initial sizing exercise the author used estimated loading values provided by a system beam model, developed by the actor with help from the project advisors. Using this information, simple calculation checks were performed, some of which are described here. When a bolt was acted on in shear (typically double shear as shown in Figure 4.18 in this application) the following formulae were applied [46]: Bolt, Single Shear: 𝜏𝑆= 𝐹𝑆 𝐴𝑆 (4.1) (4.1) Here, 𝜏𝑆 is the shear stress on the bolt, 𝐹𝑆 is the force transmitted through the joint, and 𝐴𝑆 is defined as the shear area for bolt. For the loading condition shown in Figure 4.18, the joint is subjected to double shear through the pin diameter. Thus, the equation for 𝐴𝑆 becomes: 𝐴𝑆= 𝜋𝑑𝑏 2 4 (4.2) (4.2) where the area subjected to shear is defined as the pin diameter area with diameter defined by 𝑑𝑏. Note that the value for 𝑑𝑏 shall be selected as the nominal bolt diameter, as defined in the part specification. 60 When the pin is under double shear loading conditions, illustrated in Figure 4.18, the equation for the shear stress in the pin is defined by: When the pin is under double shear loading conditions, illustrated in Figure 4.18, the equation for the shear stress in the pin is defined by: Bolt, Double Shear: Bolt, Double Shear: 𝜏𝑆= 𝐹𝑆/2 𝐴𝑆 (4.3) Figure 4.18: Section cut, illustrating a typical double shear loading on the HDWS 𝜏𝑆= 𝐹𝑆/2 𝐴𝑆 (4.3) (4.3) Figure 4.18: Section cut, illustrating a typical double shear loading on the HDWS Here the loading is simplified, assuming equal distribution acting through the centre of each of the lugs. For the piston lug, where the axial load 𝐹𝑆 is transmitted through a single spherical bearing, this loading definition can be approximated as linear. However, in practice, and as can be more clearly recognized at the trailing arm lug interface, where the loading is reacted between the two lugs, the load can be more appropriately defined by taking into account the deformation of the pin. Such considerations were applied in the detailed analyses performed by the noted stress actor and were not the subject of attention for the author during the pre-sizing of joints. Bolts Analysis: 61 Bolts that were subjected to tensile loading were checked for tensile stresses within the bolt diameter using the following equation [46]: Bolts that were subjected to tensile loading were checked for tensile stresses within the bolt diameter using the following equation [46]: Bolt Under Tensile Loading: Bolt Under Tensile Loading: Bolt Under Tensile Loading: Bolt Under Tensile Loading: 𝜎𝑇= 𝐹𝑇 𝐴𝑇 (4.4) (4.4) Here, 𝜎𝑇 is the tensile stress on the bolt, 𝐹𝑇 is the tensile force, typically due to bolt pre-loading applied at assembly, and 𝐴𝑇 is defined as the tensile area of the bolt. In practice, the portion of the pin with the smallest 𝐴𝑇 will be the likely source of failure in a bolt under tension. These minimized areas are typically found at the head of the pin, where a small undercut is typically applied at the design phase to allow for a larger transition radius, reducing stress concentrations. For the standard NAS bolts used on this project, however, the bolt head is designed to mate with a large ID washer and thus does not lose bolt area close to the head. So, for a conservative initial calculation, the minor diameter of the bolts thread was selected as the smallest area 𝐴𝑇 and therefore subject to the highest tensile stress, 𝜎𝑇. Lastly, a final check of the shear stress produced in the bolt head, while the bolt is subject to tensile loading, as shown in Figure 4.19, was conducted. The eqution is defined as follows: 𝜏𝑆= 𝐹𝑇 𝐴𝑆𝐶 (4.5) (4.5) where 𝐴𝑆𝐶 , the shear area of the pin head under tensile loading, is defined by: 𝐴𝑆𝐶= 2𝜋𝑟𝑡 (4.6) (4.6) Here the value for 𝑟 was typically chosen as 𝑑𝑏2 ⁄ , that is, the circumferential location where the shear force is maximum, and the thickness 𝑡 of the bolt head as defined by the bolt manufacturers. When combined, the value for 𝑟 and 𝑡 define shear area of the pin head, as shown in Figure 4.19. 62 Figure 4.19: Section cut, illustrating a shear stress around the circumference of the bolt head Figure 4.19: Section cut, illustrating a shear stress around the circumference of the bolt head Bush and Bearing Sizing: As discussed in Chapter 4, the flanged bushings and spherical bearings that were used for all rotational joints were of the self-lubricating type. As a preliminary sizing exercise, the guidelines illustrated in the Kamatics engineering product catalogue were used [47]. Both spherical and flanged bushings use a “projected area” method, widely used in the engineering industry when defining the initial bearing pressure 𝑃𝐵. As per Kamatics, the equation for 𝑃𝐵 can be written as [47]: 𝑃𝐵= 𝐹𝐴 𝐴𝐵 (4.7) 𝑃𝐵= 𝐹𝐴 𝐴𝐵 (4.7) (4.7) Here 𝐹𝐴 is defined as the applied load on the bearing originating from a pin or bolt. The force 𝐹𝐴 is then assumed to be distributed linearly across the bearing ID. As recommended by Kamatics, the value of bushing area 𝐴𝐵 is defined by the projected area (see Figure 4.20) using the equation [47]: 𝐴𝐵= 𝐿𝐵𝑥𝐼𝐷𝐵 (4.8) (4.8) 63 Figure 4.20: Flanged bush (left), illustrating a projected bearing area (right) used for the preliminary calculation of the bearing stress Figure 4.20: Flanged bush (left), illustrating a projected bearing area (right) used for the preliminary calculation of the bearing stress Following insights from the HDWS project industrial advisors and taking note of the discussions found in reference [47], equation (4.8) is further refined to remain conservative. That is, the useable length of the bearing 𝐿𝐵 is reduced by the machining of chamfers at each end of the bushing ID. Referencing Figure 4.21, the equation for 𝐴𝐵 becomes: Referencing Figure 4.21, the equation for 𝐴𝐵 becomes: 𝐴𝐵= (𝐿𝐵−𝐶𝐻1 + 𝐶𝐻2)𝑥𝐼𝐷𝐵 (4.9) (4.9) Figure 4.21: Flanged bush illustrating the features involved in a bearing length refinement Figure 4.21: Flanged bush illustrating the features involved in a bearing length refinement 64 The approach used here for the bearing pressure calculations is valid; however, it is a simplified approach. A wide variety of factors contribute to a more refined bearing pressure calculation, which is known to be a non-linear distribution along the length, 𝐿𝐵, principally due to pin bending. Other elements of bearing design and selection, such as the pressure on the flange face, the pressure induced by the fitting of the bushing into a housing, and thermal stresses induced on the fit as a product of the thermal dilatation coefficients are known and were checked using proprietary design guide tools provided by Safran Landing Systems Canada Inc. [48]. For this reason, they are not available for outside distribution. 4.3.2 Finite Element Analyses and Applications FEA was used for components with more complex geometrical features and complex combined loading. The software used by the author and actor for the FEA was performed using the analysis capabilities within ANSYS® [49], CATIA® V5 [38] and solidThinking Inspire© [50]. For the sake of brevity and to remain focused on the author’s contributions to the HDWS, only a sample of the actor’s FEA is presented in Figure 4.22 below and the rest are not discussed in this thesis. Figure 4.22: Sample FEA result from ANSYS®, trailing arm lug analysis [40] Figure 4.22: Sample FEA result from ANSYS®, trailing arm lug analysis [40] The author had the opportunity to use the analysis and design software solidThinking Inspire© that enabled an FEA-based topology optimization study on the BLDC motor mounting bracket as 65 shown in Figure 4.23. The bracket was to be machined from a solid aluminum block in its baseline configuration; however, the application of topology optimization enabled the production of a design concept that was additive manufacturing (AM) friendly and highly mass optimized. The finished topology optimized design concept for the motor bracket is shown in Figure 4.24 and a sample of the FEA analysis is shown in Figure 4.25. Figure 4.23: HDWS motor mount bracket, machine from solid variant (maroon colored) [17] Figure 4.23: HDWS motor mount bracket, machine from solid variant (maroon colored) [17] The optimized bracket was subsequently manufactured using an additive metal manufacturing (AMM) process. The process and the finished product are shown in Section 4.5.3. Readers interested in further details regarding the topology optimization methodology (design for addictive manufacturing) used in the present work may refer to reference [17]. 66 Figure 4.24: Final bracket design, produced from an FEA based optization [17] Figure 4.24: Final bracket design, produced from an FEA based optization [17] Figure 4.25: Example of a bracket displacement analysis, gravity load applied [17] Figure 4.25: Example of a bracket displacement analysis, gravity load applied [17] 67 Figure 4.28: Typical design notes used for the HDWS aluminum parts After being submitted through a formal drawing check process conducted by project advisors, the drawings produced by the author for the HDWS’s detail parts were transmitted to the manufacturing company contracted for the project, Durham Precision Machine CNC Inc. For this project, the 2D drawings were considered the master plan for manufacturing and the 3D models, if supplied to the manufacturer, were for reference use only. This practice is still very common in the industry today to ensure that the notes on the drawing, material choice, surface finishes and tolerances are closely followed. However, the author is aware of a new model-based definition (MBD) design concept [52], where no 2D drawing is produced and all the typical drawing notes, tolerancing and feature identifiers are defined in the 3D model. The assembly drawings produced by the author were used for the assembly of the HDWS, the installation of bushings and the pressure seals. All drawings used in the manufacturing and assembly of the HDWS for the components discussed throughout the entirety of Chapter 4 are included in the Appendix A. 4.4 Part and Assembly Engineering Drawings The HDWS detail design process followed a standard industry design approach were concept level sketches, typically hand-drawn, are first drafted for visualization purposes and then transferred to computer aided design (CAD) software. CATIA® V5, widely used in the aerospace and automotive industry, was used for the design of the HDWS. As per modern design methodology, 3D models of the HDWS parts, sub-assemblies and final assembly were produced using CATIA® V5. After the 3D models were finished the models were drawn using CATIA® V5’s Drafting Workbench. In order to follow the latest industry standards and best practices as they pertain to draftsmanship, the ANSI/ASME Y14.5M-1994 Geometric Dimensioning and Tolerancing (GD&T) [51] standard was followed where applicable. Ensuring an industry standard level of quality for the finished drawings, the author utilized an industry based template, developed with assistance from Safran Landing Systems Canada Inc. [48], for the HDWS’s drawings. An example of the drawing template is shown in Figure 4.26. Figure 4.26: Sample of a typical HDWS engineering drawing, key elements encircled Figure 4.26: Sample of a typical HDWS engineering drawing, key elements encircled 68 The template features the basic elements required to adequately communicate the design to a reviewer and most importantly the manufacturer. In particular, the HDWS drawing template, shown in Figure 4.26, incorporates: part specific design notes (typically included in the top left corner); a drawing title block (bottom right); 3D view(s) to provide an overview of the finished component or assembly and the body of the drawing containing views only as is required to clearly convey the design features. An example of the drawing title block and typical part notes can be seen in Figure 4.27 and Figure 4.28, respectively. Figure 4.27: Sample of title block layout used for the HDWS Figure 4.27: Sample of title block layout used for the HDWS 69 4.5 Manufacturing and Treatment Processes The following presents a summary of the different manufacturing processes used to create the HDWS product. It is important to note that each component of the HDWS was designed with the end manufacturing process in mind, e.g. cutting tool radii, tool paths, and number of operation setups. 70 4.5.1 Subtractive Manufacturing According to Kalpakijian and Schmid [53], subtractive manufacturing covers a wide variety of manufacturing process including: CNC milling, turning of parts on a lathe, drilling operations and grinding. Each of these manufacturing methods were used in creating the HDWS and several examples are described in Figure 4.29 and Figure 4.30. Figure 4.29: Images depicting from (1) through (3) the stages in the turning process performed for the manufacturing of the shock absorber outercylinder ACME 3G thread Figure 4.29: Images depicting from (1) through (3) the stages in the turning process performed for the manufacturing of the shock absorber outercylinder ACME 3G thread 71 Figure 4.30: Images (1) through (3) depict the milling process starting from a solid metal block, and image (4) depicts the internal thread screw cut performed on a lathe Figure 4.30: Images (1) through (3) depict the milling process starting from a solid metal block, and image (4) depicts the internal thread screw cut performed on a lathe 4.5.2 Protective Finishes and Processes After the manufacturing process was completed, the components that required protections, mainly the aluminum and steel components, underwent protective treatment processes. In most cases these protections were for external environmental effects (natural corrosion) and in others to prevent galvanic corrosion caused by dissimilar metals [5]. The processes used for the present work are briefly described and illustrated where possible with examples. Aluminum Parts: Although aluminum does not rust like steel, aluminum does react naturally with oxygen and overtime forms an external layer of aluminum oxide. This external layer of aluminum oxide represents a protective surface with much higher corrosion and abrasion resistance than freshly machined aluminum. The electrochemical process of anodizing pre-emptively oxidizes the surface of the aluminium part providing a finished part with a more durable and corrosion-resistant finish. 72 During the anodizing process the parts can be coloured (typically for aesthetic purposes) using organic dyes [54]. Figure 4.31 below shows the result of anodizing (blue) one of the trailing arm components. Figure 4.31: Trailing arm pictured after undergoing blue anodizing Figure 4.31: Trailing arm pictured after undergoing blue anodizing Corrosion Resistant Steel (CRES): The HDWS prototype used two different types of stainless steel– 303L for the piston and pins, and 17-4PH CRES for the KAron® [47] lined bushings. A common form of post-fabrication protection for stainless steels is passivation. As explained by Debold and Martin [56], it is a process that enhances the inherent corrosion resistant properties of stainless steels, such as 303L. In practice, the need for passivation after manufacturing the stainless part comes from the micro-deposits of iron left behind from the grinding or cutting tools and is deposited on the surface. Although microscopic, Debold and Martin notes that this frequently causes a fine layer of rust over extended periods of time [56]. For these reasons, all the HDWS 303L components were passivated. The 17-4PH bushings were treated with cadmium plating on their installation surfaces (the outer diameter) to protect the bushing and mounting part from galling (stainless on stainless) and galvanic effects [32]. Carbon Steel: For ease of manufacturing the external ACME thread and for overall cost savings, the prototype HDWS’s shock absorber outer cylinder was machined from AISI 12L14 carbon steel. Being non- corrosion resistant by nature, the steel required post treatment to protect against corrosion (rusting). Since the part was required to maintain its tight operating tolerances internally for the sealing surfaces and externally for the ACME threaded engagement, black oxide conversion coating was selected for its minimal build up (thickness) and superior abrasion resistance when compared to paint. The abrasion resistance was particularly important to consider along the ACME threaded surface, where the black oxide was expected to deteriorate overtime [55]. Figure 4.32 shows the shock strut outer cylinder assembly after going through the conversion coating process. 73 Figure 4.32: A shock strut outer cylinder shown with black oxide surface treatment Figure 4.32: A shock strut outer cylinder shown with black oxide surface treatment 4.5.3 Additive Manufacturing From the resulting topology optimization discussed in Section 4.3.2, the motor mounting bracket was selected for AMM. Thanks to Burloak Technologies Inc. [57], seven motor brackets were 74 printed from AlSi10Mg using state-of-the-art direct metal laser sintering (DMLS) processes [17]. The DMLS process uses a high-power laser to sinter the part, layer by layer, in an additive fashion [58]. After each layer is sintered, a fresh layer of metal powder is swept over the previously sintered layer, as illustrated in Figure 4.33. This process is repeated until the final part geometry has been achieved. The result, after post processing (shot peening and build support removal), is shown in Figure 4.33. Figure 4.33: Images (1) through (2) depict the DMLS process used to create the final (3) AMM part [17] Figure 4.33: Images (1) through (2) depict the DMLS process used to create the final (3) AMM part [17] 5 The Deployable and Retractable Shock Strut Following the definition of the kinematic layout, the HDWS concept was broken down into its key subsystems in Chapter 4, to familiarize the reader with the baseline concept design. As explained in Chapter 4, a significant portion of the HDWS design is comprised of the Shock Strut sub- assembly (P/N 30-1200-00) and the Retraction sub-assembly (P/N 30-1300-00). Due to their complex nature, their specific detail design and functions, separately and as a combined unit, are discussed in this chapter. 4.6 Chapter Summary The present chapter provided a detailed overview of the HDWS components and discussed some of their basic functions. After discussing the primary top-level (dressed) assemblies, the assemblies that make up the main structure (undressed assembly) were described. As it was mentioned earlier in Section 4.2.2, the detailed design and performance characteristics of the shock strut, fitted within the retraction assembly, is one the most complex components on the HDWS. For this reason, the detailed design of the shock strut and its operation within the retraction and extension framework, defined in Section 3.5, will be the primary subject of Chapter 5. 75 5.1 System Configuration The Deployable and Retractable Shock Strut [9] configuration is itself the most important component of the HDWS and was designed to address the unique kinematic requirements of the Hyperloop, as defined within Chapter 3. Utilizing the present design, it is now possible to achieve shock absorption, retraction and extension capabilities within one combined unit (Figure 5.1), also proven through testing. By design, the system includes a retract housing (fixed to the pod and free to rotate), where at least one motor is attached, and a classic oleo-pneumatic shock absorber internal configuration is retained but has its exterior modified to define a power lead screw, specifically ACME based. The benefit here, as compared to Waide [8] and Sharples [25], is that the shock absorber can linearly translate through the trunnion pin mounted retract housing, using an ACME gear nut mounted between two bearings and free to rotate. This generates translational motion and allows the system to fold into a vertically compact stowed position. As is designed here, the system can be optimally configured (kinematically) when utilized with a classic trailing arm configuration, requiring only two total pod interface points (at the retract housing and trailing arm) and an electrical connection to the pod. The use of torque links here to react the rotational forces generated by friction in the ACME threads is particularly robust when compared to the proposal discussed in Chapter 2 by Waide [8]. As an added benefit, using externally mounted sensors and those typically pre-installed within a BLDC motor (hall sensor), the position of the system can be monitored and managed in real time. 76 Figure 5.1: Deployable and retractabe shock strut system configuration, partial cross-section Figure 5.1: Deployable and retractabe shock strut system configuration, partial cross-section This electrically operated arrangement came as an alternate solution to a standard hydraulic fluid system, typically used in the extension of landing gear, as discussed in Chapter 2. The current electric based design requires only a power supply (pre-existing on a pod) and avoids the use of a hydraulic system, which would require pumps, reservoirs, and hydraulic lines to be located throughout the pod. 77 5.2 System Operation Using the deployable and retractable shock strut configuration discussed in Section 5.1, the kinematic architecture as defined in Chapter 3, Section 3.5.2, was achieved. The extension (deployment) of the system, as shown in Figure 5.2, was accomplished in-part using an electronics and control system developed by Shonibare [16] and Adhikari [40]. The same system was used for the retraction of the system, by reversing the torque on the motor shaft, and performing height maintenance activities. By utilizing two linear potentiometers and four pod mounted lasers (one in each corner), the deployable and retractable shock strut was demonstrated to provide active feedback response, when the pod mass was disturbed by external sources (i.e. the change of the pod CG, for testing purposes, using a moving mass). Figure 5.2: HDWS extension and retraction process, some parts hidden for clarity Figure 5.2: HDWS extension and retraction process, some parts hidden for clarity Now that the reader is familiar with the overall HDWS concept and has been introduced to the deployable and retractable shock strut concept, the detail design considerations of the primary elements will be discussed. 78 5.3 HDWS Shock Absorber Design This section presents the preliminary and detail design of the HDWS shock absorber (or shock strut) component. The shock strut provides energy dissipation on extension (at touchdown) and ensures no loads are directly transferred to the Hyperloop pod vehicle frame. An oleo-pneumatic shock strut architecture was selected, which is the typical choice for aircraft landing gear due to their light-weight design and superior energy dissipation characteristics [5]. The preliminary design of the shock strut, particularly the design of an oleo-pneumatic shock strut, is covered by the works of Currey [5] and Conway [6] and was used here for design reference. Industry specifications and standards were used for the detail design process, the most applicable of which are tabulated here in Table 5.1 for convenience. Table 5.1: Shock strut design standards and specifications referenced Standard/Specification Document Description ASME/ANSI B1.5-1997 General Purpose Acme Thread Form SAE AIR1362B Aerospace Hydraulic Fluid Physical Properties SAE AS28889A Valve, Air, High Pressure Charging, 5000 PSI SAE AS8934/2 Bearing, Sleeve, Flanged, Self-lubricating SAE AS8879D Screw Threads – UNJ Profile, Inch SAE AS4716B Gland Design, O-ring and Other Elastomeric Seals SAE AS5857A Gland Design, O-ring and Other Elastomeric Seals, Static Applications MIL-L-8552C Landing Gear, Aircraft Shock Absorber (Air-Oil Type) MIL-T-6053C Tests, Impact, Shock Absorber Landing Gear, Aircraft MIL-PRF-5606H Hydraulic Fluid, Petroleum Base (Red Oil) Table 5.1: Shock strut design standards and specifications referenced 5.3.1 Oleo-Pneumatic Shock Absorber Introduction Oleo-pneumatic shock absorbers consist of oil, typically petroleum based as specified by MIL- PRF-5606H, and a gas, typically dry nitrogen. Although it is possible to use air as the gas, modern designs use dry nitrogen gas due to its inert properties, therefore inhibiting corrosion [5]. An example of a single acting shock absorber, typically comprised of a piston, shock absorber cylinder and orifice, is shown in Figure 5.3. In this design, when the shock strut is compressed, oil is forced through a controlled area (typically a dampening orifice) and into the pressurized gas spring to dissipate energy in a controlled manor. In most designs, the shock absorber’s rate of motion under 79 dynamic loading is limited by the damping action induced by the oil flowing through a controlled area [24]. As shown in Figure 5.3, there are many ways in which a designer can configure the internals of a shock strut to achieve the desired performance characteristics, with no one way being the best solution in all conditions. Figure 5.3: An example of an oleo-pneumatic shock strut with oil and gas free to mix [5] Figure 5.3: An example of an oleo-pneumatic shock strut with oil and gas free to mix [5 The first stage in the shock absorber design process is typically to understand the energy dissipation requirements at landing. This is ordinarily the scenario in which an aircraft shock strut sees its worst-case loading and requirement for energy dissipation. This provides a rough estimate for the required landing stroke (distance through which the kinetic energy is dissipated). As is demonstrated by Currey [5], this preliminary exercise usually considers, at minimum, a rate of descent (sink speed) of 6ft/s (1.8m/s) for aircraft. In the case of the HDWS, which is deployed in a controlled manor onto the sub-track surface, the equivalent sink speed provided by the 80 deployment actuation unit is approximately 0.11 ft/s (0.035 m/s). For this reason, it was expected that this method would be unsuccessful for the HDWS design. Hence, other preliminary design methods and assumptions outlined by Currey were used in its place [5]. In this case, a mix of gas spring properties, preliminary design guidelines and geometric constraints imposed by the prescribed operational space were applied. 5.3.1 Oleo-Pneumatic Shock Absorber Introduction For the design of a shock absorber gas spring for a wheeled vehicle, the effects of tire compression under load should also theoretically be considered [5], however, the polyurethane wheel discussed in Chapter 4 was assumed to be sufficiently rigid and its compression was subsequently ignored for this work. 5.3.2 Shock Absorber Gas Spring Preliminary Design After a general layout of the shock strut arrangement was created for reference, the next step was to begin assigning values to variables defined in Table 5.5. The process for selecting the final numbers was highly iterative but began first, as recommended in Currey [5], by defining a pressure at static stroke. Turning to statics, the following equation is defined [46]: 𝑃𝑆= 𝐹𝑆 𝐴𝑃 (5.1) (5.1) where 𝑃𝑆 is defined as the pressure at static piston stroke, 𝐹𝑆 is the static axial loading on the shock strut and 𝐴𝑃 is the area of the piston. The equation for the area of the piston is circular and is defined by the piston rod outer diameter, 𝑂𝐷𝑃𝑅. This is used to determine the total volume change within the shock absorber throughout stroking operations. where 𝑃𝑆 is defined as the pressure at static piston stroke, 𝐹𝑆 is the static axial loading on the shock strut and 𝐴𝑃 is the area of the piston. The equation for the area of the piston is circular and is defined by the piston rod outer diameter, 𝑂𝐷𝑃𝑅. This is used to determine the total volume change within the shock absorber throughout stroking operations. At this point, there are three unknowns in equation (5.1). A key piece of information required from the pod manufacturer is an estimate of the entire system’s final weight. Based on initial estimates of the U of T pod architecture, a pod mass was selected that enabled the determination of the force, 𝐹𝑆, at a static stroke. Using Figure 5.4 as an example, assume a pod exists with a known mass 𝑚 and CG, an optimal positioning of the HDWS’s within the pod frame can be determined. For the purposes of the present discussion an equal distribution of the pod mass, 750 lbm (340 kg) was assumed. Note that if detailed pod CG and inertia was known, the distribution of the HDWS’s 81 throughout the pod could be determined for best performance and stability during all projected loading scenarios (e.g. dynamic braking, accelerating). This process would normally be done in practice at the early stages of HDWS subsystem design and pod frame design. 5.3.2 Shock Absorber Gas Spring Preliminary Design Figure 5.4: Example of a hypothetical pod layout, loads applied to the HDWS in a static position Figure 5.4: Example of a hypothetical pod layout, loads applied to the HDWS in a static position As shown in Figure 5.4, the HDWS reaches a static stroke when the vertical load transferred through the trailing arm axle is equivalent to the weight of the pod divided equally among the systems. Since the system architecture is of the trailing arm type, the load through the shock strut is not equivalent to the load at the wheel axle. In Figure 5.5 the reference key points for the HDWS kinematics are shown in its Cartesian coordinates as defined in Chapter 3. These key points – (1 through 5) – are used to mathematically determine the force in shock strut member (4) – (5), defined as 𝐹45. As shown in Figure 5.5, the loading in 𝐹45 was calculated by finding the sum of the moments about point (1). To do so, the perpendicular distance (shortest distance) between member (4) – (5) and pivot point (1) was determined. By the same token, the horizontal distance (shortest distance) between point (1) and line of action of the force at (2), where a portion of the pod weight is reacted, was also determined. 82 Figure 5.5: The HDWS preliminary configuration, stick diagram Figure 5.5: The HDWS preliminary configuration, stick diagram It was found that the moment arm distances for each force about point (1) were best determined from the kinematic models within CATIA® V5. This was advantageous in the early stages of design when the positions of the key points defined in Figure 5.5 were subject to change. The mathematical definition is shown here for reference. Let 𝑎 be the horizontal distance along the x-axis between points (1) and (2) such that: 𝑎= \|𝑥2 −𝑥1\| (5.2) (5.2) where 𝑥1 and 𝑥2 are the 𝑥-coordinates relative to a specific reference point, namely point (1). where 𝑥1 and 𝑥2 are the 𝑥-coordinates relative to a specific reference point, namely point (1). 5.3.2 Shock Absorber Gas Spring Preliminary Design Let 𝑏 be the perpendicular distance from the force vector 𝐹45 defined as: Let 𝑏 be the perpendicular distance from the force vector 𝐹45 defined as: Let 𝑏 be the perpendicular distance from the force vector 𝐹45 defined as: 𝑏= \|[(𝑥4 −𝑥5)(𝑦4 −𝑦1)] −[(𝑥4 −𝑥1)(𝑦5 −𝑦4)]\| √(𝑥4 −𝑥5)2 + (𝑦4 −𝑦5)2 (5.3) (5.3) Finally, the force in the shock strut 𝐹45 can be determined. In this case, the value for 𝐹𝑆 is desired to assist in the initial shock strut sizing. Thus, the values utilized for the 𝑥 and 𝑦 coordinates of the points (2) and (4) shall be those approximating the position of static stroke. As a first approximation, Currey [5] provides a data table consisting of historic static stroke positions as a 83 percentage of maximum stroke while Conway [6] suggested that a maximally loaded vehicle should have a compressed stroke position of not more than two-thirds total stroke. Acknowledging the preceding, the HDWS’s unique kinematic motion taking place between the retracted state (shock strut fully extended, stroke equal to zero) and the static stroke state has no effect on the values of 𝑎 and 𝑏 defined in equations (5.2) and (5.3). It can now be stated that the values of 𝑎 and 𝑏 change with the extension and retraction of the HDWS, rotating about point (1), up to WOW (touchdown). After WOW is achieved, the system compresses the oleo-pneumatic shock strut piston until reaching the static load of the pod. During the compression, the motion of the shock strut is linear – that is, the perpendicular distance defined by 𝑏 remains the same. The assumption here holds true if the pod is levitating at a planned altitude throughout the extension operation. Furthermore, when the HDWS performs a height adjustment of the pod relative to the ground, the values of 𝑎 and 𝑏 defined in equations (5.2) and (5.3) change as the pod is raised. This can be easily understood by imagining points (1) and (5), fixed to a pod raising in the vertical direction, while point (4) swings CW about point (5) and point (2) remains “fixed” at a radial distance about the tube sub-track. Bearing all the preceding information in mind, it can be appreciated that by modifying the component geometry it is possible to control the value for 𝐹45 while maintaining the kinematic arrangement within the specified area. 5.3.2 Shock Absorber Gas Spring Preliminary Design Thus, the equation governing the value for 𝐹45, referencing Figure 5.5, is defined by: +𝐶𝐶𝑊∑𝑀𝐴= 0 ; (𝑊𝑃𝑜𝑑 4 ) (𝑎) − 𝐹45𝑏= 0 (5.4) (5.4) Solving equation (5.4) for 𝐹45: Solving equation (5.4) for 𝐹45: Solving equation (5.4) for 𝐹45: 𝐹45 = (𝑊𝑃𝑜𝑑 4 ) (𝑎) 𝑏 (5.5) (5.5) As discussed earlier in this report, equation (5.5) is only valid for solving the force 𝐹45 at the static stroke of the piston and at any new static stroke defined by a height adjustment thereafter. Since the present calculations were based on a pod mass of 750lbm (340kg), a value for 𝐹45 can be determined using the kinematic arrangements determined in Chapter 3. 84 Referring to equation (5.1), two terms remain unknown – the area of the piston 𝐴𝑃 and the pressure at static stroke 𝑃𝑆. It was decided that the minimum permissible pressure required would be used to overcome the seal friction (sticksion) stated by Currey [5] to be 60 psi (413.7 kPa). As noted by Currey, this initial pressure assures that the shock absorber can fully extend when unloaded. Thus, using 60 psi as the initial pressure, a spread sheet partially shown in Table 5.2 was created by the author based on the ideal gas law (isothermal) formulae presented by Currey [5]. This tool enabled the calculation of an initial gas spring utilizing the preliminary compression ratios defined by Currey and based on the kinematic geometry required to achieve the static force required to balance the pod weight, that is 𝐹𝑆= 𝐹45(𝑊𝑃𝑜𝑑, 𝑎, 𝑏). The initial key results of the iteration, as displayed in the shock strut design excel sheet, are shown in Table 5.2 and Table 5.3 for reference. Table 5.2: Shock strut design tool developed by the author using equations derived in Currey [5] Table 5.2: Shock strut design tool developed by the author using equations derived in Cu Table 5.2: Shock strut design tool developed by the author using equations derived in Currey [5] LEGEND Input Output Gas Spring (Isothermal) Stroke (in) V (in3) P (psi) Load (lb) Required Info/Design Parameter Value 0 2.775 60.000 52.849 Pod Max Weight (lb) 750.000 0.5 2.334 71.321 62.820 No. Table 5.3: Key shock strut stroke position outputs (ideal gas law, isothermal assumption) Extended Load (lb) 52.9 P1 (psi) 60 V1 (in3) 2.775 Stroke (in) 0.00 % Stroke 0.00 Static Load (lb) 222.2 P2 (psi) 252 V2 (in3) 0.661 Stroke (in) 2.40 % Stroke 80.00 Compressed Load (lb) 1111.2 P3 (psi) 1260 V3 (in3) 0.132 Stroke (in) 3.00 % Stroke 100.00 5.3.2 Shock Absorber Gas Spring Preliminary Design of Struts 4.000 1 1.894 87.907 77.429 Load per Strut, Static Case (lb) 187.500 1.5 1.453 114.545 100.893 Length, Trailing Arm (in) 10.000 2 1.013 164.348 144.759 Postion, SS/T-Arm (in) 9.000 2.1 0.925 180.000 158.546 Radial offset of SS/T-Arm Lug (in) 2.500 2.2 0.837 198.947 175.235 Static Trailing Arm Position (rad) -0.305 2.3 0.749 222.353 195.850 Static SS Position (rad) 2.069 2.4 0.661 252.000 221.964 Static Axial Loading (lb) 222.249 2.5 0.573 290.769 256.112 Static to Extended Ratio 4.200 2.6 0.484 343.636 302.678 Compressed to Static Ratio 5.000 2.7 0.396 420.000 369.940 Total Stroke (in) 3.000 2.8 0.308 540.000 475.637 Charge Pressure(psi) 60.000 2.9 0.220 756.000 665.892 Total Gas Displacement (V1-V3) (in3) 2.642 3 0.132 1260.000 1109.819 Table 5.2: Shock strut design tool developed by th LEGEND Input Output Required Info/Design Parameter Value Pod Max Weight (lb) 750.000 No. of Struts 4.000 Load per Strut, Static Case (lb) 187.500 Length, Trailing Arm (in) 10.000 Postion, SS/T-Arm (in) 9.000 Radial offset of SS/T-Arm Lug (in) 2.500 Static Trailing Arm Position (rad) -0.305 Static SS Position (rad) 2.069 Static Axial Loading (lb) 222.249 Static to Extended Ratio 4.200 Compressed to Static Ratio 5.000 Total Stroke (in) 3.000 Charge Pressure(psi) 60.000 Total Gas Displacement (V1-V3) (in3) 2.642 Gas Spring (Isothermal) Stroke (in) V (in3) P (psi) Load (lb) 0 2.775 60.000 52.849 0.5 2.334 71.321 62.820 1 1.894 87.907 77.429 1.5 1.453 114.545 100.893 2 1.013 164.348 144.759 2.1 0.925 180.000 158.546 2.2 0.837 198.947 175.235 2.3 0.749 222.353 195.850 2.4 0.661 252.000 221.964 2.5 0.573 290.769 256.112 2.6 0.484 343.636 302.678 2.7 0.396 420.000 369.940 2.8 0.308 540.000 475.637 2.9 0.220 756.000 665.892 3 0.132 1260.000 1109.819 Table 5.3: Key shock strut stroke position outputs (ideal gas law, isothermal assumption) Extended Load (lb) 52.9 P1 (psi) 60 V1 (in3) 2.775 Stroke (in) 0.00 % Stroke 0.00 Static Load (lb) 222.2 P2 (psi) 252 V2 (in3) 0.661 Stroke (in) 2.40 % Stroke 80.00 Compressed Load (lb) 1111.2 P3 (psi) 1260 V3 (in3) 0.132 Stroke (in) 3.00 % Stroke 100.00 Table 5.3: Key shock strut stroke position outputs (ideal gas law, isothermal assumption) 85 Using the theory presented by Currey [5] as a preliminary calculation, the initial gas spring calculation and its results shown in Table 5.2 and Table 5.3 were generated based on an isothermal simplification of the ideal gas law (thermodynamics). 5.3.2 Shock Absorber Gas Spring Preliminary Design The equations used to generate the results shown in Table 5.2 and Table 5.3 are now defined with reference to Figure 5.6. Figure 5.6: Illustration of oleo-pneumatic shock strut key operational states Figure 5.6: Illustration of oleo-pneumatic shock strut key operational states 86 As an approximation of the static gas spring behaviour at the states illustrated in Figure 5.6, the ideal gas law is applied under isothermal assumptions, where the temperature 𝑇 is assumed constant such that 𝑇1 = 𝑇2 = 𝑇3 resulting in the relationship: 𝑃1𝑉1 = 𝑃2𝑉2 = 𝑃3𝑉3 (5.6) (5.6) Referencing equation (5.6) and Figure 5.6, the following definitions apply: Referencing equation (5.6) and Figure 5.6, the following definitions apply: Referencing equation (5.6) and Figure 5.6, the following definitions apply: Table 5.4: Shock strut gas spring design features and definitions per Currey [5] Table 5.4: Shock strut gas spring design features and definitions per Currey [5] Symbol Description 𝑃1 Gas pressure at full extension, 0% stroke 𝑃2 Gas pressure at static stroke under static load 𝑃3 Gas pressure in the compressed position, 100% stroke 𝑉1 Gas volume at full extension, 0% stroke 𝑉2 Gas volume at static stroke under static load 𝑉3 Gas volume in the compressed position, 100% stroke Since 𝑃1 was selected to be 60 psi the next step was to determine 𝑃2 and 𝑃3 using the recommended pressure ratios provided by Currey [5]. These ratios were selected to help ensure floor height variation between static and compressed positions during dynamic operation is kept to a minimum. This was an important design factor to consider when designing the gas spring that ensured the pod’s primary levitation systems or any part of the pod frame would not crash into the sub-track. F li i i i th ti tili d C [5] For preliminary sizing purposes, the ratios utilized per Currey [5] were: Static to extended = 4/1 (5.7) Compressed to static = 3/1 (5.8) Static to extended = 4/1 (5.7) Compressed to static = 3/1 (5.8) Static to extended = 4/1 (5.7) (5.7) Compressed to static = 3/1 (5.8) (5.8) (5.8) Note: the above compression ratios should be used with caution as they do not account for the compressibility of the oil [5]. 5.3.2 Shock Absorber Gas Spring Preliminary Design 87 Based on the ratio defined by (5.7) the pressure 𝑃2 is found by: Based on the ratio defined by (5.7) the pressure 𝑃2 is found by: 4 1 = 𝑃2 𝑃1 → 𝑃2 = 4𝑃1 (5.9) (5.9) (5.9) Similarly, the pressure at the compressed position (maximum stroke) 𝑃3 is estimated using (5.8) and found by: 3 1 = 𝑃3 𝑃2 → 𝑃3 = 3𝑃2 (5.10) Now, using a known value of 𝐹𝑆 and by substituting the result of equation (5.9), the area of the piston is determined as follows: Now, using a known value of 𝐹𝑆 and by substituting the result of equation (5.9), the area of the piston is determined as follows: 𝑃2 = 4𝑃1 = 𝐹𝑆 𝐴𝑃 (5.11) 𝑃2 = 4𝑃1 = 𝐹𝑆 𝐴𝑃 (5.11) Re-arranging equation (5.11) for the piston area 𝐴𝑃 gives: Re-arranging equation (5.11) for the piston area 𝐴𝑃 gives: 𝐴𝑃= 𝐹𝑆 4𝑃1 (5.12) (5.12) This enabled a preliminary sizing for the shock strut piston outer diameter, 𝑂𝐷𝑃𝑅, which was solved by: 𝑂𝐷𝑃𝑅= √4𝐴𝑃 𝜋 (5.13) (5.13) Note: The significance of equation (5.13) will not be discussed here but will play a key role in determining the final gas spring properties of the shock strut and will thus be referred to at the end of this section prior to defining the internal geometries at a later stage. Knowing the area 𝐴𝑃 by which the internal volume of the shock strut is compressed, the total change in volume from extended stroke to compressed stroke can be found by: ∆𝑉= 𝑉3 −𝑉1 = 𝐴𝑃 𝑆𝑀𝑎𝑥 (5.14) (5.14) 88 where 𝑆𝑀𝑎𝑥 is defined as the total permissible stroke of the shock strut. At this stage, it was necessary to define a maximum stroke which was selected based on the kinematic studies highlighted in Chapter 3. For the present work, 𝑆𝑀𝑎𝑥= 3. In general, solving equation (5.14) for 𝑉3 and substituting the result into equation (5.6) gives: In general, solving equation (5.14) for 𝑉3 and substituting the result into equation (5.6) gives: 𝑉1 = 𝑉3𝑃3 𝑃1 = 𝑃3𝐴𝑃 𝑆𝑀𝑎𝑥+ 𝑉1𝑃3 𝑃1 (5.15) (5.15) After solving for 𝑉1, the ideal gas law was used to find 𝑉2 and 𝑉3. Using these results a shock absorber load-stroke curve shown in Figure 5.7 was produced. 5.3.2 Shock Absorber Gas Spring Preliminary Design Figure 5.7: Graphic representation of the isothermal oleo-pneumatic gas spring curve produced using the results in Table 5.2 0.0 50.0 100.0 150.0 200.0 250.0 300.0 350.0 400.0 450.0 500.0 550.0 600.0 650.0 700.0 750.0 800.0 850.0 900.0 950.0 1000.0 1050.0 1100.0 1150.0 1200.0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 Shock Strut Load [lb] Piston Stroke [in] Load vs. Piston Stroke Curve 0.0 50.0 100.0 150.0 200.0 250.0 300.0 350.0 400.0 450.0 500.0 550.0 600.0 650.0 700.0 750.0 800.0 850.0 900.0 950.0 1000.0 1050.0 1100.0 1150.0 1200.0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 Shock Strut Load [lb] Piston Stroke [in] Load vs. Piston Stroke Curve Load vs. Piston Stroke Curve Load vs. Piston Stroke Curve Figure 5.7: Graphic representation of the isothermal oleo-pneumatic gas spring curve produced using the results in Table 5.2 By examining Figure 5.7 it can be said that a shock strut whose static stroke is in the range of 2.2 to 3 inches will be best able to resist the effects of dynamic loading during pod travel. This is an 89 important consideration for the HDWS shock strut design to ensure that any dynamic loading, which by design causes the shock strut to compress, does not allow rapid and large displacements that could damage the pods exterior elements close to the tube sub-track. As the details of the shock strut internals were further defined geometrically the final internal gas and oil volumes were determined with the assistance of CATIA® V5. Before moving to the next section on the geometric sizing of the shock strut, it is important to recognize the following points, summarized in list form, to be addressed in the detailed gas spring design. 1) The preceding gas spring design was developed under isothermal assumptions. It is known that as substance compresses, particularly at high speeds and pressures, that the effect of temperature should not be ignored. 2) By the same token, the present process has not yet considered the compressibility of the fluid utilized, industry standard MIL-H-5606 red mineral oil. The bulk modulus of the fluid, as defined by AIR 1362B, was used to determine the final gas spring. 3) The present gas spring has only considered static loading, which, per Currey [5], is sufficient for normal, low-speed disturbances. 5.3.2 Shock Absorber Gas Spring Preliminary Design The final gas spring will consider both dynamic and static loading using an isentropic (adiabatic) gas coefficient suitable for a mixed oil and gas shock strut configuration, defined by γ. 4) The initial pressure ratios defined by (5.7) and (5.8) were iteratively varied to meet geometric requirements, specifically for the sealing surface on the piston outer diameter (𝑂𝐷𝑃𝑅) defined by equation (5.13). Using the ratio specified by Currey [5] as a guideline, the ratio was varied, keeping a 60-psi charge pressure constant, to converge on a value for 𝑂𝐷𝑃𝑅 near a standard sealing OD, specified by SAE AS4716B for dynamic sealing surfaces. 5.3.3 Internal Geometry Definition This section will present an overview of the design factors from a mechanical and performance point of view. Figure 5.8 highlights the fundamental shock absorber components whose relevant geometric features are described in Table 5.5 and further defined in Figure 5.9. Table 5.5 provides definitions for the key geometrical elements that were considered during the design phase and later used to size the final detail parts. 90 Figure 5.8: A simplified oleo-pneumatic strut, key preliminary design components labeled for clarity Figure 5.8: A simplified oleo-pneumatic strut, key preliminary design components labeled for clarity Figure 5.8: A simplified oleo-pneumatic strut, key preliminary design components labeled for clarity Table 5.5: Shock strut mechanical design features and definitions Symbol Description and Sizing Considerations 𝑅𝑃𝐿 Piston Lug Radius – defined to ensure an adequate lug thickness, typically < ODPR for lower bearing assembly purposes. 𝐼𝐷𝑃𝐿 Piston Lug Inner Diameter – typically sized based on the interfacing pin connection, or bush install (in this case, a spherical bearing). This joint diameter was sized for a swaged spherical bearing that was installed with a small interference fit and swaged after installation. 𝑂𝐷𝑃𝑅 Piston Rod Outer Diameter – defined as the running surface against the dynamic seals located within the lower bearing (gland nut), typically adjusted to meet closest standard seal sizing per AS4716B after preliminary pressure/volume sizing. 𝐼𝐷𝑃𝑅 Piston Rod Inner Diameter – is sized preliminarily based on required wall thicknesses to withstand hoop stress and compression stresses (buckling) at bottoming. The inner diameter can be further refined based on internal configuration requirements, e.g. the addition of an orifice support tube with externally mounted dynamic seal (Figure 5.3). 𝑂𝐷𝑃𝑈𝐵 Piston Upper Bearing Outer Diameter – typically sized in conjunction with the lower bearing gland nut and the outer cylinder inner diameter (𝐼𝐷𝑂𝐶). The final sizing typically accounts for additional flow regulation, as will be shown in the detail design discussion. 𝐿𝑃𝑈𝐵 Piston Upper Bearing Length – preliminarily sized based on the total bearing overlap length (𝐿𝐵𝑂) and then further refined based on the bearing pressures and material selection (typically a bronze for good wear resistance and lubricity). Table 5.5: Shock strut mechanical design features and definitions Table 5.5: Shock strut mechanical design features and definitions Symbol Description and Sizing Considerations 𝑅𝑃𝐿 Piston Lug Radius – defined to ensure an adequate lug thickness, typically < ODPR for lower bearing assembly purposes. 5.3.3 Internal Geometry Definition 𝐼𝐷𝑃𝐿 Piston Lug Inner Diameter – typically sized based on the interfacing pin connection, or bush install (in this case, a spherical bearing). This joint diameter was sized for a swaged spherical bearing that was installed with a small interference fit and swaged after installation. 𝑂𝐷𝑃𝑅 Piston Rod Outer Diameter – defined as the running surface against the dynamic seals located within the lower bearing (gland nut), typically adjusted to meet closest standard seal sizing per AS4716B after preliminary pressure/volume sizing. 𝐼𝐷𝑃𝑅 Piston Rod Inner Diameter – is sized preliminarily based on required wall thicknesses to withstand hoop stress and compression stresses (buckling) at bottoming. The inner diameter can be further refined based on internal configuration requirements, e.g. the addition of an orifice support tube with externally mounted dynamic seal (Figure 5.3). 𝑂𝐷𝑃𝑈𝐵 Piston Upper Bearing Outer Diameter – typically sized in conjunction with the lower bearing gland nut and the outer cylinder inner diameter (𝐼𝐷𝑂𝐶). The final sizing typically accounts for additional flow regulation, as will be shown in the detail design discussion. 𝐿𝑃𝑈𝐵 Piston Upper Bearing Length – preliminarily sized based on the total bearing overlap length (𝐿𝐵𝑂) and then further refined based on the bearing pressures and material selection (typically a bronze for good wear resistance and lubricity). 91 𝐿𝐿𝐵 Lower Bearing Length – the final sizing of the lower bearing length is a highly dependent on the utilized engagement method (e.g. threading to the ID or OD of the outer cylinder) and the quantity and size of the seals installed within its volume. 𝐿𝐵𝑂 Total Bearing Overlap Length – (𝐿𝐵𝑂> 1.25𝑥𝑂𝐷𝑃𝑅 per MIL-L-8552C and Currey [5]) 𝐼𝐷𝑂𝐶 Outer Cylinder, Inner Diameter – typically sized based on required piston head area for optimal P/A load bearing capabilities. The inner diameter typically directly engages with the Piston upper bearing outer diameter (𝑂𝐷𝑃𝑈𝐵) and thus shall be sized in parallel with the outer diameter of the cylinder (𝑂𝐷𝑂𝐶) based on the required wall thickness. 𝑂𝐷𝑂𝐶 Outer Cylinder, Outer Diameter – the outer cylinder is typically sized to minimize cylinder growth, possible at high pressures, and to withstand bending loads. The present outer cylinder was sized based on the minor diameter of the ACME 3G thread machined onto its OD. 𝑆𝑀𝑎𝑥 Max Piston Stroke – the max stroke is the total amount of length adjustment a shock strut piston can undergo during operation. 5.3.3 Internal Geometry Definition Typically, pre-sized based on the energy dissipation requirements at touchdown. After an initial sizing, typically from expected landing loads, the max stroke is tweaked to ensure adequate ground clearance while deployed and meet kinematic requirements. It is important to note, as mentioned by Currey [5], that the piston stroke is not a function of the supporting structure (e.g. Hyperloop pod) weight. 𝐿𝑃𝐿𝑂 Piston Lug Centre Offset – this feature is typically defined in the detail design phase to ensure that the piston OD is clear of the interfacing geometry at the piston/trailing arm lug. The offset is typically at least equal to the Piston Lug Radius (𝑅𝑃𝐿). 𝐿𝐿𝐵 Lower Bearing Length – the final sizing of the lower bearing length is a highly dependent on the utilized engagement method (e.g. threading to the ID or OD of the outer cylinder) and the quantity and size of the seals installed within its volume. 𝐿𝐵𝑂 Total Bearing Overlap Length – (𝐿𝐵𝑂> 1.25𝑥𝑂𝐷𝑃𝑅 per MIL-L-8552C and Currey [5]) 𝐼𝐷𝑂𝐶 Outer Cylinder, Inner Diameter – typically sized based on required piston head area for optimal P/A load bearing capabilities. The inner diameter typically directly engages with the Piston upper bearing outer diameter (𝑂𝐷𝑃𝑈𝐵) and thus shall be sized in parallel with the outer diameter of the cylinder (𝑂𝐷𝑂𝐶) based on the required wall thickness. 𝑂𝐷𝑂𝐶 Outer Cylinder, Outer Diameter – the outer cylinder is typically sized to minimize cylinder growth, possible at high pressures, and to withstand bending loads. The present outer cylinder was sized based on the minor diameter of the ACME 3G thread machined onto its OD. 𝑆𝑀𝑎𝑥 Max Piston Stroke – the max stroke is the total amount of length adjustment a shock strut piston can undergo during operation. Typically, pre-sized based on the energy dissipation requirements at touchdown. After an initial sizing, typically from expected landing loads, the max stroke is tweaked to ensure adequate ground clearance while deployed and meet kinematic requirements. It is important to note, as mentioned by Currey [5], that the piston stroke is not a function of the supporting structure (e.g. Hyperloop pod) weight. 𝐿𝑃𝐿𝑂 Piston Lug Centre Offset – this feature is typically defined in the detail design phase to ensure that the piston OD is clear of the interfacing geometry at the piston/trailing arm lug. The offset is typically at least equal to the Piston Lug Radius (𝑅𝑃𝐿). 5.3.3 Internal Geometry Definition 𝐿𝐿𝐵 Lower Bearing Length – the final sizing of the lower bearing length is a highly dependent on the utilized engagement method (e.g. threading to the ID or OD of the outer cylinder) and the quantity and size of the seals installed within its volume. 𝐿𝑃𝐿𝑂 Piston Lug Centre Offset – this feature is typically defined in the detail design phase to ensure that the piston OD is clear of the interfacing geometry at the piston/trailing arm lug. The offset is typically at least equal to the Piston Lug Radius (𝑅𝑃𝐿). 𝐿𝑃𝐿𝑂 Piston Lug Centre Offset – this feature is typically defined in the detail design phase to ensure that the piston OD is clear of the interfacing geometry at the piston/trailing arm lug. The offset is typically at least equal to the Piston Lug Radius (𝑅𝑃𝐿). Figure 5.9: An example of an oleo-pneumatic shock strut highlighting preliminary geometric features Figure 5.9: An example of an oleo-pneumatic shock strut highlighting preliminary geometric features Figure 5.9: An example of an oleo-pneumatic shock strut highlighting preliminary geometric features 92 92 Based on the preliminary gas spring calculations performed and discussed in Section 5.3.2, the sizing of the piston rod outer diameter (𝑂𝐷𝑃𝑅) is used to determine the behavior of the gas spring. For this reason, the sizing of 𝑂𝐷𝑃𝑅 was incorporated into a shock strut design spread sheet, as shown in Table 5.6, and calculated from the piston area, 𝐴𝑃, determined by the ratio of 4:1 for static to extended, as defined by Currey [5]. Utilizing a spread sheet format with key terms defined enabled quick iterations to be performed. The result of one calculation, shown in Table 5.6, indicates that the dimension for 𝑂𝐷𝑃𝑅 was to be 1.086-in. Using this result, the available sealing sizes were referred to and helped to refine the calculated value, if necessary. 5.3.3 Internal Geometry Definition Table 5.6: Piston detail design template, as defined in the author’s shock strut design tool LEGEND Input Output Detail Design of Piston Value Piston D/t (for steel) (<15) 10.000 Calculated Piston Wall Thickness (in) 0.109 Resulting Piston Area (in2) 0.926 Resulting Piston Outer Diameter (in) 1.086 Required Piston Rod OD (see gland table), B (in) 1.059 Required Piston Area (in2) 0.881 Estimated Piston Inner Diameter (in) 0.869 Required ID (see gland table), A (in) 0.864 Piston Thickness (based on Gland Req.) (in) 0.098 Table 5.6: Piston detail design template, as defined in the author’s shock strut design tool As shown in Table 5.7, the closest possible piston rod bore dimension (B), defined by the sealing design specification SAE AS4716B, called for a 𝑂𝐷𝑃𝑅 of 1.059-in. To accommodate the standard fitting size based on the Rod Bore dimension shown in Table 5.7, the ratio of static to extended stroke, defined by equation (5.7), was modified. Table 5.7: Piston rod dynamic seal gland dimensions, per Greene Tweed [59], based on SAE AS4716B Piston Dynamic Seal GLAND # Rod Bore, (B) [in] Tolerance [in] Gland ID [in] Gland Width, G [in] -215 1.06 (-) 0.002 1.302 0.304 Piston rod dynamic seal gland dimensions, per Greene Tweed [59], based on SAE AS4716 93 For the reference of the reader, Figure 5.10 illustrates an example of a typical gland design, the feature that is used to house a seal. A similar design trade-off process, as was discussed here, was followed for all sealing surfaces during the design of the shock strut. For the reference of the reader, Figure 5.10 illustrates an example of a typical gland design, the feature that is used to house a seal. A similar design trade-off process, as was discussed here, was followed for all sealing surfaces during the design of the shock strut. Figure 5.10: Piston rod gland design, key dimensions labeled, as defined by Greene Tweed [59] Figure 5.10: Piston rod gland design, key dimensions labeled, as defined by Greene Tweed [59] 5.3.4 Detailed Gas Spring Design The calculation to determine the final gas spring was accomplished using a Safran Landing Systems Inc. proprietary gas spring software, Dynatool© Version 2.1 [60]. Fundamentally, Dynatool© provides a means to: 1) Apply the compressible polytropic (adiabatic) and isothermal ideal gas theory to produce dynamic and static gas spring curves using nitrogen specific volume data presented by Din [61] on the behaviour of real gases 2) Account for the compressibility of the fluid (oil) by incorporating the fluids bulk modulus, defined in AIR 1362B [62] as the ability of a fluid to resist changes in volume under pressure, and provided a means to account for the dynamic effects by utilizing the bulk modulus as a function of pressure and temperature (in both isothermal and adiabatic calculations). 3) Account for the thermal expansion of the oil using the coefficient of thermal expansion 𝛼 94 4) Account for the mechanical behaviour of the structural elements, i.e. the piston and cylinder components and their respective tendencies expand and stretch under pressure. 4) Account for the mechanical behaviour of the structural elements, i.e. the piston and cylinder components and their respective tendencies expand and stretch under pressure. 4) Account for the mechanical behaviour of the structural elements, i.e. the piston and cylinder components and their respective tendencies expand and stretch under pressure. Finally, considering points 1) through 4) noted above, the tool obeys the continuity equation (no oil or gas is destroyed) such that: Finally, considering points 1) through 4) noted above, the tool obeys the continuity equation (no oil or gas is destroyed) such that: Finally, considering points 1) through 4) noted above, the tool obeys the continuity equation (no oil or gas is destroyed) such that: 0 = 𝑉𝑐−𝑉𝑐𝑜−𝐴𝑃𝑆−(𝑉𝑔−𝑉𝑔𝑜) −(𝑉𝑓−𝑉𝑓𝑜) (5.16) (5.16) where: 𝑉𝑐 is the total volume of the shock strut at operating temperature and pressure; 𝑉𝑐𝑜 is the total volume of shock strut at its initial servicing (filling) conditions; 𝐴𝑃 is the area of the piston found using equation (5.12); 𝑆 is defined as the stroke of the shock strut piston; 𝑉𝑔 is defined as the volume of nitrogen gas at operating temperature and pressure; 𝑉𝑔𝑜 is the volume of nitrogen gas at servicing conditions; 𝑉𝑓 is the volume of oil at operating temperature and pressure; and 𝑉𝑓𝑜 is the initial volume of oil supplied at shock strut servicing conditions. 5.3.4 Detailed Gas Spring Design The results of the Dynatool© gas spring curve are shown in Figure 5.11 and Figure 5.12 representing the load without friction vs. stroke and pressure vs. stroke respectively. A sample of the output data is also shown in Appendix D. Table 5.8: Sample set of the Dynatool© program interface inputs required from the user Input Description Value Input Description Value Cylinder ID (in) 1.492 Nitrogen Charge Pressure, P1 (psi) 60 Cylinder OD (in) 1.709 Final (Servicing) Volume, V1 (in3) 0.106 Cylinder Poisson Ratio 0.32 Dynamic Polytropic Exponent N/A Cylinder Elastic Modulus (psi) 290000 Max Piston Stroke (in) 3 Piston ID (in) 0.864 Cylinder Load at Static (lb) 222.5 Piston OD (in) 1.059 Operating Temperature (°C) 23 Piston Poisson Ratio 0.32 Oil Bulk Modulus (psi) 260000 Piston Elastic Modulus (psi) 290000 Change in Bulk Modulus with pressure (adiabatic) (psi/psi) 6.1 Total Volume (oil+gas) at fully extended stroke (in3) 9.174 Oil Thermal Expansion Coefficient (1/1/°C) 0.000864 Servicing Temperature (°C) 23 Static Stroke (est.) 2.26 Oil Bulk Modulus, isothermal (psi) 200000 Change in Bulk Modulus with pressure (isothermal) (psi/psi) 5.1 Table 5.8: Sample set of the Dynatool© program interface inputs required from the user 95 Figure 5.11: Dynatool© HDWS gas spring curve (static and dynamic), load vs. stroke Figure 5.11: Dynatool© HDWS gas spring curve (static and dynamic), load vs. stroke Figure 5.11: Dynatool© HDWS gas spring curve (static and dynamic), load vs. stroke Figure 5.12: Dynatool© HDWS gas spring curve (static and dynamic), pressure vs. stroke Figure 5.12: Dynatool© HDWS gas spring curve (static and dynamic), pressure vs. stroke Figure 5.12: Dynatool© HDWS gas spring curve (static and dynamic), pressure vs. strok 96 To provide the most accurate possible values for the input volumes required to run Dynatool©, the CATIA© V5 Part Design Workbench was used and the results are shown in Figure 5.13 through Figure 5.15. To provide the most accurate possible values for the input volumes required to run Dynatool©, the CATIA© V5 Part Design Workbench was used and the results are shown in Figure 5.13 through Figure 5.15. 5.3.4 Detailed Gas Spring Design Figure 5.13: Cross-section view showing the shock strut while fully compressed at servicing, total oil volume (red) 6.426 in3 Figure 5.13: Cross-section view showing the shock strut while fully compressed at servicing, total oil volume (red) 6.426 in3 Figure 5.14: Cross-section view showing the total volume when extended (purple) 9.174 in3, gas+oil Figure 5.14: Cross-section view showing the total volume when extended (purple) 9.174 in3, gas+oil Figure 5.14: Cross-section view showing the total volume when extended (purple) 9.174 in3, gas+oil Figure 5.15: Cross-section view of the shock strut trapped air volume (white) 0.106 in3, max piston compression Figure 5.15: Cross-section view of the shock strut trapped air volume (white) 0.106 in3, max piston compression 97 5.3.5 Shock Absorber Detail Design After determining the basic internal dimensions discussed in Section 5.3.2 and Section 5.3.3 required to create a functioning shock absorber, the detail geometric design process commenced. In reality, the close relationship between the seal gland sizing and interfacing surfaces forced the consideration of design details throughout the preliminary design phase. Some of the key details are now briefly discussed. Lower Bearing (Gland Nut): The shock strut lower bearing (Figure 5.16) was designed to serve three purposes: to act as a seal carrier for the dynamic, scraper and static seals; to act as a bearing surface keeping the piston rod axially aligned with the shock strut during stroke; and to act as an out-stop surface retaining the piston while under extended pressure (60 psi). Figure 5.16: Cross-section, lower bearing, key design details labeled Figure 5.16: Cross-section, lower bearing, key design details labeled Figure 5.16: Cross-section, lower bearing, key design details labeled 98 98 The gland nut was fitted with wrench slots, as defined in MIL-S-8552 and recommended by Currey [5], for ease of assembly. Specifically, this enabled the use of standard spanner wrench tooling. The sizes of the wrench slots were defined with reference to MIL-S-8552. The same wrench slots also provided an interface for a lock tab, labeled in Figure 4.14, prohibiting the gland nut from unscrewing. Furthermore, the threads on the outer diameter of the lower bearing were sized to handle at least two times the maximum pressure loading induced by oil and gas volume at maximum compression. In order prevent damage to the static seal during assembly, the threading was sufficiently offset to guarantee seal protection on assembly. As alluded to earlier, the seal glands were sized with reference to the SAE AS4176B and AS5857A specifications for the dynamic and static seal gland designs respectively. Custom installation tooling was designed to protect the seals during installation and, in some cases, provide a pre- squeeze to enable a smoother installation with a mating part. The tool drawings can be found in Appendix B. Upper Bearing, Piston Head: The piston head upper bearing, as can be seen in Figure 5.16, was designed to serve two purposes: to act as a flow regulator under fast compressions speeds, in theory providing additional dampening, by limiting flow rate of the oil during stroke; and to act in tandem with the lower bearing keeping the piston rod axially aligned with the shock strut during stroke. The design is a split ring configuration, attached on assembly within grooves machined into the shock strut piston outer diameter, prior to install into the shock strut outer cylinder. Filler Tube and Servicing Valve: The shock strut filler tube (Figure 5.17) was designed to serve two purposes: to act as a seal carrier for the static seal interfacing between the outer diameter of the filler tube and inner diameter of the cylinder; to enable the filling of the shock strut volume with oil and gas through the use of a standard valve designed per AS28889A in accordance with MIL-S-8552 [5]. As can be deduced from observing Figure 5.17 the filler tube must be assembled through the bottom of the shock strut 99 prior to installing the piston and lower bearing. The flange head within the interior of the cylinder utilizes a machined hexagonal socket interface for installation and to enable secure fitting of the filler tube through the tightening of the jam nut disposed below the AS28889A valve. The valve is mounted to the inner diameter of the filler tube using threads designed by the AS8879D UNJF thread specification per the matting requirements specified in AS28889A. Figure 5.17: 3D cross-section of the filler tube, key design details labeled Figure 5.17: 3D cross-section of the filler tube, key design details labeled As can be seen in Figure 5.17, the filler tube flange head was sized to act as a stoppage feature when the piston reaches maximum stroke (max compression). An additional important feature, highlighted in Figure 5.18, is the small bore machined perpendicular to the main filler bore axis. The positioning of this bore helps to regulate the amount of trapped gas in the shock strut during compression and thus plays a role in determining the stiffness of the gas spring. As can be seen in Figure 5.18, when the shock strut is compressed, the gas (colored in turquoise) tends to become trapped within the volume available above the small bore. The positioning of this feature allows for the softening of the gas spring, that is, it directly relates to the positioning of the static stroke along a given gas spring curve. 100 Figure 5.18: 3D cross-section view of the filler tube with trapped gas illustrated Figure 5.18: 3D cross-section view of the filler tube with trapped gas illustrated Piston: The piston (Figure 5.19) was manufactured from 303L structural stainless steel for corrosion resistance and to obtain a tough running surface for the seals. To meet specifications provided by the seal manufacturer, the outer diameter of the piston rod was ground to a specified surface roughness of 12 µin Ra or better. The thickness of the piston was pre-sized using diameter to thickness (D/t) ratios provided for reference by Safran Landing Systems Canada Inc. [48], not available for outside distribution. Finally, the piston was fit with a spherical bearing at the lug rod- end to limit the loading transferred from the trailing arm to axial loads only. The lug at the rod- end was subsequently sized to ensure that it could be passed through the lower bearing during installation. Figure 5.19: 3D view of the piston, spherical bearing installed, key features labeled Figure 5.19: 3D view of the piston, spherical bearing installed, key features labeled 101 101 Outer Cylinder: These lugs, when attached with the mating torque link assembly routed through the trailing arm, prevents the shock strut from rotating with the spur gear driven ACME retraction nut by removing the torque due to friction and enables the translational motion of the shock strut. Outer Cylinder: Outer Cylinder: The shock absorber outer cylinder (Figure 5.20) was designed to enable the use of typical internal shock absorber components, as discussed in the present chapter. In addition, the shock absorber for the HDWS is also designed to enable the extension and retraction of the HDWS system when equipped to the Retraction sub-assembly housing through the ACME teeth on its exterior. Figure 5.20: 3D cross-sectional view of the shock strut outer cylinder, key features labeled Figure 5.20: 3D cross-sectional view of the shock strut outer cylinder, key features labeled In particular, the thread was designed per the ASME/ANSI B1.5-1997 [63] General Purpose Acme thread form design specification. Per the information provided by Oberg et al. [64] within the Machinery’s Handbook, General Purpose threads are classified into three classes: 2G, 3G, and 4G. These three classes define the pitch diameter tolerances of the thread form. Due to the tight control required to maintain the height of the pod and reduce gaps in the actuation that could allow for vibration movement, a 3G ACME thread was selected, as recommended in [64], for use in cases where less backlash or “end-play” is desired. Based on the geometrical stack up imposed on the design by the sizing of the shock strut gas spring and internal features described here, a 2.250-3 ACME-3G external thread was selected for the shock strut. This size provided a sufficient outer cylinder wall thickness, which was closely monitored due to the stress concentration prone zones at the thread root. The sizing here played a key role in sizing the mating ACME nut, discussed in the next section. 102 The last feature of interest is the set of lugs (partially hidden in Figure 5.20) protruding from the bottom of the shock strut outer cylinder. These lugs, when attached with the mating torque link assembly routed through the trailing arm, prevents the shock strut from rotating with the spur gear driven ACME retraction nut by removing the torque due to friction and enables the translational motion of the shock strut. The last feature of interest is the set of lugs (partially hidden in Figure 5.20) protruding from the bottom of the shock strut outer cylinder. 5.4 HDWS Actuation Mechanism Design The Retraction sub-assembly was produced to provide a trunnion mounted configuration, with pod mounting pins disposed on either side of the retraction housing component, allowing the space between the mounting points to be utilized for functional features as can be seen in Figure 5.21. The Retraction sub-assembly performs three major functions that enable the HDWS’s operation. These functions include: acting as a pod interface structure by transmitting the load from the shock strut directly to the pod frame; housing an ACME geared retract nut responsible for transmission of the motor torque to the shock strut ACME threaded outer cylinder; and acting as the support structure for the mounting of the BLDC motor and sensor equipment described in Chapter 4’s Sections 4.1.2 and 4.1.4 respectively. Figure 5.21: 3D cross-sectional view of the retract assembly, trunnion pins labeled Figure 5.21: 3D cross-sectional view of the retract assembly, trunnion pins labeled 103 Retract Housing (Bushed): Fabricated from light-weight 6061-T6 aluminum the retract housing (Figure 5.22) is the primary structural element of the Retraction sub-assembly housing all the transmission components within its inner volume. The housing was fitted with AS8879D UNJF threads on the center bore, for engaging with a packing nut (not shown in Figure 5.22) during assembly. The body was machined from solid and includes a flange for mounting the motor bracket, using four 0.25-in bolt holes, and provided threaded pickup points for the cable potentiometer bracket, described in Section 4.1.4. Figure 5.22: Retract housing (bushed), key design features labeled Figure 5.22: Retract housing (bushed), key design features labeled To provide a bearing surface with excellent wear properties, the trunnion bores (Figure 5.21) were fitted with custom aluminum bronze (Al-Br) flanged bushings. To fix the rotation of the pin within the housing trunnion bore, the bushings and housing frame utilize cross bolts as an anti-rotation feature for the installed trunnion pins shown in Figure 5.21. Because of the anti-rotation feature, the inner diameter of the flanged bush (press-fit) does not require lubrication after initial installation since there is no relative rotation between the pin and the bushing. Lastly, the wear due 104 to low speed rotation is limited to the bushing outer flange, sized to mate with self-lubricating KAron® lined bushings at the pod interface. Packing Nut: Finally, with reference to Figure 5.24, a packing nut was designed equipped with wrench slots, as defined in MIL-S-8552 for ease of assembly. Specifically, this enabled the use of standard spanner wrench tooling. The same wrench slots also provided an interface for a lock tab, which is mounted to the retract housing via two NAS drilled head cap screws which are locked on assembly with lock-wiring (not shown). ACME Retract Nut: The ACME retract nut as shown in Figure 5.23 was designed to interface with the shock absorber outer cylinder, as shown in Figure 5.20. Consequently, the inner diameter of the rotatable nut was fitted with a 2.250-3 ACME-3G internal thread as specified by the ASME/ANSI B1.5-1997 [63] General Purpose Acme thread form design specification. To ensure no clashing or thread-end binding occurs between the transition radius (shown above the lugs in Figure 5.20) of the mating external ACME thread and the ACME nut, the nut’s design incorporated a modest thread offset, shown in Figure 5.23, providing clearance between the ACME nut and the cylinder lug transition feature. Figure 5.23: 3D cross-section view, ACME retract nut, key design features labeled Figure 5.23: 3D cross-section view, ACME retract nut, key design features labeled By design, the use of ACME threads provides a self-locking characteristic [65] that is considered to be a built-in safety feature for the HDWS. As an example, an extended and loaded HDWS will not retract (back-drive) if a power failure occurs on the pod since its locked position is not held in place by torque from the BLDC motor. Instead, the high friction of the threads is used, which does result in low thread efficiency [65]. The efficiency is then maximized in operation using Al-Br 105 (good friction and wear characteristics) as the ACME gear nut material and by applying vacuum grease lubrication (Dow Corning® [36] high-vacuum silicone grease) to provide a well-lubricated interface between the Al-Br nut and the steel cylinder. The ACME nut shown in Figure 5.23 has two machined interface grooves sized for an interference fit with mating deep groove angular contact bearings, supplied by SKF [42]. The bearings are installed on the outer diameter of the ACME nut, shown in Figure 5.24, prior to installation into the retract housing. The type of interference fit was selected based on the recommendations from the supplier and ensured that the inner race (ring) of the ball bearing was fixed from rotating relative to the ACME nut but that the internals of the ball bearing were free to rotate. Since angular contact bearings are specifically designed for this type of combined loading (thrust and radial) they were selected in place of thrust bearings. Figure 5.24: Retract assembly 3D exploaded view, key design features labeled Figure 5.24: Retract assembly 3D exploaded view, key design features labeled 106 6 Experimental Results This chapter provides a brief overview of the testing that took place on the HDWS’s starting in August of 2016 and concluding in January 2017. The testing of the HDWS was split up into component testing and full system testing. Using the testing capabilities and facilities provided by aircraft landing gear manufacturer Safran Landing Systems Canada Inc. [48] testing was performed on: the polyurethane wheel subject to high speeds and loading, the shock strut gas spring to verify the gas spring performance, and a full HDWS system. 5.5 Chapter Summary In this chapter, the Deployable and Retractable Shock Strut, as equipped to the HDWS, has been presented and its operation defined. The key elements of the Deployable and Retractable Shock Strut have been discussed and a detail design summary for the shock strut has been provided. After completing the detail design phase, five HDWSs were manufactured and assembled for testing and demonstration purposes. The next chapter will briefly discuss these tests and provide insights into both observed and measured results. 107 107 6.1 Polyurethane Wheel Performance Testing As discussed in Chapter 4, the HDWS prototypes used 95A polyurethane wheels for initial proof of concept testing and to meet the development deadlines set by SpaceX [3]. Testing was initiated in August 2016 to confirm the viability of the wheel proposed by Sunray Inc. [44]. The purpose of these tests was to confirm the safety and performance of the polyurethane wheel. The tests were designed to submit the polyurethane wheel to the expected Hyperloop competition speeds and projected loading conditions, based on the U of T pod concept. For the test to be deemed a success, the wheel was required to demonstrate that its performance (including wheel balance, roundness and resistance to wear) will not deteriorate under the specified loads and rotational speeds. To perform this test, a fixture was designed to house a single polyurethane wheel, the detail design drawings of which are included in the Appendix C. A wheel spin-up machine, consisting of an electric motor, drive shaft, and fly-wheel, developed by Safran Landing Systems Canada Inc. [48], was used to simulate the required testing speed of 180 mph (290 kph). The spin-up machine was linearly retractable and extendable, capable of engaging and disengaging with the fixed polyurethane wheel. The spin-up machine was positioned at engagement to exert an average load of 200lb, close to the load seen in the designed U of T configuration, and was measured using a load cell. The typical test setup, prior to engagement, is shown in Figure 6.1. 108 Figure 6.1: Wheel test configuration Figure 6.1: Wheel test configuration Figure 6.1: Wheel test configuration The testing procedure used is generally summarized here. The testing procedure used is generally summarized here. Test Summary: 1) The wheel (test article) was installed on the test fixture frame 2) The test fixture frame was fitted with an photo reflector to measure the speed (RPM) of the test article 3) The test article was equipped with reflective tape used to trigger the photo reflector sensor and provide feedback on the wheel RPM (Figure 6.2) 4) A thermal gun (Figure 6.1.) was used to measure in real-time the temperature of the wheel surface, placed 180 degrees from the contact point, clear of the engaging spin-up fixture (Figure 6.2) 109 109 5) Prior to engagement with the test article, the spin-up machine accelerated its Al 6061-T6 fly wheel to approximately 3400RPM, simulating a contact speed equivalent to a pod traversing the sub-track at 180 mph. 5) Prior to engagement with the test article, the spin-up machine accelerated its Al 6061-T6 fly wheel to approximately 3400RPM, simulating a contact speed equivalent to a pod traversing the sub-track at 180 mph. 6) The spin-up machine engaged the test article with a relative velocity of 180 mph, simulating a “touchdown” loading condition at contact spin-up machine contact, shown at 15s in Figure 6.3. 6) The spin-up machine engaged the test article with a relative velocity of 180 mph, simulating a “touchdown” loading condition at contact spin-up machine contact, shown at 15s in Figure 6.3. It was determined that this test would provide a worst case loading condition, applying approximately 200 lb plus high speeds instantaneously. In reality, the shock strut will begin to slowly compress at “touch-down” and the load will gradually increase on the wheel from 0 – 200 lb+ over time. This more accurate loading case was achieved during full-system testing. Test data, as recorded by the data acquisition unit, is shown in Figure 6.3. Here, the RPM of the fly-wheel (blue) is plotted along with the RPM as measured by the photo reflector of the polyurethane wheel (red). For convenience, the RPM of the polyurethane wheel was also shown as a linear velocity (green), defined as the pod speed (mph). Test Summary: Figure 6.2: Reflector tape positioning and wheel temperature measurement region shown Figure 6.2: Reflector tape positioning and wheel temperature measurement region shown 110 Figure 6.3: Plot illustrating Test #1, the pre-spin of the spin-up machine, engaging the stationary pod mounted wheel at 180 mph, and braking to stop at a decceleration rate of 0.6g Figure 6.3: Plot illustrating Test #1, the pre-spin of the spin-up machine, engaging the stationary pod mounted wheel at 180 mph, and braking to stop at a decceleration rate of 0.6g Because of the aggressive acceleration, shown graphically in Figure 6.3, and instantaneous loading conditions described above, the following results were observed: Because of the aggressive acceleration, shown graphically in Figure 6.3, and instantaneous loading conditions described above, the following results were observed: 1) Examining the wheel outer diameter before and after engagement it was noticed that localized, and “trailing” wear marking appeared. These markings appeared to be most significant at one localized point, and trailed off across the rest of the wheel diameter, shown in Figure 6.4. This would seem to indicate the wear is caused at the point of touchdown, when the relative velocities are furthest apart. This localized “spot” wearing is likely to have been caused in part by the “instantaneous” application of 200lb on the wheel outer diameter. It was noted that the temperature reading on the outer diameter, as measured by the thermal gun, indicated a peak temperature of over 176°F (80°C), close to the 195°F (90°C) operational performance limit of the material specified by Sunray Inc. [44]. 2) After an initial marking was formed, the tests were repeated, and it was found that after the first surface damage had occurred, the wear on the wheel outer diameter significantly 2) After an initial marking was formed, the tests were repeated, and it was found that after the first surface damage had occurred, the wear on the wheel outer diameter significantly 111 increased. This was observed during the second test when a thin layer of polyurethane was “shed” from the surface of the wheel and deposited on the spin-up fixture’s aluminum fly- wheel, shown in Figure 6.5. This event was also observed using a high-speed camera. Test Summary: Figure 6.4: Localized wear marking, followed by a trail-off effect, observed after Test #1 Figure 6.4: Localized wear marking, followed by a trail-off effect, observed after Test # Figure 6.5: Thin polyurethane deposited on the aluminum fly-wheel observed after Test #2, wheel deterioration increased under same loading and speeds Figure 6.5: Thin polyurethane deposited on the aluminum fly-wheel observed after Test #2, wheel deterioration increased under same loading and speeds 112 3) In subsequent testing, the wheel was accelerated in the same fashion as Figure 6.3 and after reaching 180 mph, the spin-up machine was “instantaneously” disengaged from the polyurethane wheel. This was meant to simulate the wheel “lifting off” from the pod sub-track once the pod has reached the usable speed of its primary levitation system. The test showed that the sudden disengagement caused the wheel to ovalize immediately after the disengagement of the load, and expand and contract in an oscillatory manor. This was recorded via the use of a high-speed camera and its effects were further documented through before and after photos, shown in Figure 6.6, indicating an expansion and contraction had occurred. Furthermore, subsequent tests of the same nature, but at speeds of 220 mph, showed signs of roller bearing rotation within its housing and loss of grease through the bearing seal cover due to the high rotation speeds of the bearing, shown in Figure 6.7. 3) In subsequent testing, the wheel was accelerated in the same fashion as Figure 6.3 and after reaching 180 mph, the spin-up machine was “instantaneously” disengaged from the polyurethane wheel. This was meant to simulate the wheel “lifting off” from the pod sub-track once the pod has reached the usable speed of its primary levitation system. The test showed that the sudden disengagement caused the wheel to ovalize immediately after the disengagement of the load, and expand and contract in an oscillatory manor. This was recorded via the use of a high-speed camera and its effects were further documented through before and after photos, shown in Figure 6.6, indicating an expansion and contraction had occurred. Furthermore, subsequent tests of the same nature, but at speeds of 220 mph, showed signs of roller bearing rotation within its housing and loss of grease through the bearing seal cover due to the high rotation speeds of the bearing, shown in Figure 6.7. Test Summary: Figure 6.6: Shifting of the reflective tape caused by the expanding and contracting outer diameter of the wheel after disengagement from the spin-up machine at 180 mph Figure 6.6: Shifting of the reflective tape caused by the expanding and contracting outer diameter of the wheel after disengagement from the spin-up machine at 180 mph In conclusion, the testing performed on the polyurethane wheel suggested that the solid polyurethane frame was unstable at high speeds and moderate loading. The disengagement at retraction should not cause the wheel to oscillate violently and cause bearing dislodgement from its housing frame, in this case, the solid polyurethane frame. Hence, the aluminum framed wheel discussed in Section 4.1.3, should be considered for future designs. On the other hand, a decision regarding the wear properties was not made at this time, as it was believed the addition of the shock absorbing unit would reduce the wear at touchdown caused by 113 a rigidly fixed wheel acted on by an “instantaneous” contact load. Furthermore, tests conducted on the spin-up machines load application, graphed in Figure 6.8, indicate the possibility of a large load spike at initial contact due to the spin-up machines forward inertia, before stabilizing at 200 lb. lb. Figure 6.7: Grease splatter and roller rotation evident as traced by the orange anti-rotation paint Figure 6.7: Grease splatter and roller rotation evident as traced by the orange anti-rotation paint Figure 6.8: Graph displaying the average load measured by the load cell as applied by the spin machine, load spikes at initial contact encircled in red Figure 6.8: Graph displaying the average load measured by the load cell as applied by the spin machine, load spikes at initial contact encircled in red 114 6.2 Gas Spring Testing Testing was performed on the shock strut to verify the behaviour of its gas spring, as defined using Dynatool© and the preliminary isothermal calculations discussed in Chapter 5. The testing was performed a full day after servicing (filling the strut with oil and nitrogen) to allow the gas and oil to mix and settle. The internal pressure was measured using a pressure transducer mounted at the top of the shock strut through its filler valve prior to testing. In each case, for all five shock absorbers (HDWS#1 through HDWS#5), the measured values were compared to the theoretical values generated by Dynatool©. The test fixture was configured as shown in Figure 6.9 and included a hydraulic actuator to apply the compressive force on the shock strut measured through a load cell mounted between the shock strut and the hydraulic actuator rod-end. The test setup, as shown in Figure 6.9, was re-oriented vertically, as shown in Figure 6.10 for the final testing to ensure that the oil remained at the lower half of the strut and the gas in the upper half. Figure 6.9: Shock absorber gas spring verification initial, initial setup Figure 6.9: Shock absorber gas spring verification initial, initial setup 115 Figure 6.10: Shock absorber gas spring verification test, final setup Figure 6.10: Shock absorber gas spring verification test, final setup Using a pressure control valve on the hydraulic load input actuator, the piston was compressed and the load values as a function of stroke were measured and recorded. Since the measuring operation was done manually, using a digital Vernier caliper and reference points on the bottom of the shock strut cylinder and top of the load cell, only specific strokes were identified to be measured in the interest of time. These key strokes are defined in Table 6.1 and their data for HDWS #4 is recorded 116 for reference. As can be seen by the comparison of the collected data and the theoretical data, some variation exists. It was observed that the vertical configuration of the test stand, shown in Figure 6.10, induces a pre-load on the load cell equal to the weight of the shock strut, filled with oil and nitrogen, was measured to be 9.385 lbm for HDWS #4. This adjustment in the load is accounted for in Table 6.1 and its value is compared to the theoretical data produced in Dynatool©. 6.2 Gas Spring Testing Table 6.1: Gas spring test verification results vs. Dynatool© theoretical data 1 97 87.615 83.7 4.468412943 2 189 179.615 168.8 6.021212037 2.1 202 192.615 186.2 3.330477896 2.2 219 209.615 207.3 1.104405696 2.3 248 238.615 236.3 0.970182092 2.4 277 267.615 266.3 0.491377539 2.5 314 304.615 309.2 1.505178668 2.6 364 354.615 367.2 3.54891925 2.7 429 419.615 449.6 7.145836064 2.8 516 506.615 574.4 13.37998283 2.9 663 653.615 780.7 19.44340323 3 1077 1067.615 1159.9 8.644033664 Static curve, Dynatool©, (lb) % Deviation Measured Load (lb) Adjusted Load (lb) Stroke (in) HDWS # 4 Table 6.1: Gas spring test verification results vs. Dynatool© theoretical data It can be said that the deviation in the measured values to that of the theoretical one is largely related to the human error caused by the manual measuring of the stroke using a Vernier caliper. This is a reasonable assumption when one examines the initial and final measurements and recognize the relatively small percent deviation. Another source of error can be related to the servicing process. During this process, it is imperative that the strut be filled with oil and it be serviced so that all trapped air is removed from the cylinder. This is especially important here since the impact of a discrepancy in the initial volume of nitrogen vs. oil can have a significant effect when dealing with the relatively small-scale of the design. Lastly, the theoretical gas spring curve utilizes nominal geometrical values for each of the input features, so a part used with a dimension 117 other than nominal, but still within the specified tolerances, could in theory produce a slightly different gas spring curve. For further reference, the measured data is recorded in the Appendix D for the reader’s interest. This data is in an “as-recorded” state and has not been adjusted to account for the weight of the shock strut which, as mentioned previously, creates an initial offset in the load cell due to the vertical testing position. 6.3 Full Single-System Testing The purpose of the full single-system testing, shown in Figure 6.11, was to confirm the performance of the HDWS when subjected to high-speeds and representative loading conditions. To perform this, it was necessary to impose a typical Hyperloop pod mission profile as defined within the framework of the SpaceX Hyperloop Competition, for which the present prototype was designed. The goal of these tests was to verify: the performance of the Deployable and Retractable Shock Strut; that the wear on the polyurethane wheels would be reduced when used in combination with the shock strut; and that the sensor hardware bracketry was adequately designed to support the equipment under vibration loading. Figure 6.11: HDWS full system test configuration (temperature sensors out of view) Figure 6.11: HDWS full system test configuration (temperature sensors out of view) 118 The testing was further defined by the following success criteria: Polyurethane Wheel Inspection: Polyurethane wheel inspection indicating minor distributed wear was deemed acceptable. Any wear causing a visual rotational imbalance to the polyurethane wheel when spun, or surface temperatures exceeding 195°F (80°C), during nominal mission profile conditions, causing the polyurethane wheel to melt, was considered failure. The wheel assemblies bearing-wheel interface was to be examined and documented prior to each test to monitor the unseating of the bearing from the wheel bore shown in the wheel component tests. Fastener Inspections: Visual inspections were performed and documented at the start and completion of every test. Fasteners, locked and marked with torque seal (orange paint marked, Figure 6.12), were monitored exclusively. If an inspection reveals that a fastener has come lose then that fastener must be documented with the data corresponding to that test (pod speed, G’s, loading). Figure 6.12: Example of torque paint (orange) applied, used to track loosening of the hardware 12: Example of torque paint (orange) applied, used to track loosening of the hardware 119 External Bracketry Inspections: Visual inspections were preformed and documented at the start and completion of every test. Brackets used to mount and route sensor equipment (potentiometers, limit switch) were inspected for visual damage (e.g. delamination, thread shearing). With all the above-mentioned success criteria in mind, the testing mission profile is now defined as was designed for the testing. Referencing Figure 6.13, the mission profile consists of an acceleration phase (A), cruise phase (C) and deceleration phase (D) which ends with successful stoppage of the pod’s forward motion. The HDWS begins the mission profile in a deployed and locked position defined by static stroke. As the pod moves through the mission profile the HDWS goes through a retraction phase (R), that occurs part way through the acceleration and an extension phase (E) during deceleration. The HDWS remains deployed and locked at static load stroke at completion of the mission profile. The major phases of the mission profile, described above, were further defined in the testing procedures to include: variations of the acceleration and declaration rate, varying the load applied to the wheel system (generated by the wheel system compressing to a desired stroke correlating with a theoretical load as found on the gas spring curve), and varying the pod speed at which the HDWS would extend and retract to and from the sub-track (in this case aluminum fly-wheels as shown in Figure 6.11). For the sake of brevity, further details are omitted here and a sample is shown. Figure 6.13: General pod mission profile inidcating key regions/events of interest (not to scale) Figure 6.13: General pod mission profile inidcating key regions/events of interest (not to scale) 120 As an example, a test result from the HDWS single unit test, Test Case #4, is shown in Figure 6.14. The graph illustrates the input command from the spin-up machine’s command module, shown in the graph legend as CMD (black). This represents the “commanded” profile for the spin-up machine to follow, where as the actual profile followed by the spin-up machine is defined by SM (purple). The difference between the commanded CMD and the actual SM can be caused by data lag but was likely to have been caused by the inertia of the motor and fly-wheel assembly. External Bracketry Inspections: The graph shows that the HDWS (red) accelerated, approximately linearly, up to a pod speed (blue) of approximately 130 mph and proceeded to retract from the sub-track (fly-wheels) while the pod accelerated up to approximately 190 mph, before beginning to decelerate at 18s. At approximately 78 mph the HDWS touched down on the sub-track (aluminum fly-wheels) and was decelerated while engaged until braking. Figure 6.14: Graph of the data recorded during Test Case #4, with the HDWS subjected to 2.4g acceleration and decceleration, key events labeled for clarity Figure 6.14: Graph of the data recorded during Test Case #4, with the HDWS subjected to 2.4g acceleration and decceleration, key events labeled for clarity 121 As was hypothesized, the wear on the wheels (Figure 6.15) was reduced to a slight “polish” due to the “gradual” loading and unloading capabilities of the retracting and extending shock strut. The temperature, monitored on the outer diameter of the wheel, was found to peak 79.2°F (26.2°C) from a laboratory temperature of 70.5°F (21.4°C). This was a significant change from the 176°F (80°C) peek temperature measured during the wheel component testing without the shock absorber. Figure 6.15: An example of typical wheel wear as shown after full systems testing Figure 6.15: An example of typical wheel wear as shown after full systems testing In all cases, it was shown that the spinning of the unloaded polyurethane wheel after being disengaged from the fly-wheel, caused a rotation of the wheel bearings relative to its installed position in the wheel housing. This confirmed that future considerations shall include the design of a custom wheel with a metallic frame but has not ruled out the use of a polyurethane liner on the exterior of a custom wheel, as proposed in Chapter 4. 122 6.4 Dummy Pod Test (4 Units) To address the need for a demonstration pod after the U of T departure, a testing chassis or “dummy pod” was developed. The dummy pod was equipped with mounting points for four HDWSs and provided a flat bed (at the top of the dummy pod) where a 200 lbm dummy mass was located. This dummy mass, equipped with wheels, was traversable on the flat bed providing the effect of CG imbalance and thus altering the distribution of the pod loading. The testing pod was further equipped with four laser sensors distributed at each corner of the pod. These sensors provided accurate and active feedback for the pod height above the dummy sub-track. The dummy pod’s features enabled the successful development and testing of the HDWS electronic and control system as conceived by Adhikari [40] and developed Shonibare [16]. Figure 6.16: Dummy pod test bench, control system testing fixture with 200 lbm mobile dummy mass to simulate shifts in CG Figure 6.16: Dummy pod test bench, control system testing fixture with 200 lbm mobile dummy mass to simulate shifts in CG 123 7.1 Conclusion The present work set out to design and develop a deployable wheel system for Elon Musk’s proposed high-speed transportation system, the Hyperloop. Since modern wheeled systems of the retractable and extendable type operate within relatively large spaces, the challenge was to develop a new method of performing these operations, electrically, within a Hyperloop pod framework. In doing so, the requirements, guidelines and constraints imposed on the design of a Hyperloop subsystem were explored. These subjects were then addressed using the most up-to-date Hyperloop research and, where applicable, the knowledge gap was filled by the author with applicable aerospace engineering design and development practices and standards. By applying key aspects of landing gear design theory to the Hyperloop, a unique, electrically based, system architecture was proposed to address the gap in existing retractable and extendable wheel system design. In addressing the technical design challenges associated with the development of a Hyperloop wheel system architecture, a highly iterative engineering design process was employed demonstrating the multi-disciplinary aspects of the present engineering problem. Engineering design management was used to establish a product organization structure, defining the baseline numerical configuration and enabling the mixing and matching of components while tracing the configuration with ease – extremely important in a research and development project. The detail design of the system’s shock absorbing mechanism utilized compressible adiabatic and isothermal gas theory, in combination with geometric, material selection and structural analysis considerations, all resulting in the fabrication of a complete working product. This work led to an innovative, patent-pending deployable and retractable shock strut, built for the author’s Hyperloop Deployable Wheel System baseline architecture and capable of addressing the need for small volume operation, utilizing only electrical power to operate, and incorporating active control features enabling active height stabilization. 124 7.2 Future Work Ideas for areas of further development and prototype research can be defined within three broad categories: Wheels and brakes, Hybrid Extension and Retraction, and Electronics and Controls. Hybrid Extension and Retraction System 1. Investigate the custom design of a compact, dual redundancy motor for safety a 2. Re-design the retract housing for integrated sensors, increasing sensor robustness, for measuring stroke in real-time, indicating full retraction, stowage, WOW, etc. 3. Explore the addition of a contractible and extendable bellow sleeve, as demonstrated by Bradford [66], to protect the ACME cylinder from dust and debris. Wheels and Brakes 1. Investigate the possibility of integrating an electric braking system directly into the wheels 2 i i d h l/ i f l k d i di i 1. Investigate the possibility of integrating an electric braking system directly into the wheels 2. Design a customized wheel/tire for Hyperloop track and operation conditions 3. Investigate the possibility of direct drive technology integration into the axle, enabling low speed propulsion 4. Green power generation methods for a sustainable power mode, utilizing regenerative breaking 4. Green power generation methods for a sustainable power mode, utilizing regenerative breaking Hybrid Extension and Retraction System Electronics and Controls 1. Refine and develop a commercially viable control and electronics system for single unit and multi-unit operation 2. Design electronic systems for operation in a near-vacuum environment (utilize pre-existing methods, standards, etc.) 2. Design electronic systems for operation in a near-vacuum environment (utilize pre-existing methods, standards, etc.) 125 This page was intentionally left blank 126 126 16 15 14 13 12 11 10 9 8 7 6 5 4 3 17 18 19 20 21 22 23 24 25 26 27 28 H G F E D C B A K L M N P Q R S READ, ALL OVER, USING SILICONE BASED HIGH VACUUM GREASE PRIOR TO INSTALLATION. S REQUIRED TO ACHIEVE COTTER PIN INSTALLATION. COTTER PIN TO BE SEATED 75% TO 100% WITHIN NY COMBINATION OF NAS1149C0416R AND NAS1149C0432R TO ACHIEVE FIT. UIRED USING VARIOUS P/N 40-1108-00 SHIM STOCK THICKNESSES. INSTALL SHIMS EQUALLY ACROSS UIRED. UIRED USING VARIOUS P/N 40-1210-00 SHIM STOCK THICKNESSES. INSTALL SHIMS EQUALLY ACROSS UIRED. AFTER INSTALLATION. P DEPLOYABLE WHEEL SYSTEM INSTALLATION GUIDE FOR DETAILED ASSEMBLY PROCEDURES. 0 1 0 0 m m P R O D U C T A C C E P T A N C E T E S T S C H E D U L E I N A C C O R D A N C E W I T H P A R T N U M B E R : C O N D I T I O N S D E R E C E P T I O N S U I V A N T R E F E R E N C E : P R O G R A M M E P R O G R A M M E P A R T N U M B E R O F U N I T D R A W I N G R E F E R E N C E D U D E S S I N D E D E F I N I T I O N M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T S C A L E E C H E L L E S I Z E F O R M A T S H E E T P L A N C H E T I T L E T I T R E P A R T N U M B E R R E F E R E N C E 3 5 9 6 2 E d . I s . This page was intentionally left blank C 2 0 1 6 1 : 1 A M E L I O R A T I O N A M E N D M E N T A M E N D E M E N T D E S I G N O F F I C E B U R E A U D ' E T U D E S D R A W N B Y D E S S I N E P A R C H E C K E D B Y V E R I F I E P A R D A T E D E S I G N E T U D E S U N I T U N I T E S T R E S S C A L C U L (sauf indications particulières): (unless otherwise stated): Unit / Unité KLIM G - - KLIM G 1/12/2016 23.9 lbs TORONTO HYPERLOOP - 1 UNDRESSED ASSEMBLY - HDWS 20-1000-00 1 : 1 10-1000-00 M A X N O M I N A L C A L C U L A T E D W E I G H T M A S S E C A L C U L E E J D E S I G N E T U D E S S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R - - - 1 in D A T E C L A S S I F I C A T I O N 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION AMENDMENT AMENDEMENT MODIFICATION APPROVAL FORM / EDES FICHE D'APPROBATION DE MODIFICATION Y E S O U I N O N O N X K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 0 1 0 0 m m P A R T N U M B E R R E F E R E N C E I s s u e E d i t i o n S H E E T P L A N C H E V I E W V U E 20-1000-00 1 1 2 1 2 P/N 30-1500-02, AXLE SUB-ASSEMBLY N P/N 40-1305-01, STBD PINTLE PIN P/N 30-1200-21, SHOCK ABSORBER SUB-ASSEMBLY P/N NAS6204-32D, HEX BOLT, QTY 1 P/N NAS1149C0432R, WASHER, QTY 2 P/N MS14145L4, CASTELLATED NUT, QTY 1 P/N MS24665-151, COTTER PIN, QTY 1 2X 1 2 5 B C DETAIL B SCALE 2:1 P/N 40-1211-00, INTERFACE BRACKET, ACME POT. This page was intentionally left blank P/N NAS6204-34D, HEX BOLT, QTY 1 P/N NAS1149C0432R, WASHER, QTY 3 P/N MS14145L4, CASTELLATED NUT, QTY 1 P/N MS24665-151, COTTER PIN, QTY 1 P/N 40-1210-00, SHIM WASHER (BOTH SIDES) P/N 40-1202-00, GUIDE BRACKET, OLEO POT. 2 4 5 DETAIL C SCALE 2:1 P/N NAS6205-41D, HEX BOLT, QTY 1 P/N NAS1149C0532R, WASHER, QTY 3 P/N MS14145L5, CASTELLATED NUT, QTY 1 P/N MS24665-151, COTTER PIN, QTY 1 P/N 40-1108-00, SHIM WASHER (BOTH SIDES) 2 5 3 REF ONLY SCALE: NONE 3D VIEW 1 REF ONLY SCALE: NONE 3D VIEW 2 P/N 30-1100-20, TRAILING ARM SUB-ASSEMBLY P/N 30-1400-01, TORQUE LINK SUB-ASSEMBLY P/N 30-1300-02, RETRACTION SUB-ASSEMBLY P/N NAS6204-34D, HEX BOLT, QTY 1 P/N NAS1149C0432R, WASHER, QTY 1 P/N MS14145L4, CASTELLATED NUT, QTY 1 P/N MS24665-151, COTTER PIN, QTY 1 P/N 40-1404-00, INTERFACE BRACKET, OLEO POT. 2 5 A A PARTIAL SECTION A-A SCALE 2:1 P/N 40-1107-00, TAPERED WASHER P/N NAS6204-33D, HEX BOLT, QTY 1 P/N NAS1149C0432R, WASHER, QTY 2 P/N MS14145L4, CASTELLATED NUT, QTY 1 P/N MS24665-151, COTTER PIN, QTY 1 2 5 PARTIAL SECTION A-A SCALE 2:1 P/N 40-1107-00, TAPERED WASHER P/N NAS6204-33D, HEX BOLT, QTY 1 P/N NAS1149C0432R, WASHER, QTY 2 P/N MS14145L4, CASTELLATED NUT, QTY 1 P/N MS24665-151, COTTER PIN, QTY 1 2 5 1 LUBRICATE ACME THREAD, ALL OVER, USING SILICONE BASED HIGH VACUUM GREASE PRIOR TO INSTALLATION. 5 DEFORM COTTER PIN AFTER INSTALLATION. PARTIAL SECTION A-A SCALE 2:1 P/N 30-1200-21, SHOCK ABSORBER SUB-ASSEMBLY REF ONLY SCALE: NONE 3D VIEW 1 REF ONLY SCALE: NONE 3D VIEW 2 C P/N 40-1404-00, INTERFACE BRACKET, OLEO POT. P/N 30-1100-20, TRAILING ARM SUB-ASSEMBLY P/N 30-1100-20, TRAILING ARM SUB-ASSEMBLY P/N 30-1500-02, AXLE SUB-ASSEMBLY 1 0 A R T N U M B E R E F E R E N C E s s u e E d i t i o n 20-1000-00 1 A 1 LUBRICATE THREADS WITH MIL-PRF-23827 AEROSHELL 33 OR PETROLEUM JELLY OR EQUIVALENT LUBRICANT PRIOR TO ASSEMBLY. REMOVE EXCESS LUBRICANT. 2 APPLY LOCTITE (THREADLOCKER) OR EQUIVALENT TO THREAD SURFACE PRIOR TO INSTALLATION. 5. REFER TO HYPERLOOP DEPLOYABLE WHEEL SYSTEM ASSEMBLY GUIDE FOR DETAILED ASSEMBLY PROCEDURES. This page was intentionally left blank C 2 0 1 6 1 : 1 A M E L I O R A T I O N A M E N D M E N T A M E N D E M E N T D E S I G N O F F I C E B U R E A U D ' E T U D E S D R A W N B Y D E S S I N E P A R C H E C K E D B Y V E R I F I E P A R D A T E D E S I G N E T U D E S U N I T U N I T E S T R E S S C A L C U L (sauf indications particulières): (unless otherwise stated): Unit / Unité KLIM G SKA, MHS - KLIM G 7/26/2016 4.4 lbs TORONTO HYPERLOOP - 1 TRAILING ARM SUB-ASSY, - HDWS 30-1100-20 1 : 1 20-1000-00 M A X N O M I N A L C A L C U L A T E D W E I G H T M A S S E C A L C U L E E X E D E S I G N E T U D E S S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R - - - 1 in D A T E C L A S S I F I C A T I O N 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 7/26/2016 KLIM G - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION AMENDMENT AMENDEMENT MODIFICATION APPROVAL FORM / EDES FICHE D'APPROBATION DE MODIFICATION Y E S O U I N O N O N X K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 REF ONLY SCALE: NONE 3D VIEW A B B 1.0015 1.0005 2X IN-LINE 4.265 4.235 -A P/N 40-1106-00, AXLE BUSH, QTY 2 1 2 32 A 63 63 .002 A .002 A D .010 D .3140 .3130 4X IN-LINE .400 ( ) 1.740 ( ) SECTION B-B VIEW SIMPLIFIED FOR CLARITY SCALE: 2:1 P/N KRJ5-UDSVC-008, KARON LINED JOURNAL BEARING, QTY 4 1 3 C .002 C TYPICAL 4 PL. NOTES: 1 DIMENSION APPLIES AFTER INSTALLATION. HONE OR HAND REAM ONLY. 2 INSTALL CUSTOM BUSHINGS USING PRESS FIT METHOD ONLY. 3 INSTALL KAMATICS BUSHINGS USING PRESS FIT OR COOLING METHOD 4. BUSHINGS TO BE INSTALLED AFTER ANNODIZING OF TRAILING ARM COMPONENT. 5. REFER TO THE KAMATICS KARON DESIGN GUIDE (08-21-14) FOR RECOMENDED INSTALLATION PRACTICES. This page was intentionally left blank 11 10 9 8 7 6 5 4 3 2 12 13 14 15 16 17 18 0 1 0 0 m m P R O D U C T A C C E P T A N C E T E S T S C H E D U L E I N A C C O R D A N C E W I T H P A R T N U M B E R : C O N D I T I O N S D E R E C E P T I O N S U I V A N T R E F E R E N C E : P R O G P R O G M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T S C A L E E C H E L L E S I Z E F O R M A T S H E E T P L A N C H E T I T L E T I T R E P A R T N U M B E R R E F E R E N C E 3 5 9 6 2 C 2 0 1 6 1 : 1 A M E L I O R A T I O N A M E N D M E N T A M E N D E M E N T D E S I G N O F F I C E B U R E A U D ' E T U D E S D R A W N B Y D E S S I N E P A R C H E C K E D B Y V E R I F I E P A R D A T E D E S I G N E T U D E S U N I T U N I T E S T R E S S C A L C U L (sauf indications particulières): (unless otherwise stated): Unit / Unité KLIM G N/A N/A KLIM G 6/6/2017 N/A lbs TORONTO HYPERL - ELECTRIC MOTO HD 20-20 1 : 1 M A X N O M I N A L C A L C U L A T E D W E I G H T M A S S E C A L C U L E E E 1 in C L A S S I F I C A T I O N NA - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION Y E S O U I N O N O N X K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 P/N 40-2102-00, PINNION GEAR (SPUR) P/N 40-2101-00, BLYSG34 BRUSHLESS DC MOTOR P/N 40-2103-03, MOTOR MOUNT BRACKET (AMM) B B P/N 40-2104-00, PINNION GEAR SHAFT COVER (AM) 4 X P/N M6x1-25, LOW-PROFILE SS CAP SCREWS (METRIC) P/N NAS1149C0432R, FLAT WASHER P/N MS35338-139, SPLIT LOCK WASHER REF ONLY SCALE: NONE 3D VIEW PARTIAL SECTION B-B P/N 90291A190, SOFT NYLON-TIP SET SCREW, QTY 2 4 2 P/N 40-2103-03, MOTOR MOUNT BRACKET (AMM) B PARTIAL SECTION B-B 4 X D A T E 20-2000-00 11 11 10 10 9 9 12 13 14 15 16 17 18 19 20 21 22 12 13 14 15 16 17 18 H G F E D C B A K L M N P Q R S ACTICES. This page was intentionally left blank 0 1 0 0 m m P R O D U C T A C C E P T A N C E T E S T S C H E D U L E I N A C C O R D A N C E W I T H P A R T N U M B E R : C O N D I T I O N S D E R E C E P T I O N S U I V A N T R E F E R E N C E : P R O G R A M M E P R O G R A M M E P A R T N U M B E R O F U N I T D R A W I N G R E F E R E N C E D U D E S S I N D E D E F I N I T I O N M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T S C A L E E C H E L L E S I Z E F O R M A T S H E E T P L A N C H E T I T L E T I T R E P A R T N U M B E R R E F E R E N C E 3 5 9 6 2 E d . I s . NOTES: C 2 0 1 6 2 : 1 A M E L I O R A T I O N A M E N D M E N T A M E N D E M E N T D E S I G N O F F I C E B U R E A U D ' E T U D E S D R A W N B Y D E S S I N E P A R C H E C K E D B Y V E R I F I E P A R D A T E D E S I G N E T U D E S U N I T U N I T E S T R E S S C A L C U L (sauf indications particulières): (unless otherwise stated): Unit / Unité KLIM G - - KLIM G 9.4 lbs TORONTO HYPERLOOP - 1 SHOCK ABSORBER SUB-ASSY, - HDWS 30-1200-21 2 : 1 - M A X N O M I N A L C A L C U L A T E D W E I G H T M A S S E C A L C U L E E J D E S I G N E T U D E S S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R - - - 1 in D A T E C L A S S I F I C A T I O N 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION AMENDMENT AMENDEMENT MODIFICATION APPROVAL FORM / EDES FICHE D'APPROBATION DE MODIFICATION Y E S O U I N O N O N X K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 0 1 0 0 m m P A R T N U M B E R R E F E R E N C E I s s u e E d i t i o n S H E E T P L A N C H E V I E W V U E 30-1200-21 1 1 2 1 2 P/N 35-1202-21, SHOCK ABSORBER CYLINDER SUB-ASSEMBLY P/N NAS1352C08H8, CAP SCREW (DRILLED HEAD), QTY 2 1 4 A B SECTION A-A P/N 35-1201-02, PISTON SUB-ASSEMBLY P/N 40-1205-02, PISTON UPPER BEARING (SPLIT RING) P/N 40-1209-00, FILLER TUBE P/N 40-1207-02, GLAND NUT P/N R2361-215E344, MSE SCRAPER (GREENE TWEED) P/N 7215FT-964-P17, AGT RING DYNAMIC SEAL (GREENE TWEED) P/N 7215MT-964-P15, AGT RING STATIC SEAL (GREENE TWEED) MTE-161-P15, AGT RING STATIC SEAL (GREENE TWEED) 1 2 2 2 2 2 2 3 2 FULLY EXTENDED, RETRACTED POSITION 4.000 ( ) P/N 40-1208-02, LOCK TAB P/N NAS1149CN816R, WASHER, QTY 2 3 REF ONLY SCALE: NONE 3D VIEW 1.000 ( ) SECTION B-B FULLY COMPRESSED STROKE, 3 in 4 TORQUE CAPSCREW TIGHT, ADD 1/4 TURN, INSTALL LOCKWIRE. NOTES: 1 LUBRICATE THREADS WITH MIL-PRF-23827 AEROSHELL 33 OR PETROLEUM JELLY OR EQUIVALENT LUBRICANT PRIOR TO ASSEMBLY. REMOVE EXCESS LUBRICANT. 2 LUBRICATE SEAL AND SEALING SURFACES THOROUGHLY, ALL SURFACES, WITH MIL-PRF-5606 RED OIL OR PETROLEUM JELLY OR EQUIVALENT PRIOR TO SEAL INSTALLATION. 2 LUBRICATE SEAL AND SEALING SURFACES THOROUGHLY, ALL SURFACES, WITH MIL-PRF-5606 RED OIL OR PETROLEUM JELLY OR EQUIVALENT PRIOR TO SEAL INSTALLATION. 3 TORQUE GLAND NUT TIGHT, BACK OFF TO NEAREST CASTELLATION OF PISTON GLAND NUT AND LOCK WITH LOCKING TAB. 16 15 14 13 12 11 10 9 8 7 6 17 18 19 20 21 22 23 24 25 26 27 28 H G F E D C K L M N P Q R S LOCKWIRE. 4 TURN, LOCKWIRE FROM SHOCK ABSORBER TO JAM NUT, NSTALLATION GUIDE FOR ASSEMBLY PROCEDURES. NOTES: 0 1 0 0 m m P R O D U C T A C C E P T A N C E T E S T S C H E D U L E I N A C C O R D A N C E W I T H P A R T N U M B E R : C O N D I T I O N S D E R E C E P T I O N S U I V A N T R E F E R E N C E : P R O G R A M M E P R O G R A M M E P A R T N U M B E R O F U N I T D R A W I N G R E F E R E N C E D U D E S S I N D E D E F I N I T I O N M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T S C A L E E C H E L L E S I Z E F O R M A T S H E E T P L A N C H E T I T L E T I T R E P A R T N U M B E R R E F E R E N C E 3 5 9 6 2 E d . I s . NOTES: 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 17 18 19 20 21 22 23 , , 5 TORQUE JAMNUT AND CHARGE VALVE TIGHT, ADD 1/4 TURN, LOCKWIRE FROM SHOCK ABSORBER TO JAM NUT, FROM JAM NUT TO CHARGE VALVE. 6. REFER TO HYPERLOOP DEPLOYABLE WHEEL SYSTEM INSTALLATION GUIDE FOR ASSEMBLY PROCEDURES. 0 1 0 0 m m P A R T N U M B E R R E F E R E N C E I s s u e E d i t i o n S H E E T P L A N C H E V I E W V U E 30-1200-21 1 1 2 1 2 P/N 35-1202-21, SHOCK ABSORBER CYLINDER SUB-ASSEMBLY P/N NAS1423-14, JAM NUT P/N NAS1149C1432R, WASHER P/N MS28889-2, CHARGE VALVE P/N NAS1352C08H8, CAP SCREW (DRILLED HEAD), QTY 2 1 5 1 4 1 5 A A B B SECTION A-A P/N 35-1201-02, PISTON SUB-ASSEMBLY P/N 40-1205-02, PISTON UPPER BEARING (SPLIT RING) P/N 40-1209-00, FILLER TUBE P/N 40-1207-02, GLAND NUT P/N R2361-215E344, MSE SCRAPER (GREENE TWEED) P/N 7215FT-964-P17, AGT RING DYNAMIC SEAL (GREENE TWEED) P/N 7215MT-964-P15, AGT RING STATIC SEAL (GREENE TWEED) P/N 711D6MTE-161-P15, AGT RING STATIC SEAL (GREENE TWEED) 1 2 2 2 2 2 2 2 3 2 FULLY EXTENDED, RETRACTED POSITION 4.000 ( ) 1.000 ( ) SECTION B-B FULLY COMPRESSED STROKE, 3 in 5 TORQUE JAMNUT AND CHARGE VALVE TIGHT, ADD 1/4 TURN, LOCKWIRE FROM SHOCK ABSORBER TO JAM NUT, FROM JAM NUT TO CHARGE VALVE. P/N 35 1202 21 SHOCK ABSORBER CYLINDER SUB ASSEMBLY 6. REFER TO HYPERLOOP DEPLOYABLE WHEEL SYSTEM INSTALLATION GUIDE FOR ASSEMBLY PROCEDURES. P/N 35-1202-21, SHOCK ABSORBER CYLINDER SUB-ASSEMBLY 6. REFER TO HYPERLOOP DEPLOYABLE WHEEL SYSTEM INSTALLATION GUIDE FOR ASSEMBLY PROCEDURES. H G K L M N P Q R S 0 1 0 0 m m P R O D U C T A C C E P T A N C E T E S T S C H E D U L E I N A C C O R D A N C E W I T H P A R T N U M B E R : C O N D I T I O N S D E R E C E P T I O N S U I V A N T R E F E R E N C E : P R O G R A M M E P R O G R A M M E P A R T N U M B E R O F U N I T D R A W I N G R E F E R E N C E D U D E S S I N D E D E F I N I T I O N M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T S C A L E E C H E L L E S I Z E F O R M A T S H E E T P L A N C H E T I T L E T I T R E P A R T N U M B E R R E F E R E N C E 3 5 9 6 2 E d . I s . C 2 0 1 6 1 : 1 A M E L I O R A T I O N A M E N D M E N T A M E N D E M E N T D E S I G N O F F I C E B U R E A U D ' E T U D E S D R A W N B Y D E S S I N E P A R C H E C K E D B Y V E R I F I E P A R D A T E D E S I G N E T U D E S U N I T U N I T E S T R E S S C A L C U L (sauf indications particulières): (unless otherwise stated): Unit / Unité KLIM G SKA, MHS - KLIM G 7/26/2016 4.4 lbs TORONTO HYPERLOOP - 1 TRAILING ARM SUB-ASSY, - HDWS 30-1100-20 1 : 1 20-1000-00 M A X N O M I N A L C A L C U L A T E D W E I G H T M A S S E C A L C U L E E X E D E S I G N E T U D E S S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R - - - 1 in D A T E C L A S S I F I C A T I O N 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 7/26/2016 KLIM G - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION AMENDMENT AMENDEMENT MODIFICATION APPROVAL FORM / EDES FICHE D'APPROBATION DE MODIFICATION Y E S O U I N O N O N X K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 P/N 40-1101-66, TRAILING ARM A A B B 1.0015 1.0005 2X IN-LINE 1.0015 1.0005 2X IN-LINE 4.130 4.110 4.265 4.235 SECTION A-A P/N 40-1105-00, PIVOT PIN BUSH, QTY 2 P/N 40-1106-00, AXLE BUSH, QTY 2 1 2 2 1 32 32 A B 63 63 63 63 .002 A .002 B .002 B .002 A D E .005 E .010 D .3140 .3130 4X IN-LINE .400 ( ) 1.740 ( ) SECTION B-B VIEW SIMPLIFIED FOR CLARITY SCALE: 2:1 P/N KRJ5-UDSVC-008, KARON LINED JOURNAL BEARING, QTY 4 1 3 C .002 C TYPICAL 4 PL. .3140 .3130 4X IN-LINE .400 ( ) 1.740 ( ) SECTION B-B VIEW SIMPLIFIED FOR CLARITY SCALE: 2:1 P/N KRJ5-UDSVC-008, KARON LINED JOURNAL BEARING, QTY 4 1 3 C .002 C TYPICAL 4 PL. P/N 40-1101-66, TRAILING ARM A A B B 1.0015 1.0005 2X IN-LINE 1.0015 1.0005 2X IN-LINE 4.130 4.110 4.265 4.235 SECTION A-A P/N 40-1105-00, PIVOT PIN BUSH, QTY 2 P/N 40-1106-00, AXLE BUSH, QTY 2 1 2 2 1 32 32 A B 63 63 63 63 .002 A .002 B .002 B .002 A D E .005 E .010 D .400 ( ) S A A P/N KRJ5-UDSVC-008, KARON LINED JOURNAL BEARING, QTY 4 SECTION B-B VIEW SIMPLIFIED FOR CLARITY SCALE: 2:1 VIEW SIMPLIFIED FOR CLARITY SCALE: 2:1 P/N 40-1105-00, PIVOT PIN BUSH, QTY 2 63 4.265 4.235 .010 D .002 A NOTES: P/N NAS1352C08H8, CAP SCREW (DRILLED HEAD), QTY 2 1 4 P/N NAS1352C08H8, CAP SCREW (DRILLED HEAD), QTY 2 1 4 P/N 7215MT-964-P15, AGT RING STATIC SEAL (GREENE TWEED) 2 P/N 40-1208-02, LOCK TAB 3 P A R T N U M B E R R E F E R E N C E I s s u e E d i t i o n S H E E T P L A N C H E V I E W V U E 30-1200-21 1 1 2 1 2 P A R T N U M B E R R E F E R E N C E I s s u e E d i t i o n 30-1200-21 1 H E V I E W V U E 1 2 2 1 LUBRICATE THREADS WITH MIL-PRF-23827 AEROSHELL 33 OR PETROLEUM JELLY OR EQUIVALENT LUBRICANT PRIOR TO ASSEMBLY. REMOVE EXCESS LUBRICANT. 2 LUBRICATE BEARING, INNER AND OUTER RACE DIAMETER FACES, WITH MIL-PRF-23827 AEROSHELL 33 OR PETROLEUM JELLY OR EQUIVALENT LUBRICANT PRIOR TO ASSEMBLY. REMOVE EXCESS LUBRICANT. 4 TORQUE PACKING NUT TIGHT, BACK OFF TO NEAREST CASTELLATION AND LOCK WITH LOCKING TAB. 5. ENSURE RETRACT NUT ROTATES FREELY WITHOUT BINDING AFTER TORQUING THE PACKING NUT. 5. ENSURE RETRACT NUT ROTATES FREELY WITHOUT BINDING AFTER TORQUING THE PACKING NUT. 6 TORQUE CAPSCREW TIGHT, ADD 1/4 TURN, INSTALL LOCKWIRE. 7. REFER TO HYPERLOOP DEPLOYABLE WHEEL SYSTEM ASSEMBLY GUIDE FOR DETAILED ASSEMBLY PROCEDURES. NOTES: COTTER PIN TO BE SEATED 75% TO 100% WITHIN NUT CASTLE. USE ANY COMBINATION OF NAS1149C0416R AND NAS1149C0432R TO ACHIEVE FIT. 3. REFER TO HYPERLOOP DEPLOYABLE WHEEL SYSTEM INSTALLATION GUIDE FOR DETAILED ASSEMBLY PROCEDURES. NOTES: 11 10 9 8 7 6 5 4 3 2 12 13 14 15 16 17 18 0 1 0 0 m m P R O D U C T A C C E P T A N C E T E S T S C H E D U L E I N A C C O R D A N C E W I T H P A R T N U M B E R : C O N D I T I O N S D E R E C E P T I O N S U I V A N T R E F E R E N C E : P R O G P R O G R M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T S C A L E E C H E L L E S I Z E F O R M A T S H E E T P L A N C H E T I T L E T I T R E P A R T N U M B E R R E F E R E N C E 3 5 9 6 2 C 2 0 1 6 3 : 2 A M E L I O R A T I O N A M E N D M E N T A M E N D E M E N T D E S I G N O F F I C E B U R E A U D ' E T U D E S D R A W N B Y D E S S I N E P A R C H E C K E D B Y V E R I F I E P A R D A T E D E S I G N E T U D E S U N I T U N I T E S T R E S S C A L C U L (sauf indications particulières): (unless otherwise stated): Unit / Unité KLIM G SKA - KLIM G 8/15/2016 7.2 lbs TORONTO HYPERL - RETRACTION SU - HD 30-130 3 : 2 M A X N O M I N A L C A L C U L A T E D W E I G H T M A S S E C A L C U L E E E D E S C C V 1 in C L A S S I F I C A T I O N 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION Y E S O U I N O N O N X K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 P/N 35-1300-02, RETRACT HOUSING SUB-ASSY A A SECTION A-A P/N 40-1303-01, PACKING NUT 1 P/N 40-1302-02, RETRACT NUT P/N 61813-2RZ, DEEP GROOVE BALL BEARING (LOWER) P/N 61813-2RZ, DEEP GROOVE BALL BEARING (LOWER) 3 3 2 2 P/N 40-1304-00, LOCK TAB P/N NAS1149CN816R, WASHER , QTY 2 P/N NAS1352C0H8, CAP SCREW (DRILLED HEAD), QTY 2 6 1 4 REF ONLY SCALE: NONE 3D VIEW P/N 35-1300-02, RETRACT HOUSING SUB-ASSY A A P/N 40-1303-01, PACKING NUT 1 P/N 40-1302-02, RETRACT NUT P/N 61813-2RZ, DEEP GROOVE BALL BEARING (LOWER) P/N 61813-2RZ, DEEP GROOVE BALL BEARING (LOWER) 3 3 2 2 P/N 40-1302-02, RETRACT NUT P/N 61813-2RZ, DEEP GROOVE BALL BEARING (LOWER) 3 2 P/N 35-1300-02, RETRACT HOUSING SUB-ASSY P/N 61813-2RZ, DEEP GROOVE BALL BEARING (LOWER) 3 2 P/N 40-1303-01, PACKING NUT 1 P/N 40-1303-01, PACKING NUT 1 D A T E S 30-1300-02 1 INSTALL WASHERS AS REQUIRED TO ACHIEVE COTTER PIN INSTALLATION. 3. REFER TO HYPERLOOP DEPLOYABLE WHEEL SYSTEM INSTALLATION GUIDE FOR DETAILED ASSEMBLY PROCEDURES. NOTES: 1 0 0 m m P R O D U C T A C C E P T A N C E T E S T S C H E D U L E I N A C C O R D A N C E W I T H P A R T N U M B E R : C O N D I T I O N S D E R E C E P T I O N S U I V A N T R E F E R E N C E : P R O G R A M M E P R O G R A M M E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T S C A L E E C H E L L E S I Z E F O R M A T S H E E T P L A N C H E T I T L E T I T R E P A R T N U M B E R R E F E R E N C E 3 5 9 6 2 C 2 0 1 6 3 : 1 A M E L I O R A T I O N D E S I G N O F F I C E B U R E A U D ' E T U D E S D R A W N B Y D E S S I N E P A R C H E C K E D B Y V E R I F I E P A R D A T E D E S I G N E T U D E S U N I T U N I T E S T R E S S C A L C U L (sauf indications particulières): (unless otherwise stated): Unit / Unité KLIM G - - KLIM G 8/15/2016 0.6 lbs TORONTO HYPERLOOP - TORQUE LINK ASS - HDWS 30-1400-0 3 : 1 M A X N O M I N A L C A L C U L A T E D W E I G H T M A S S E C A L C U L E E E D E S I G N E T U D E S S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R 1 in C L A S S I F I C A T I O N 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION AMENDMENT AMENDEMENT Y E S O U I N O N O N X K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 P/N 35-1402-01, UPPER TORQUE LINK SUB-ASSEMBLY P/N 35-1401-01, LOWER TORQUE LINK SUB-ASSEMBLY 2.500 ( ) 4.000 ( ) P/N MS24665-151, COTTER PIN P/N MS14145L4, HEX NUT, CASTALLATED P/N NAS1149C0432R, WASHER P/N NAS6204-22D, HEX BOLT P/N NAS1149C0432R, WASHER 2 1 1 SCALE: NONE 1 0 0 m m P R O D U C T A C C E P T A N C E T E S T S C H E D U L E I N A C C O R D A N C E W I T H P A R T N U M B E R : C O N D I T I O N S D E R E C E P T I O N S U I V A N T R E F E R E N C E : P R O G R A M M E P R O G R A M M E P A R T N U M B E R R E F E R E N C E D U D M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T S C A L E E C H E L L E T I T L E T I T R E 3 5 9 6 2 C 2 0 1 6 A M E L I O R A T I O N D E S I G N O F F I C E B U R E A U D ' E T U D E S D R A W N B Y D E S S I N E P A R C H E C K E D B Y V E R I F I E P A R D A T E D E S I G N E T U D E S U N I T U N I T E S T R E S S C A L C U L (sauf indications particulières): (unless otherwise stated): Unit / Unité KLIM G - - KLIM G 8/15/2016 0.6 lbs TORONTO HYPERLOOP - TORQUE LINK ASSEMBLY 20-10 M A X N O M I N A L C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N E T U D E S S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R - - - in C L A S S I F I C A T I O N 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION AMENDMENT AMENDEMENT MODIFICATION APPRO FICHE D'APPROBATION Y E S O U I N O N O N X K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S P/N MS24665-151, COTTER PIN P/N MS14145L4, HEX NUT, CASTALLATED P/N NAS1149C0432R, WASHER P/N NAS6204-22D, HEX BOLT P/N NAS1149C0432R, WASHER 2 1 1 2.500 ( ) P/N NAS6204-22D, HEX BOLT P/N MS14145L4, HEX NUT, CASTALLATED P/N 35-1401-01, LOWER TORQUE LINK SUB-ASSEMBLY 4.000 ( ) D A T E 30-1400-01 NOTES: 1. CUSTOM WHEEL/BEARING CONFIGURATION PROCURED FROM SUNRAY INC. NOTES: 10.020 9.980 P/N 40-3100-00, POLYURETHANE WHEEL A A 2.005 1.995 SECTION A-A 1.010 .990 2X .505 .495 2X P/N R16-2RS, BALL BEARING P/N R16-2RS, BALL B H G F E K L M N P Q R 1 0 0 m m P R O D U C T A C C E P T A N C E T E S T S C H E D U L E I N A C C O R D A N C E W I T H P A R T N U M B E R : C O N D I T I O N S D E R E C E P T I O N S U I V A N T R E F E R E N C E : P R O G R A M M E P R O G R A M M E P A R T N U M B E R O F U N I T D R A W I N G R E F E R E N C E D U D E S S I N D E D E F I N I T I O N A M E L I O R A T I O N D R A W N B Y D E S S I N E P A R KLIM G HYPERLOOP - 10-1000-00 C L A S S I F I C A T I O N 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 9/9/2016 - - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION AMENDMENT AMENDEMENT MODIFICATION APPROVAL FORM / EDES FICHE D'APPROBATION DE MODIFICATION Y E S O U I N O N O N X K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 10.020 9.980 P/N 40-3100-00, POLYURETHANE WHEEL A A 2.005 1.995 SECTION A-A 1.010 .990 2X .505 .495 2X P/N R16-2RS, BALL BEARING P/N R16-2RS, BALL BEARING REF ONLY SCALE: NONE 3D VIEW P/N 40-3100-00, POLYURETHANE WHEEL SECTION A-A A NOTES: 1 DIMENSION APPLIES AFTER INSTALLATION. HONE OR HAND REAM ONLY. 3. BUSHINGS TO BE INSTALLED AFTER ANNODIZING OF MATING COMPONENT. 3. BUSHINGS TO BE INSTALLED AFTER ANNODIZING OF MATING COMPONENT. 4. REFER TO THE KAMATICS KARON DESIGN GUIDE (08-21-14) FOR RECOMENDED INSTALLATION PRACTICES. 4. REFER TO THE KAMATICS KARON DESIGN GUIDE (08-21-14) FOR RECOMENDED INSTALLATION PRACTICES. P/N 40-1201-02, SHOCK ABSORBER PISTON A A .3125 .3120 SECTION A-A P/N KR5-CNGVP, KARON LINED SPHERICAL BEARING 1 2 REF ONLY SCALE : NONE P/N 40-1201-02, SHOCK ABSORBER PISTON A A A A NOTES: 1 DIMENSION APPLIES AFTER INSTALLATION. HONE OR HAND REAM ONLY. 3. BUSHINGS TO BE INSTALLED AFTER ANNODIZING OF MATING COMPONENT. 3. BUSHINGS TO BE INSTALLED AFTER ANNODIZING OF MATING COMPONENT. 4. REFER TO THE KAMATICS KARON DESIGN GUIDE (08-21-14) FOR RECOMENDED INSTALLATION PRACTICES. 4. REFER TO THE KAMATICS KARON DESIGN GUIDE (08-21-14) FOR RECOMENDED INSTALLATION PRACTICES. 11 10 9 8 7 6 5 4 12 13 14 15 16 17 18 H K L M N P Q R S 0 1 0 0 m m P R O D U C T A C C E P T A N C E T E S T S C H E D U L E I N A C C O R D A N C E W I T H P A R T N U M B E R : C O N D I T I O N S D E R E C E P T I O N S U I V A N T R E F E R E N C E : P R O G R A M M E P R O G R A M M E P A R T N U M B E R O F U N I T D R A W I N G R E F E R E N C E D U D E S S I N D E D E F I N I T I O N M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T S C A L E E C H E L L E S I Z E F O R M A T S H E E T P L A N C H E T I T L E T I T R E P A R T N U M B E R R E F E R E N C E 3 5 9 6 2 E d . I s . NOTES: HONE OR HAND REAM ONLY. INSTALL CUSTOM BUSHINGS USING PRESS FIT METHOD OR EQUIVALENT. BUSHINGS TO BE INSTALLED AFTER ANNODIZING OF RETRACT HOUSING COMPONENT. 0 0 m m P R O D U C T A C C E P T A N C E T E S T S C H E D U L E I N A C C O R D A N C E W I T H P A R T N U M B E R : C O N D I T I O N S D E R E C E P T I O N S U I V A N T R E F E R E N C E : P R O G R A M M E P R O G R A M M E P A R T N U M B E R E F E R E N C E D U A M E L I O R A T I O N C L A S S I F I C A T I O N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION AMENDMENT AMENDEMENT MODIFICATION APPRO FICHE D'APPROBATION P/N 40-1301-02, RETRACT HOUSING A A SECTION A-A P/N 40-1308-00, TRUNION BUSH, QTY 2 63 63 A .005 A 32 32 .002 B B .002 C C 1 1 2 2 .002 B-C .002 B-C 7.635 7.605 1.0015 1.0005 1.0015 1.0005 B B REF ONLY SCALE: NONE 3D VIEW SECTION B-B PARTIAL VIEW ONLY UP TO 5 DEGREES MAXIMUM CROSS BOLT BORE MISSALIGNMENT ALLOWED AFTER BUSH INSTALLATION. CHECK FIT AFTER INSTALLATION, REAM IF REQUIRED. NOTES: C 2 0 1 6 2 : 1 A M E L I O R A T I O N A M E N D M E N T A M E N D E M E N T D E S I G N O F F I C E B U R E A U D ' E T U D E S D R A W N B Y D E S S I N E P A R C H E C K E D B Y V E R I F I E P A R D A T E D E S I G N E T U D E S U N I T U N I T E S T R E S S C A L C U L (sauf indications particulières): (unless otherwise stated): Unit / Unité KLIM G - - KLIM G 5/7/2016 7.6 lbs TORONTO HYPERLOOP - 1 SHOCK ABSORBER CYLINDER SUB-ASSEMBLY - HDWS 35-1202-21 2 : 1 30-1202-21 M A X N O M I N A L C A L C U L A T E D W E I G H T M A S S E C A L C U L E E X E D E S I G N E T U D E S S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R - - - 1 in D A T E C L A S S I F I C A T I O N 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 5/7/2016 KLIM G - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION AMENDMENT AMENDEMENT MODIFICATION APPROVAL FORM / EDES FICHE D'APPROBATION DE MODIFICATION Y E S O U I N O N O N X K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 P/N 40-1202-21, SHOCK ABSORBER CYLINDER A A REF ONLY SCALE: NONE 3D VIEW 1.198 1.174 .2515 .2500 2x IN-LINE SECTION A-A P/N KRJ4-USDVC-008, KARON LINED JOURNAL BEARING, QTY 2 1 2 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 5/7/2016 KLIM G - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION AMENDMENT AMENDEMENT MODIFICATION APPROVAL FORM / EDES FICHE D'APPROBATION DE MODIFICATION P/N 40-1202-21, SHOCK ABSORBER CYLINDER A A REF ONLY SCALE: NONE 3D VIEW 1.198 1.174 .2515 .2500 2x IN-LINE SECTION A-A P/N KRJ4-USDVC-008, KARON LINED JOURNAL BEARING, QTY 2 1 2 P/N KRJ4-USDVC-008, KARON LINED JOURNAL BEARING, QTY 2 P/N 40-1202-21, SHOCK ABSORBER CYLINDER A A 1.198 1.174 P/N 40-1202-21, SHOCK ABSORBER CYLINDER 1 2 P A R T N U M B E R O F U N I T D R A W I N G R E F E R E N C E D U D E S S I N D E D E F I N I T I O 30-1202-21 D A T E 35-1202-21 ES: DIMENSION APPLIES AFTER INSTALLATION. NOTES: 1 DIMENSION APPLIES AFTER INSTALLATION. HONE OR HAND REAM ONLY. 1 DIMENSION APPLIES AFTER INSTALLATION. HONE OR HAND REAM ONLY. 2 INSTALL CUSTOM BUSHINGS USING PRESS FIT METHOD OR EQUIVALENT. UP TO 5 DEGREES MAXIMUM CROSS BOLT BORE MISSALIGNMENT ALLOWED AFTER BUSH INSTALLATION. CHECK FIT AFTER INSTALLATION, REAM IF REQUIRED. UP TO 5 DEGREES MAXIMUM CROSS BOLT BORE MISSALIGNMENT ALLOWED AFTER BUSH INSTALLATION. CHECK FIT AFTER INSTALLATION, REAM IF REQUIRED. UP TO 5 DEGREES MAXIMUM CROSS BOLT BORE MISSALIGNMENT ALLOWED AFTER BUSH INSTALLATION CHECK FIT AFTER INSTALLATION, REAM IF REQUIRED. 3. BUSHINGS TO BE INSTALLED AFTER ANNODIZING OF RETRACT HOUSING COMPONENT. P/N 40-1301-02, RETRACT HOUSING A H G K L M N P Q - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P/N 40-1301-02, RETRACT HOUSING A A SECTION A-A P/N 40-1308-00, TRUNION BUSH, QTY 2 63 63 A .005 A 32 32 .002 B B .002 C C 1 1 2 2 .002 B-C .002 B-C 7.635 7.605 1.0015 1.0005 1.0015 1.0005 B B B 63 A 35-1300-02 H G F E D C B A K L M N P Q R S HONE OR HAND REAM ONLY. FIT OR COOLING METHOD.. IZING OF MATING COMPONENT. UIDE (08-21-14) FOR RECOMENDED INSTALLATION PRACTICES. 0 1 0 0 m m P R O D U C T A C C E P T A N C E T E S T S C H E D U L E I N A C C O R D A N C E W I T H P A R T N U M B E R : C O N D I T I O N S D E R E C E P T I O N S U I V A N T R E F E R E N C E : P R O G R A M M E P R O G R A M M E P A R T N U M B E R O F U N I T D R A W I N G R E F E R E N C E D U D E S S I N D E D E F I N I T I O N M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T S C A L E E C H E L L E S I Z E F O R M A T S H E E T P L A N C H E T I T L E T I T R E P A R T N U M B E R R E F E R E N C E 3 5 9 6 2 E d . I s . C 2 0 1 6 2 : 1 A M E L I O R A T I O N A M E N D M E N T A M E N D E M E N T D E S I G N O F F I C E B U R E A U D ' E T U D E S D R A W N B Y D E S S I N E P A R C H E C K E D B Y V E R I F I E P A R D A T E D E S I G N E T U D E S U N I T U N I T E S T R E S S C A L C U L (sauf indications particulières): (unless otherwise stated): Unit / Unité KLIM G MHS ELAHI M KLIM G 6/28/2016 0.365 lbs TORONTO HYPERLOOP - 1 LOWER TORQUE LINK SUB-ASSEMBLY - HDWS 35-1401-01 2 : 1 30-1400-01 M A X N O M I N A L C A L C U L A T E D W E I G H T M A S S E C A L C U L E E E D E S I G N E T U D E S S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R - - - 1 in D A T E C L A S S I F I C A T I O N 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 7/4/2016 KLIM G - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION AMENDMENT AMENDEMENT MODIFICATION APPROVAL FORM / EDES FICHE D'APPROBATION DE MODIFICATION Y E S O U I N O N O N X K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 P/N 40-1401-01, LOWER TORQUE LINK A A REF ONLY SCALE: NONE 3D VIEW .708 .688 1.808 1.788 .2515 .2500 2X IN-LINE .3140 .3130 2X IN-LINE SECTION A-A P/N KRJ4-UDSVC-008, KARON LINED JOURNAL BEARING, QTY 2 P/N KRJ5-UDSVC-008, KARON LINED JOURNAL BEARING, QTY 2 A B .002 A .002 A .002 B .002 B 1 1 2 2 1 DIMENSION APPLIES AFTER INSTALLATION. HONE OR HAND REAM ONLY. 3. BUSHINGS TO BE INSTALLED AFTER ANNODIZING OF MATING COMPONENT. 3. BUSHINGS TO BE INSTALLED AFTER ANNODIZING OF MATING COMPONENT. 4. REFER TO THE KAMATICS KARON DESIGN GUIDE (08-21-14) FOR RECOMENDED INSTALLATION PRACTICES. 4. REFER TO THE KAMATICS KARON DESIGN GUIDE (08-21-14) FOR RECOMENDED INSTALLATION PRACTICES. P/N 40-1401-01, LOWER TORQUE LINK A A .708 .688 1.808 1.788 .2515 .2500 2X IN-LINE .3140 .3130 2X IN-LINE SECTION A-A P/N KRJ4-UDSVC-008, KARON LINED JOURNAL BEARING, QTY 2 P/N KRJ5-UDSVC-008, KARON LINED JOURNAL BEARING, QTY 2 A B .002 A .002 A .002 B .002 B 1 1 2 2 P/N 40-1401-01, LOWER TORQUE LINK P/N 40-1401-01, LOWER TORQUE LINK A A A P/N KRJ5-UDSVC-008, KARON LINED JOURNAL BEARING, QTY 2 P/N KRJ4-UDSVC-008, KARON LINED JOURNAL BEARING, QTY 2 NOTES: 1 DIMENSION APPLIES AFTER INSTALLATION. HONE OR HAND REAM ONLY. 3. BUSHINGS TO BE INSTALLED AFTER ANNODIZING OF MATING COMPONENT. 3. BUSHINGS TO BE INSTALLED AFTER ANNODIZING OF MATING COMPONENT. 4. REFER TO THE KAMATICS KARON DESIGN GUIDE (08-21-14) FOR RECOMENDED INSTALLATION PRACTICES. 4. REFER TO THE KAMATICS KARON DESIGN GUIDE (08-21-14) FOR RECOMENDED INSTALLATION PRACTICES. 11 10 9 8 7 6 12 13 14 15 16 17 18 H G F E D K L M N P Q R S 0 1 0 0 m m P R O D U C T A C C E P T A N C E T E S T S C H E D U L E I N A C C O R D A N C E W I T H P A R T N U M B E R : C O N D I T I O N S D E R E C E P T I O N S U I V A N T R E F E R E N C E : P R O G R A M M E P R O G R A M M E P A R T N U M B E R O F U N I T D R A W I N G R E F E R E N C E D U D E S S I N D E D E F I N I T I O N M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T S C A L E E C H E L L E S I Z E F O R M A T S H E E T P L A N C H E T I T L E T I T R E P A R T N U M B E R R E F E R E N C E 3 5 9 6 2 E d . I s . C 2 0 1 6 3 : 1 A M E L I O R A T I O N A M E N D M E N T A M E N D E M E N T D E S I G N O F F I C E B U R E A U D ' E T U D E S D R A W N B Y D E S S I N E P A R C H E C K E D B Y V E R I F I E P A R D A T E D E S I G N E T U D E S U N I T U N I T E S T R E S S C A L C U L (sauf indications particulières): (unless otherwise stated): Unit / Unité KLIM G MHS ELAHI M KLIM G 6/28/2016 0.205 lbs TORONTO HYPERLOOP - 1 UPPER TORQUE LINK SUB-ASSEMBLY - HDWS 35-1402-02 3 : 1 30-1400-01 M A X N O M I N A L C A L C U L A T E D W E I G H T M A S S E C A L C U L E E E D E S I G N E T U D E S S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R - - - 1 in D A T E C L A S S I F I C A T I O N 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 7/4/2016 KLIM G - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION AMENDMENT AMENDEMENT MODIFICATION APPROVAL FORM / EDES FICHE D'APPROBATION DE MODIFICATION Y E S O U I N O N O N X K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 P/N 40-1402-01, UPPER TORQUE LINK A A .2515 .2500 2X .2515 .2500 2X 1.172 1.152 .687 .667 SECTION A-A P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 1 1 2 2 REF ONLY SCALE: NONE 3D VIEW E d . 11 10 9 8 7 6 12 13 14 15 16 17 18 H G F E D K L M N P Q R S 0 1 0 0 m m P R O D U C T A C C E P T A N C E T E S T S C H E D U L E I N A C C O R D A N C E W I T H P A R T N U M B E R : C O N D I T I O N S D E R E C E P T I O N S U I V A N T R E F E R E N C E : P R O G R A M M E P R O G R A M M E P A R T N U M B E R O F U N I T D R A W I N G R E F E R E N C E D U D E S S I N D E D E F I N I T I O N M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T S C A L E E C H E L L E S I Z E F O R M A T S H E E T P L A N C H E T I T L E T I T R E P A R T N U M B E R R E F E R E N C E 3 5 9 6 2 E d . I s . C 2 0 1 6 3 : 1 A M E L I O R A T I O N A M E N D M E N T A M E N D E M E N T D E S I G N O F F I C E B U R E A U D ' E T U D E S D R A W N B Y D E S S I N E P A R C H E C K E D B Y V E R I F I E P A R D A T E D E S I G N E T U D E S U N I T U N I T E S T R E S S C A L C U L (sauf indications particulières): (unless otherwise stated): Unit / Unité KLIM G MHS ELAHI M KLIM G 6/28/2016 0.205 lbs TORONTO HYPERLOOP - 1 UPPER TORQUE LINK SUB-ASSEMBLY - HDWS 35-1402-02 3 : 1 30-1400-01 M A X N O M I N A L C A L C U L A T E D W E I G H T M A S S E C A L C U L E E E D E S I G N E T U D E S S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R - - - 1 in D A T E C L A S S I F I C A T I O N 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 7/4/2016 KLIM G - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION AMENDMENT AMENDEMENT MODIFICATION APPROVAL FORM / EDES FICHE D'APPROBATION DE MODIFICATION Y E S O U I N O N O N X K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 P/N 40-1402-01, UPPER TORQUE LINK A A .2515 .2500 2X .2515 .2500 2X 1.172 1.152 .687 .667 SECTION A-A P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 1 1 2 2 REF ONLY SCALE: NONE 3D VIEW 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 2 6/17/2016 KLIM G - 1 16/06/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 119 117 2 I s . 11 10 9 8 7 6 12 13 14 15 16 17 18 H G F E D K L M N P Q R S 0 1 0 0 m m P R O D U C T A C C E P T A N C E T E S T S C H E D U L E I N A C C O R D A N C E W I T H P A R T N U M B E R : C O N D I T I O N S D E R E C E P T I O N S U I V A N T R E F E R E N C E : P R O G R A M M E P R O G R A M M E P A R T N U M B E R O F U N I T D R A W I N G R E F E R E N C E D U D E S S I N D E D E F I N I T I O N M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T S C A L E E C H E L L E S I Z E F O R M A T S H E E T P L A N C H E T I T L E T I T R E P A R T N U M B E R R E F E R E N C E 3 5 9 6 2 E d . I s . C 2 0 1 6 3 : 1 A M E L I O R A T I O N A M E N D M E N T A M E N D E M E N T D E S I G N O F F I C E B U R E A U D ' E T U D E S D R A W N B Y D E S S I N E P A R C H E C K E D B Y V E R I F I E P A R D A T E D E S I G N E T U D E S U N I T U N I T E S T R E S S C A L C U L (sauf indications particulières): (unless otherwise stated): Unit / Unité KLIM G MHS ELAHI M KLIM G 6/28/2016 0.205 lbs TORONTO HYPERLOOP - 1 UPPER TORQUE LINK SUB-ASSEMBLY - HDWS 35-1402-02 3 : 1 30-1400-01 M A X N O M I N A L C A L C U L A T E D W E I G H T M A S S E C A L C U L E E E D E S I G N E T U D E S S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R - - - 1 in D A T E C L A S S I F I C A T I O N 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 7/4/2016 KLIM G - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION AMENDMENT AMENDEMENT MODIFICATION APPROVAL FORM / EDES FICHE D'APPROBATION DE MODIFICATION Y E S O U I N O N O N X K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 P/N 40-1402-01, UPPER TORQUE LINK A A .2515 .2500 2X .2515 .2500 2X 1.172 1.152 .687 .667 SECTION A-A P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 1 1 2 2 REF ONLY SCALE: NONE 3D VIEW H G F E K L M N P Q P/N 40-1402-01, UPPER TORQUE LINK A A .2515 .2500 2X .2515 .2500 2X 1.172 1.152 .687 .667 SECTION A-A P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 1 1 2 2 P/N 40-1402-01, UPPER TORQUE LINK A A .2515 .2500 2X .2515 .2500 2X 1.172 1.152 .687 .667 SECTION A-A P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 1 1 2 2 P/N 40-1402-01, UPPER TORQUE LINK A A .2515 .2500 2X .2515 .2500 2X 1.172 1.152 .687 .667 SECTION A-A P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 1 1 2 2 P/N 40-1402-01, UPPER TORQUE LINK A A P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 .2515 .2500 2X 1 2 35-1402-02 14 15 16 17 18 19 20 21 22 14 15 16 17 18 H G F E D C B A K L M N P Q R S T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R E X N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 1 2 30-1100-20 HYPERLOOP 16/6/2016 KLIM G ELAHI M, KLIM G SKA KLIM G 3.9 lbs in TORONTO X X X 40-1101-66 T-ARM ASSEMBLY -HDWS TRAILING ARM MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O M A M E L I O R A T I O N 1 : 1 2 0 1 6 C I s . 11 10 9 8 7 6 12 13 14 15 16 17 18 H G F E D K L M N P Q R S 0 1 0 0 m m P R O D U C T A C C E P T A N C E T E S T S C H E D U L E I N A C C O R D A N C E W I T H P A R T N U M B E R : C O N D I T I O N S D E R E C E P T I O N S U I V A N T R E F E R E N C E : P R O G R A M M E P R O G R A M M E P A R T N U M B E R O F U N I T D R A W I N G R E F E R E N C E D U D E S S I N D E D E F I N I T I O N M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T S C A L E E C H E L L E S I Z E F O R M A T S H E E T P L A N C H E T I T L E T I T R E P A R T N U M B E R R E F E R E N C E 3 5 9 6 2 E d . I s . C 2 0 1 6 3 : 1 A M E L I O R A T I O N A M E N D M E N T A M E N D E M E N T D E S I G N O F F I C E B U R E A U D ' E T U D E S D R A W N B Y D E S S I N E P A R C H E C K E D B Y V E R I F I E P A R D A T E D E S I G N E T U D E S U N I T U N I T E S T R E S S C A L C U L (sauf indications particulières): (unless otherwise stated): Unit / Unité KLIM G MHS ELAHI M KLIM G 6/28/2016 0.205 lbs TORONTO HYPERLOOP - 1 UPPER TORQUE LINK SUB-ASSEMBLY - HDWS 35-1402-02 3 : 1 30-1400-01 M A X N O M I N A L C A L C U L A T E D W E I G H T M A S S E C A L C U L E E E D E S I G N E T U D E S S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R - - - 1 in D A T E C L A S S I F I C A T I O N 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 7/4/2016 KLIM G - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION AMENDMENT AMENDEMENT MODIFICATION APPROVAL FORM / EDES FICHE D'APPROBATION DE MODIFICATION Y E S O U I N O N O N X K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 P/N 40-1402-01, UPPER TORQUE LINK A A .2515 .2500 2X .2515 .2500 2X 1.172 1.152 .687 .667 SECTION A-A P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 1 1 2 2 REF ONLY SCALE: NONE 3D VIEW E d . 11 10 9 8 7 6 12 13 14 15 16 17 18 H G F E D K L M N P Q R S 0 1 0 0 m m P R O D U C T A C C E P T A N C E T E S T S C H E D U L E I N A C C O R D A N C E W I T H P A R T N U M B E R : C O N D I T I O N S D E R E C E P T I O N S U I V A N T R E F E R E N C E : P R O G R A M M E P R O G R A M M E P A R T N U M B E R O F U N I T D R A W I N G R E F E R E N C E D U D E S S I N D E D E F I N I T I O N M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T S C A L E E C H E L L E S I Z E F O R M A T S H E E T P L A N C H E T I T L E T I T R E P A R T N U M B E R R E F E R E N C E 3 5 9 6 2 E d . I s . C 2 0 1 6 3 : 1 A M E L I O R A T I O N A M E N D M E N T A M E N D E M E N T D E S I G N O F F I C E B U R E A U D ' E T U D E S D R A W N B Y D E S S I N E P A R C H E C K E D B Y V E R I F I E P A R D A T E D E S I G N E T U D E S U N I T U N I T E S T R E S S C A L C U L (sauf indications particulières): (unless otherwise stated): Unit / Unité KLIM G MHS ELAHI M KLIM G 6/28/2016 0.205 lbs TORONTO HYPERLOOP - 1 UPPER TORQUE LINK SUB-ASSEMBLY - HDWS 35-1402-02 3 : 1 30-1400-01 M A X N O M I N A L C A L C U L A T E D W E I G H T M A S S E C A L C U L E E E D E S I G N E T U D E S S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R - - - 1 in D A T E C L A S S I F I C A T I O N 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 7/4/2016 KLIM G - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION AMENDMENT AMENDEMENT MODIFICATION APPROVAL FORM / EDES FICHE D'APPROBATION DE MODIFICATION Y E S O U I N O N O N X K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 P/N 40-1402-01, UPPER TORQUE LINK A A .2515 .2500 2X .2515 .2500 2X 1.172 1.152 .687 .667 SECTION A-A P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 1 1 2 2 REF ONLY SCALE: NONE 3D VIEW MATERIAL: ALUMINUM 6061-T6511 (BAR) PER AMS QQ-A-200/8. 11 10 9 8 7 6 12 13 14 15 16 17 18 H G F E D K L M N P Q R S 0 1 0 0 m m P R O D U C T A C C E P T A N C E T E S T S C H E D U L E I N A C C O R D A N C E W I T H P A R T N U M B E R : C O N D I T I O N S D E R E C E P T I O N S U I V A N T R E F E R E N C E : P R O G R A M M E P R O G R A M M E P A R T N U M B E R O F U N I T D R A W I N G R E F E R E N C E D U D E S S I N D E D E F I N I T I O N M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T S C A L E E C H E L L E S I Z E F O R M A T S H E E T P L A N C H E T I T L E T I T R E P A R T N U M B E R R E F E R E N C E 3 5 9 6 2 E d . I s . C 2 0 1 6 3 : 1 A M E L I O R A T I O N A M E N D M E N T A M E N D E M E N T D E S I G N O F F I C E B U R E A U D ' E T U D E S D R A W N B Y D E S S I N E P A R C H E C K E D B Y V E R I F I E P A R D A T E D E S I G N E T U D E S U N I T U N I T E S T R E S S C A L C U L (sauf indications particulières): (unless otherwise stated): Unit / Unité KLIM G MHS ELAHI M KLIM G 6/28/2016 0.205 lbs TORONTO HYPERLOOP - 1 UPPER TORQUE LINK SUB-ASSEMBLY - HDWS 35-1402-02 3 : 1 30-1400-01 M A X N O M I N A L C A L C U L A T E D W E I G H T M A S S E C A L C U L E E E D E S I G N E T U D E S S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R - - - 1 in D A T E C L A S S I F I C A T I O N 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 7/4/2016 KLIM G - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION AMENDMENT AMENDEMENT MODIFICATION APPROVAL FORM / EDES FICHE D'APPROBATION DE MODIFICATION Y E S O U I N O N O N X K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 P/N 40-1402-01, UPPER TORQUE LINK A A .2515 .2500 2X .2515 .2500 2X 1.172 1.152 .687 .667 SECTION A-A P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 1 1 2 2 REF ONLY SCALE: NONE 3D VIEW 11 10 9 8 7 6 5 4 3 2 12 13 14 15 10.020 9.980 1.1881 1.1875 2X THRU ALL 2.510 2.490 1.010 .990 .280 .220 R 2X .570 .550 R 2X .626 .586 2X (INT) 1.1881 1.1875 2X THRU ALL 1.080 1.020 R 2X 1.080 1.020 R 2X .730 .670 R B A R .625" TRANSITION TO R .125" IN THIS AREA, BOTH SIDES B B .510 .490 R 4X 4.010 3.990 2X .790 .710 R 4X ALL AROUND .291 .279 THRU ALL .860 .840 .385 .365 .780 .720 2.010 1.990 2X .680 .620 .335 .315 .810 .790 46 44 4X .060 .040 4X .010 A A A 1.260 1.240 119 117 77 73 2X 1.110 1.090 8.680 8.640 SECTION A-A .010 A-B .655 .595 R 4X 1.650 1.590 2.130 2.070 .820 .800 1.060 1.040 SECTION B-B 10.020 9.980 1.1881 1.1875 2X THRU ALL 2.510 2.490 1.010 .990 .280 .220 R 2X .570 .550 R 2X .626 .586 2X (INT) 1.1881 1.1875 2X THRU ALL 1.080 1.020 R 2X 1.080 1.020 R 2X .730 .670 R B A R .625" TRANSITION TO R .125" IN THIS AREA, BOTH SIDES B B .510 .490 R 4X 4.010 3.990 2X .790 .710 R 4X ALL AROUND .291 .279 THRU ALL .860 .840 .385 .365 .780 .720 2.010 1.990 2X .680 .620 .335 .315 .810 .790 46 44 4X .060 .040 4X .010 A A A .655 .595 R 4X 1.650 1.590 2.130 2.070 .820 .800 1.060 1.040 SECTION B-B A .291 .279 THRU ALL .010 A .291 .279 THR .010 A .560 .540 2X .525 .515 .291 .279 2X THRU ALL .790 .710 R 2X ALL AROUND .260 .240 R 2X .145 .105 R 2X ALL AROUND .4379 .4369 2X THRU ALL 46 44 4X .050 .030 4X .010 B .005 B 4 .655 .595 R 4X 1.650 1.590 2.130 2.070 .820 .800 1.060 1.040 SECTION B-B R .625" TRANSITION TO R .125" IN THIS AREA, BOTH SIDES .280 .220 B 4 P Q R S T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R E X N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 1 2 30-1100-20 HYPERLOOP 16/6/2016 KLIM G ELAHI M, KLIM G SKA KLIM G 3.9 lbs in TORONTO X X X 40-1101-66 T-ARM ASSEMBLY -HDWS TRAILING ARM MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O M A M E L I O R A T I O N 1 : 1 2 0 1 6 C I s . 11 10 9 8 7 6 12 13 14 15 16 17 18 H G F E D K L M N P Q R S 0 1 0 0 m m P R O D U C T A C C E P T A N C E T E S T S C H E D U L E I N A C C O R D A N C E W I T H P A R T N U M B E R : C O N D I T I O N S D E R E C E P T I O N S U I V A N T R E F E R E N C E : P R O G R A M M E P R O G R A M M E P A R T N U M B E R O F U N I T D R A W I N G R E F E R E N C E D U D E S S I N D E D E F I N I T I O N M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T S C A L E E C H E L L E S I Z E F O R M A T S H E E T P L A N C H E T I T L E T I T R E P A R T N U M B E R R E F E R E N C E 3 5 9 6 2 E d . I s . C 2 0 1 6 3 : 1 A M E L I O R A T I O N A M E N D M E N T A M E N D E M E N T D E S I G N O F F I C E B U R E A U D ' E T U D E S D R A W N B Y D E S S I N E P A R C H E C K E D B Y V E R I F I E P A R D A T E D E S I G N E T U D E S U N I T U N I T E S T R E S S C A L C U L (sauf indications particulières): (unless otherwise stated): Unit / Unité KLIM G MHS ELAHI M KLIM G 6/28/2016 0.205 lbs TORONTO HYPERLOOP - 1 UPPER TORQUE LINK SUB-ASSEMBLY - HDWS 35-1402-02 3 : 1 30-1400-01 M A X N O M I N A L C A L C U L A T E D W E I G H T M A S S E C A L C U L E E E D E S I G N E T U D E S S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R - - - 1 in D A T E C L A S S I F I C A T I O N 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 7/4/2016 KLIM G - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION AMENDMENT AMENDEMENT MODIFICATION APPROVAL FORM / EDES FICHE D'APPROBATION DE MODIFICATION Y E S O U I N O N O N X K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 P/N 40-1402-01, UPPER TORQUE LINK A A .2515 .2500 2X .2515 .2500 2X 1.172 1.152 .687 .667 SECTION A-A P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 1 1 2 2 REF ONLY SCALE: NONE 3D VIEW 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 2 6/17/2016 KLIM G - 1 16/06/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 .1881 .1875 THRU ALL 2.510 2.490 1.080 1.020 R X .510 .490 R X .385 .365 .780 .720 46 44 0 0 A 1.110 1.090 REF ONLY SCALE: NONE 3D VIEW .560 .540 2X .525 .515 .291 .279 2X THRU ALL .790 .710 R 2X ALL AROUND .260 .240 R 2X .145 .105 R 2X ALL AROUND .4379 .4369 2X THRU ALL 46 44 4X .050 .030 4X .010 B .005 B 4 1. NOTES: NOTES: 1. MATERIAL: CORROSION RESISTANT STEEL (303) PER ASTM A582 OR EQUIVALENT. ALL MATERIAL TO BE IN THE ANNEALED CONDITION. 2. DEBURR SHARP EDGES .005/.015, UNLESS OTHERWISE INDICATED. 3. PASSIVATION ALL OVER PER AMS 2700 METHOD 1. 4. BAG & TAG OR FIXED TAG MARKING. NOTES: 8 7 6 5 4 3 2 9 10 11 12 .291 .279 THRU ALL .019 A A A 5.295 5.275 2.653 2.633 .9995 .9985 .760 .740 .060 .040 2X .060 .040 2X 20 10 2X 31 29 2X SECTION A-A 32 A .005 A .291 .279 THRU ALL .019 A A A A A A A 5.295 5.275 2.653 2.633 .9995 .9985 .760 .740 .060 .040 2X .060 .040 2X 20 10 2X 31 29 2X SECTION A-A 32 A .005 A 5.295 5.275 H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 1 30-1100-20 HYPERLOOP 7/5/2016 KLIM G ELAHI M SKA KLIM G 0.505 lbs in TORONTO X X X 40-1103-01 - HDWS PIVOT PIN, TRAILING ARM MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 2 : 1 2 0 1 6 C I s . NOTES: E d . 11 10 9 8 7 6 12 13 14 15 16 17 18 H G F E D K L M N P Q R S 0 1 0 0 m m P R O D U C T A C C E P T A N C E T E S T S C H E D U L E I N A C C O R D A N C E W I T H P A R T N U M B E R : C O N D I T I O N S D E R E C E P T I O N S U I V A N T R E F E R E N C E : P R O G R A M M E P R O G R A M M E P A R T N U M B E R O F U N I T D R A W I N G R E F E R E N C E D U D E S S I N D E D E F I N I T I O N M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T S C A L E E C H E L L E S I Z E F O R M A T S H E E T P L A N C H E T I T L E T I T R E P A R T N U M B E R R E F E R E N C E 3 5 9 6 2 E d . I s . C 2 0 1 6 3 : 1 A M E L I O R A T I O N A M E N D M E N T A M E N D E M E N T D E S I G N O F F I C E B U R E A U D ' E T U D E S D R A W N B Y D E S S I N E P A R C H E C K E D B Y V E R I F I E P A R D A T E D E S I G N E T U D E S U N I T U N I T E S T R E S S C A L C U L (sauf indications particulières): (unless otherwise stated): Unit / Unité KLIM G MHS ELAHI M KLIM G 6/28/2016 0.205 lbs TORONTO HYPERLOOP - 1 UPPER TORQUE LINK SUB-ASSEMBLY - HDWS 35-1402-02 3 : 1 30-1400-01 M A X N O M I N A L C A L C U L A T E D W E I G H T M A S S E C A L C U L E E E D E S I G N E T U D E S S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R - - - 1 in D A T E C L A S S I F I C A T I O N 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 7/4/2016 KLIM G - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION AMENDMENT AMENDEMENT MODIFICATION APPROVAL FORM / EDES FICHE D'APPROBATION DE MODIFICATION Y E S O U I N O N O N X K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 P/N 40-1402-01, UPPER TORQUE LINK A A .2515 .2500 2X .2515 .2500 2X 1.172 1.152 .687 .667 SECTION A-A P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 1 1 2 2 REF ONLY SCALE: NONE 3D VIEW 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 2/6/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 3D VIEW REF ONLY SCALE : NONE 11 10 9 8 7 6 12 13 14 15 16 17 18 H G F E D K L M N P Q R S 0 1 0 0 m m P R O D U C T A C C E P T A N C E T E S T S C H E D U L E I N A C C O R D A N C E W I T H P A R T N U M B E R : C O N D I T I O N S D E R E C E P T I O N S U I V A N T R E F E R E N C E : P R O G R A M M E P R O G R A M M E P A R T N U M B E R O F U N I T D R A W I N G R E F E R E N C E D U D E S S I N D E D E F I N I T I O N M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T S C A L E E C H E L L E S I Z E F O R M A T S H E E T P L A N C H E T I T L E T I T R E P A R T N U M B E R R E F E R E N C E 3 5 9 6 2 E d . I s . C 2 0 1 6 3 : 1 A M E L I O R A T I O N A M E N D M E N T A M E N D E M E N T D E S I G N O F F I C E B U R E A U D ' E T U D E S D R A W N B Y D E S S I N E P A R C H E C K E D B Y V E R I F I E P A R D A T E D E S I G N E T U D E S U N I T U N I T E S T R E S S C A L C U L (sauf indications particulières): (unless otherwise stated): Unit / Unité KLIM G MHS ELAHI M KLIM G 6/28/2016 0.205 lbs TORONTO HYPERLOOP - 1 UPPER TORQUE LINK SUB-ASSEMBLY - HDWS 35-1402-02 3 : 1 30-1400-01 M A X N O M I N A L C A L C U L A T E D W E I G H T M A S S E C A L C U L E E E D E S I G N E T U D E S S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R - - - 1 in D A T E C L A S S I F I C A T I O N 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 7/4/2016 KLIM G - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION AMENDMENT AMENDEMENT MODIFICATION APPROVAL FORM / EDES FICHE D'APPROBATION DE MODIFICATION Y E S O U I N O N O N X K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 P/N 40-1402-01, UPPER TORQUE LINK A A .2515 .2500 2X .2515 .2500 2X 1.172 1.152 .687 .667 SECTION A-A P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 1 1 2 2 REF ONLY SCALE: NONE 3D VIEW E d . 11 10 9 8 7 6 12 13 14 15 16 17 18 H G F E D K L M N P Q R S 0 1 0 0 m m P R O D U C T A C C E P T A N C E T E S T S C H E D U L E I N A C C O R D A N C E W I T H P A R T N U M B E R : C O N D I T I O N S D E R E C E P T I O N S U I V A N T R E F E R E N C E : P R O G R A M M E P R O G R A M M E P A R T N U M B E R O F U N I T D R A W I N G R E F E R E N C E D U D E S S I N D E D E F I N I T I O N M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T S C A L E E C H E L L E S I Z E F O R M A T S H E E T P L A N C H E T I T L E T I T R E P A R T N U M B E R R E F E R E N C E 3 5 9 6 2 E d . I s . C 2 0 1 6 3 : 1 A M E L I O R A T I O N A M E N D M E N T A M E N D E M E N T D E S I G N O F F I C E B U R E A U D ' E T U D E S D R A W N B Y D E S S I N E P A R C H E C K E D B Y V E R I F I E P A R D A T E D E S I G N E T U D E S U N I T U N I T E S T R E S S C A L C U L (sauf indications particulières): (unless otherwise stated): Unit / Unité KLIM G MHS ELAHI M KLIM G 6/28/2016 0.205 lbs TORONTO HYPERLOOP - 1 UPPER TORQUE LINK SUB-ASSEMBLY - HDWS 35-1402-02 3 : 1 30-1400-01 M A X N O M I N A L C A L C U L A T E D W E I G H T M A S S E C A L C U L E E E D E S I G N E T U D E S S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R - - - 1 in D A T E C L A S S I F I C A T I O N 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 7/4/2016 KLIM G - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION AMENDMENT AMENDEMENT MODIFICATION APPROVAL FORM / EDES FICHE D'APPROBATION DE MODIFICATION Y E S O U I N O N O N X K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 P/N 40-1402-01, UPPER TORQUE LINK A A .2515 .2500 2X .2515 .2500 2X 1.172 1.152 .687 .667 SECTION A-A P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 1 1 2 2 REF ONLY SCALE: NONE 3D VIEW 3 14 15 16 E D C B A G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 1 30-1100-20 HYPERLOOP 7/5/2016 KLIM G ELAHI M SKA KLIM G 0.505 lbs in TORONTO X X X 40-1103-01 - HDWS PIVOT PIN, TRAILING ARM MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 2 : 1 2 0 1 6 C I s . 11 10 9 8 7 6 12 13 14 15 16 17 18 H G F E D K L M N P Q R S 0 1 0 0 m m P R O D U C T A C C E P T A N C E T E S T S C H E D U L E I N A C C O R D A N C E W I T H P A R T N U M B E R : C O N D I T I O N S D E R E C E P T I O N S U I V A N T R E F E R E N C E : P R O G R A M M E P R O G R A M M E P A R T N U M B E R O F U N I T D R A W I N G R E F E R E N C E D U D E S S I N D E D E F I N I T I O N M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T S C A L E E C H E L L E S I Z E F O R M A T S H E E T P L A N C H E T I T L E T I T R E P A R T N U M B E R R E F E R E N C E 3 5 9 6 2 E d . I s . C 2 0 1 6 3 : 1 A M E L I O R A T I O N A M E N D M E N T A M E N D E M E N T D E S I G N O F F I C E B U R E A U D ' E T U D E S D R A W N B Y D E S S I N E P A R C H E C K E D B Y V E R I F I E P A R D A T E D E S I G N E T U D E S U N I T U N I T E S T R E S S C A L C U L (sauf indications particulières): (unless otherwise stated): Unit / Unité KLIM G MHS ELAHI M KLIM G 6/28/2016 0.205 lbs TORONTO HYPERLOOP - 1 UPPER TORQUE LINK SUB-ASSEMBLY - HDWS 35-1402-02 3 : 1 30-1400-01 M A X N O M I N A L C A L C U L A T E D W E I G H T M A S S E C A L C U L E E E D E S I G N E T U D E S S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R - - - 1 in D A T E C L A S S I F I C A T I O N 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 7/4/2016 KLIM G - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION AMENDMENT AMENDEMENT MODIFICATION APPROVAL FORM / EDES FICHE D'APPROBATION DE MODIFICATION Y E S O U I N O N O N X K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 P/N 40-1402-01, UPPER TORQUE LINK A A .2515 .2500 2X .2515 .2500 2X 1.172 1.152 .687 .667 SECTION A-A P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 P/N KRJ4-USVC-008, KARON LINED JOURNAL BEARING, QTY 2 1 1 2 2 REF ONLY SCALE: NONE 3D VIEW E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 2 17/6/2016 KLIM G - 1 13/6/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 A .002 A 63 1.750 1.730 1.1910 1.1900 A A REF ONLY SCALE: NONE 3D VIEW SECTION A-A .001 A 32 63 63 1.0015 1.0005 .065 .055 .030 .010 47 43 .030 .020 R .050 .030 2X 31 29 .0625 .0575 1.020 .980 16 14 31 29 16 3 14 15 16 E D C B A G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D X N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 2 30-1100-20 HYPERLOOP 9/06/2016 KLIM G ELAHI M SKA KLIM G 0.098 lbs in TORONTO X X X 40-1105-00 TRAILING ARM ASSY -HDWS BUSH, PIVOT PIN MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 3 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 2/6/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 SECTION A-A 8 7 6 5 4 9 10 11 12 13 14 15 16 E D C B A G H J K L 5400) PER ASTM B505 OR EQUIVALENT. NOTES: /.015, UNLESS OTHERWISE INDICATED. AT TREATED CONDITION. & TAG. NOTES: T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D X N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 2 30-1100-20 HYPERLOOP 9/06/2016 KLIM G ELAHI M SKA KLIM G 0.098 lbs in TORONTO X X X 40-1105-00 TRAILING ARM ASSY -HDWS BUSH, PIVOT PIN MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 3 : 1 2 0 1 6 C I s . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 2 17/6/2016 KLIM G - 1 13/6/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 REF ONLY SCALE: NONE 3D VIEW NOTES: E D C G H J K L 4. PART MARKING USING BAG & TAG. NOTES: A .002 A 63 1.750 1.730 1.1910 1.1900 A A SECTION A-A .001 A 32 63 63 1.0015 1.0005 .065 .055 .030 .010 47 43 .030 .020 R .050 .030 2X 31 29 .0625 .0575 1.020 .980 16 14 31 29 SECTION A-A .001 A 32 63 63 1.0015 1.0005 .065 .055 .030 .010 47 43 .030 .020 R .050 .030 2X 31 29 .0625 .0575 1.020 .980 16 14 31 29 G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D X N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 2 30-1100-20 HYPERLOOP 9/06/2016 KLIM G ELAHI M SKA KLIM G 0.098 lbs in TORONTO X X X 40-1105-00 TRAILING ARM ASSY -HDWS BUSH, PIVOT PIN MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 3 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 2 17/6/2016 KLIM G - 1 13/6/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 SECTION A-A 3 14 15 16 E D C B A G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D X N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 2 30-1500-02 HYPERLOOP 6/10/2016 KLIM G ELAHI M SKA KLIM G 0.102 lbs in TORONTO X X X 40-1106-00 -HDWS AXLE BUSH, AXLE ASSEMBLY MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 3 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 2 6/17/2016 KLIM G - 1 6/10/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 REF ONLY SCALE: NONE 3D VIEW NOTES: E D C G H J K L .002 A A 63 63 1.510 1.490 .1275 .1225 1.010 .990 1.1910 1.1900 A A SECTION A-A 32 63 .001 A 47 43 1.0015 1.0005 .080 .060 .075 .065 31 29 16 14 31 29 .050 .030 2X .030 .020 R .002 A A 63 63 1.510 1.490 .1275 .1225 1.010 .990 1.1910 1.1900 A A 8 7 6 5 4 3 2 1 9 10 11 E D C G H J K L .002 A A 63 63 1.510 1.490 .1275 .1225 1.010 .990 1.1910 1.1900 A A SECTION A-A 32 63 .001 A 47 43 1.0015 1.0005 .080 .060 .075 .065 31 29 16 14 31 29 .050 .030 2X .030 .020 R H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D X N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 2 30-1500-02 HYPERLOOP 6/10/2016 KLIM G ELAHI M SKA KLIM G 0.102 lbs in TORONTO X X X 40-1106-00 -HDWS AXLE BUSH, AXLE ASSEMBLY MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 3 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: MATERIAL: ALUMINUM 6061-T6511 (BAR) PER AMS QQ-A-200/8. E D C B G H J K L 15, UNLESS OTHERWISE INDICATED. G OR PUNCH. NOTES: T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 1 20-1000-00 HYPERLOOP 7/14/2016 KLIM G - - KLIM G 0.002 lbs in TORONTO X X X 40-1107-00 -HDWS WASHER, AXLE CROSS BOLT MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 6 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 2 6/17/2016 KLIM G - 1 6/10/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 SECTION A-A 12 NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 7/14/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 A A SECTION A-A .020 .000 60 58 47 43 .270 .250 .160 .140 .520 .480 .420 .380 REF ONLY SCALE: NONE 3D VIEW E D G H J T r a Y E S O U I K E Y C H A C A R A C T E G E N E R A T O L E R A N I S O a p p l i c a b l e w i t G E O M E T R I C T O L E R A N C E M D E S B U R E A 1 0 0 m m .XX .XXX Angle Eccen A A SECTION A-A .020 .000 60 58 47 43 .270 .250 .160 .140 .520 .480 .420 .380 A A SECTION A-A .020 .000 60 58 47 43 .270 .250 .160 .140 .520 .480 .420 .380 A A A G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 1 20-1000-00 HYPERLOOP 7/14/2016 KLIM G - - KLIM G 0.002 lbs in TORONTO X X X 40-1107-00 -HDWS WASHER, AXLE CROSS BOLT MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 6 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: E D C B A G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 1 20-1000-00 HYPERLOOP 7/14/2016 KLIM G - - KLIM G 0.002 lbs in TORONTO X X X 40-1107-00 -HDWS WASHER, AXLE CROSS BOLT MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 6 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 7/14/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 REF ONLY SCALE: NONE 3D VIEW 1. NOTES: MATERIAL: BRASS SHIM STOCK. 2 MAKE QTY 20 FROM EACH OF THE FOLLOWING STOCK THICKNESSES: 3. BAG AND TAG EACH STOCK THICKNESS. PART # THICK. -01 .005 -02 .006 -03 .007 -04 .008 -05 .010 -06 .012 -07 .015 -08 .020 -09 .025 -10 .031 NOTES: 1. MATERIAL: BRASS SHIM STOCK. 2 MAKE QTY 20 FROM EACH OF THE FOLLOWING STOCK THICKNESSES: 3. BAG AND TAG EACH STOCK THICKNESS. PART # THICK. -01 .005 -02 .006 -03 .007 -04 .008 -05 .010 -06 .012 -07 .015 -08 .020 -09 .025 -10 .031 NOTES: MATERIAL: BRASS SHIM STOCK. 2 MAKE QTY 20 FROM EACH OF THE FOLLOWING STOCK THICKNESSES: 3. BAG AND TAG EACH STOCK THICKNESS. 2 MAKE QTY 20 FROM EACH OF THE FOLLOWING STOCK THICKNESSES: E D G H J K L .3380 .3180 .760 .740 2 E D G H J .3380 .3180 .760 .740 2 .3380 .3180 .760 .740 2 .3380 .3180 .760 .740 2 .3380 .3180 .760 .740 G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 1 20-1000-00 HYPERLOOP 10/4/2016 KLIM G - - KLIM G 0 lbs in TORONTO X X X 40-1108-00 -HDWS SHIM, LTL - TL JOINT MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 5 : 1 2 0 1 6 C I s . 2 MAKE QTY 20 FROM EACH OF THE FOLLOWING STOCK THICKNESSES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 3D VIEW REF ONLY SCALE: NONE 1. NOTES: E D C B A G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 1 20-1000-00 HYPERLOOP 10/4/2016 KLIM G - - KLIM G 0 lbs in TORONTO X X X 40-1108-00 -HDWS SHIM, LTL - TL JOINT MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 5 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 7/14/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 A SECTION A-A SECTION A-A 12 3 2 1 E D C B A G H J K L NOTES: 1. NOTES: 2. DEBURR SHARP EDGES .005/.015, UNLESS OTHERWISE INDICATED. 2 MAKE QTY 20 FROM EACH OF THE FOLLOWING STOCK THICKNESSES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 12 NOTES: H G F E K L M N P Q R S T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 30-1200-21 HYPERLOOP 5/27/2016 KLIM G ELAHI M SKA KLIM G 0.7 lbs in TORONTO X X X -HDWS PISTON, SHOCK ABSORBER MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O M A M E L I O R A T I O N 2 : 1 2 0 1 6 C 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R T I T L E T I T R E S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 5/27/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR D E S I G N E T U D E S 16 A 12 SMOOTH OUT EDGE 16 0.002 A TURNING CENTER OPTIONAL MAX DEPTH 0.165" 2X BREAK EDGES FOR SEAL INSTALLATION 1.010 .990 1.010 .990 1.054 1.044 .530 .470 R 5.810 5.790 .715 .695 .135 .115 .405 .395 .160 .140 1.210 1.190 2X 1.060 1.058 GROUND OD .210 .190 .529 .519 R SR 1.044 1.034 36 34 A A SECTION A-A 0.002 A 0.002 A 16 0.002 A 0.010 A .110 .090 R 2X .869 .859 1.435 1.425 5.660 5.640 .7493 .7488 .025 .020 2X .020 .010 R 4X .260 .240 R 2X 1.064 1.054 2X .291 .281 31 29 2X .060 .040 2X 46 44 2X .910 .890 2X .210 .190 136 134 .070 .030 R - - - - - - - - - - - - 16 A 12 SMOOTH OUT EDGE 16 0.002 A TURNING CENTER OPTIONAL MAX DEPTH 0.165" 2X BREAK EDGES FOR SEAL INSTALLATION 1.010 .990 1.010 .990 1.054 1.044 .530 .470 R 5.810 5.790 .715 .695 .135 .115 .405 .395 .160 .140 1.210 1.190 2X 1.060 1.058 GROUND OD .210 .190 .529 .519 R SR 1.044 1.034 36 34 A A SECTION A-A 0.002 A 0.002 A 16 0.002 A 0.010 A .110 .090 R 2X .869 .859 1.435 1.425 5.660 5.640 .7493 .7488 .025 .020 2X .020 .010 R 4X .260 .240 R 2X 1.064 1.054 2X .291 .281 31 29 2X .060 .040 2X 46 44 2X .910 .890 2X .210 .190 136 134 .070 .030 R - - - - - - - - - - - - 16 A 12 SMOOTH OUT EDGE 16 0.002 A TURNING CENTER OPTIONAL MAX DEPTH 0.165" 2X BREAK EDGES FOR SEAL INSTALLATION 1.010 .990 1.010 .990 1.054 1.044 .530 .470 R 5.810 5.790 .715 .695 .135 .115 .405 .395 .160 .140 1.210 1.190 2X 1.060 1.058 GROUND OD .210 .190 .529 .519 R SR 1.044 1.034 36 34 A A SECTION A-A 0.002 A 0.002 A 16 0.002 A 0.010 A .110 .090 R 2X .869 .859 1.435 1.425 5.660 5.640 .7493 .7488 .025 .020 2X .020 .010 R 4X .260 .240 R 2X 1.064 1.054 2X .291 .281 31 29 2X .060 .040 2X 46 44 2X .910 .890 2X .210 .190 136 134 .070 .030 R .210 .190 R 10X .605 .595 R 5x .110 .090 5X .055 .045 5X 72.5 71.5 5x 2X BREAK EDGES FOR SEAL INSTALLATION TURNING CENTER OPTIONAL MAX DEPTH 0.165" .605 .595 R 5x P Q R S T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R E N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 1 30-1200-21 HYPERLOOP 5/27/2016 KLIM G ELAHI M SKA KLIM G 0.7 lbs in TORONTO X X X 40-1201-02 -HDWS PISTON, SHOCK ABSORBER MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O M A M E L I O R A T I O N 2 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 1. MATERIAL: AISI 12L14 CARBON STEEL (UNS G12144) PER ASTM A108 OR EQUIVALENT. H G F E D C K L M N P Q R S 6. THREADS PER AS8879. 7. BLACK OXIDE CORROSION PROTECTION ON ALL EXTERNAL SURFACES AND ALL THREADS. .927/.925 AND 1.493/1.491 BORES NOT TO HAVE BLACK OXIDE. NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 5/27/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 D E S I G N E T U D E S 40-1201-02 NOTES: T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R X N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 1 30-1200-21 HYPERLOOP 9-5-2016 KLIM G ELAHI M, KLIM G SKA KLIM G, SKA 7.5 lbs in TORONTO X X X MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O M A M E L I O R A T I O N 2 0 1 6 C 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m - - - - - - - - - - - - - - - - - - - - - - - - - - - - 3 21/6/2016 KLIM G - 2 9/5/2016 KLIM G - 1 7/5/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR D E S I G N E T U D E S .005 A 2.250-3 ACME-3G EXTERNAL THREAD: MAJOR DIAMETER: 2.2500/2.2333in PITCH DIAMETER: 2.0743/2.0620in MINOR DIAMETER: 1.8967/1.8783in T.P.I.: 3 PITCH: .333333 5 .760 .740 10.280 10.220 .460 .440 R 2.180 2.160 .760 .740 .360 .340 R OR DIAMETER 2.110 2.090 2X .385 .365 2X .3753 .3743 THRU ALL .068 .058 .250 .190 .280 .220 .042 .022 R ALL AROUND R FULL RAD .184 .164 A A SECTION A-A .005 B B .005 A 32 32 32 .002 B MINOR DIAMETER 32 32 32 .002 B .005 A 1.493 1.491 1.723 1.713 .927 .925 .072 .052 .724 .704 .960 .940 .060 .040 R 2X 46 44 16 14 .180 .120 R .110 .090 INT 1.6875 -16 UN-3B 6.010 5.990 .110 .090 R .160 .140 INT .180 .120 R 16 14 .070 .050 31 29 50 40 2.052 2.032 31 29 TAP DRILL .560/470 DEEP MIN FULL THREAD .400 DEEP 1.630 1.570 R 2x .780 .720 R 2X 1.630 1.570 .155 .095 R 2X 1.316 1.296 .290 .270 2X 46 44 2X .040 .020 2X 1.230 1.210 2X .610 .590 .305 .295 .164 2X -32 UNC-3B 1.510 1.490 2.510 2.490 1.255 1.245 1.630 1.570 R 2x T TO GET DIFFERENT WITH TORQUE .125 .095 R 2X 5 10.280 10.220 46 44 B ABSORBER - HDWS , 3 I s . NOTES: 1. THIS DRAWING IS TO BE TREATED AS A SUPPLEMENT TO 40-1202-21. MANUFACTURE PER DRAWING 40-1202-21 EXCEPT AS SHOWN/NOTED. 2 SPECIFIES 40-1202-21A PART SPECIFIC DIMENSION. 3 DIMENSION FOR REFERENCE ONLY, REFER TO 40-1202-21. 4. .993/.991 AND 1.493/1.491 BORES NOT TO HAVE BLACK OXIDE. 5. IDENTIFY WITH PART MARKING OF THIS DRAWING. NOTES: 8 7 6 5 4 9 10 11 12 E D G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 1 30-1202-21A HYPERLOOP 17/6/2016 KLIM G N/A KLIM G KLIM G 7.62 lbs in TORONTO X X X 40-1202-21A ABSORBER -HDWS OUTER CYLINDER, SHOCK MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 1 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 17/6/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 A A SECTION A-A 32 32 B .005 A .002 B 32 32 3 3 2 2 2 2 3 3 32 2 2 .002 B 2 .993 .991 6.010 5.990 11 9 .320 .280 R .160 .140 INT 1.493 1.491 31 29 .070 .050 .945 .925 46 44 .850 .800 E G H J K L A A SECTION A-A 32 32 B .005 A .002 B 32 32 3 3 2 2 2 2 3 3 32 2 2 .002 B 2 .993 .991 6.010 5.990 11 9 .320 .280 R .160 .140 INT 1.493 1.491 31 29 .070 .050 .945 .925 46 44 .850 .800 A A SECTION A-A 32 32 B .005 A .002 B 32 32 3 3 2 2 2 2 3 3 32 2 2 .002 B 2 .993 .991 6.010 5.990 11 9 .320 .280 R .160 .140 INT 1.493 1.491 31 29 .070 .050 .945 .925 46 44 .850 .800 A A A G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 1 30-1202-21A HYPERLOOP 17/6/2016 KLIM G N/A KLIM G KLIM G 7.62 lbs in TORONTO X X X 40-1202-21A ABSORBER -HDWS OUTER CYLINDER, SHOCK MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 1 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 17/6/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 2 46 44 3 14 15 16 E D C B A G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D X N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 1 30-1200-21 HYPERLOOP 12/6/2016 KLIM G - SKA KLIM G 0.066 lbs in TORONTO X X X 40-1205-02 -HDWS SPLIT UPPER BEARING, PISTON MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 4 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 13/6/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 REF ONLY SCALE: NONE 3D VIEW NOTES: E D C B G H J K L 3. MACHINE IN THE FULLY HEAT TREATED CONDITION. 4. BAG AND TAG OR FIXED TAG MARKING. 5 DIMENSION, OR DATUM, TO BE MET BEFORE OPERATION THAT RESULTS IN A DISCONTINUOUS RING. A 32 5 5 1.489 1.488 .032 (SAW CUT) A A SECTION A-A 32 .002 A .002 A .005 A 5 5 .002 A 1.076 1.066 1.299 1.279 46 44 2X .040 .020 2X 61 59 2X .025 .015 2X .100 .080 2X .390 .380 .070 .030 R 2X 16 14 2X RESULTS A 32 5 5 1.489 1.488 .032 (SAW CUT) A A SECTION A-A 32 .002 A .002 A .005 A 5 5 .002 A 1.076 1.066 1.299 1.279 46 44 2X .040 .020 2X 61 59 2X .025 .015 2X .100 .080 2X .390 .380 .070 .030 R 2X 16 14 2X T RESULTS A 32 5 5 1.489 1.488 .032 (SAW CUT) A A SECTION A-A 32 .002 A .002 A .005 A 5 5 .002 A 1.076 1.066 1.299 1.279 46 44 2X .040 .020 2X 61 59 2X .025 .015 2X .100 .080 2X .390 .380 .070 .030 R 2X 16 14 2X 5 DIMENSION, OR DATUM, TO BE MET BEFORE OPERATION THAT RESULTS IN A DISCONTINUOUS RING. NOTES: REF ONLY SCALE: NONE A A A 5 H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D X N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 1 30-1200-21 HYPERLOOP 12/6/2016 KLIM G - SKA KLIM G 0.066 lbs in TORONTO X X X 40-1205-02 -HDWS SPLIT UPPER BEARING, PISTON MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 4 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 1. MATERIAL: AL BRONZE (C95400) PER ASTM B505 OR EQUIVALENT. 1. MATERIAL: AL BRONZE (C95400) PER ASTM B505 OR EQUIVALENT. 2. DEBURR SHARP EDGES .005/.015, UNLESS OTHERWISE INDICATED. 8 7 6 5 4 3 2 9 10 11 12 CHINE IN THE FULLY HEAT TREATED CONDITION. READS PER AS8879. G AND TAG OR FIXED TAG MARKING. Y M T 1 0 0 m m 0 1.323 1.321 46 44 2X 1.6875 -16 UN-3A .050 .040 R .300 .280 1.085 1.065 .060 .040 INT 1.454 1.444 21 19 .265 .255 31 29 .060 .040 R .055 .035 .891 .871 .855 .845 63 .002 A MAJOR DIAMETER .002 A 63 63 B A A 1.304 1.302 2X .314 .304 1.063 1.062 2x .245 .235 .025 .010 R 3x 1.489 1.488 2x .009 .005 R .022 .020 31 29 .060 .040 R .475 .455 .900 .880 .041 .021 R 24x .185 .165 INT 5 0 6x .015 .005 R 2X SECTION A-A 32 32 A .002 A .002 A 32 32 32 .210 .190 12x .105 .095 12x B 16 14 31 29 .060 .040 .110 .090 INT 1.605 1.595 .060 .040 R .175 .155 12x .160 .140 R SECTION B-B 32 .005 B 32 8 7 6 5 4 9 10 11 12 E D C G H J K L KING. NOTES: T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 1 30-1200-21 HYPERLOOP 12/05/2016 KLIM G ELAHI M, KLIM G SKA KLIM G 0.45 lbs in TORONTO X X X 40-1207-02 -HDWS GLAND NUT, SHOCK ABSORBER MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 2 2 0 1 6 C I s . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 13/6/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 SECTION A-A NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 12/05/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 46 44 2X 1.6875 -16 UN-3A .050 .040 R .300 .280 1.085 1.065 .060 .040 INT 1.454 1.444 21 19 .265 .255 31 29 .060 .040 R .055 .035 .891 .871 .855 .845 63 .002 A MAJOR DIAMETER A 63 63 B A A 1.304 1.302 2X .314 .304 1.063 1.062 .245 .235 .025 .010 R 3x .009 .005 R .022 .020 31 29 .060 .040 R .475 .455 .900 .880 .041 .021 R 24x .185 .165 INT 5 0 6x .015 .005 R 2X SECTION A-A 32 32 A .002 A 32 32 32 3D VIEW SCALE: NONE .210 .190 12x 30.5 29.5 12x .105 .095 12x B B 16 14 31 29 .060 .040 .110 .090 INT 1.605 1.595 .060 .040 R .175 .155 12x .160 .140 R SECTION B-B 32 .005 B 32 A B 21 19 B SECTION A-A NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 .202 .192 2X .110 .090 R 2X 83 82 48 42 4X .610 .590 .520 .480 2X .270 .230 2X .185 .175 .084 .074 INT 8 7 .602 .582 .260 .240 .120 .080 4X 4 .135 .115 .202 .192 2X .110 .090 R 2X 83 82 48 42 4X .610 .590 .520 .480 2X .270 .230 2X .185 .175 .084 .074 INT 8 7 .602 .582 .260 .240 .120 .080 4X 4 H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 1 30-1200-21 HYPERLOOP 12/6/2016 KLIM G - SKA KLIM G 0.019 lbs in TORONTO X X X 40-1208-02 ABSORBER ASSEMBLY -HDWS LOCK TAB, SHOCK MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 4 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: E D C B A G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 1 30-1200-21 HYPERLOOP 12/6/2016 KLIM G - SKA KLIM G 0.019 lbs in TORONTO X X X 40-1208-02 ABSORBER ASSEMBLY -HDWS LOCK TAB, SHOCK MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 4 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 REF ONLY SCALE: NONE 3D VIEW E D G H J K L .202 .192 2X .110 .090 R 2X 83 82 48 42 4X .610 .590 .520 .480 2X .270 .230 2X .185 .175 .084 .074 INT 8 7 .602 .582 .260 .240 .120 .080 4X 4 E D G H J .202 .192 2X .110 .090 R 2X 83 82 48 42 4X .610 .590 .520 .480 2X .270 .230 2X .185 .175 .084 .074 INT 8 7 .602 .582 .260 .240 .120 .080 4X 4 8 7 6 5 4 9 10 11 12 E D G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 1 30-1200-21 HYPERLOOP 12/6/2016 KLIM G - SKA KLIM G 0.019 lbs in TORONTO X X X 40-1208-02 ABSORBER ASSEMBLY -HDWS LOCK TAB, SHOCK MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 4 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 1 I s . NOTES: 8 7 6 5 4 3 9 10 11 12 E D C G H J K L IXED TAG MARKING T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 1 1 30-1200-21 HYPERLOOP KLIM G ELAHI M SKA KLIM G 0.34 lbs in TORONTO X X X 40-1209-00 -HDWS FILLER TUBE, SHOCK ABSORBER MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 2 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: E d . 12 NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 A-B AMETER A 32 .002 A .005 A-B .005 A-B 63 UNF-3A .923 .922 2X 5.590 5.550 .210 .190 .460 .440 .885 .865 .860 .840 .710 .690 MINIMUM FULL THREAD LENGTH .751 .749 .041 .021 R 6X .550 .530 INT .200 .180 .100 .080 A A 3D VIEW REF ONLY SCALE: NONE SECTION A-A .005 A-B 63 32 32 33651 63 32 .005 A-B 32 MINOR DIAMETER .005 A-B .4488 ( TAP DRILL) 46 44 2X .780 .720 R .015 .005 R 2X .070 .030 R 46 44 2X .040 .020 2X .045 .035 R 2X .255 .245 .375 -24 UNF-3B 61 59 31 29 .055 .045 P DRILL DEPTH .3344 ( TAP DRILL) .060 .040 .510 .490 TAP DRILL DEPTH (0.400" MINIMUM FULL THREAD DEPTH) .573 .553 .130 .120 .066 .060 THRU ALL .2365 .2265 1.310 1.290 .563 +.002 -.010 6X A/F .2915 .2715 6X 3D VIEW REF ONLY SCALE: NONE A B 63 63 .005 A-B .4488 ( TAP DRILL) 61 59 .060 .040 MINOR DIAMETER .005 A-B .375 -24 UNF-3B NOTES: 1. THIS DRAWING IS TO BE TREATED AS A SUPPLEMENT TO 40-1202-21 ISSUE 2 AND MUST ONLY BE USED IN CONJUNCTION WITH THE AFFORMENTIONED DRAWING. 2 SPECIFIES 40-1202-21A PART SPECIFIC DIMENSION. 3 DIMENSION FOR REFERENCE ONLY, REFER TO 40-1209-00. 4. IDENTIFYU WITH PART MARKING OF THIS DRAWING. NOTES: 8 7 6 5 4 9 10 11 12 E D C G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 1 1 30-1200-21A HYPERLOOP 17/6/2016 KLIM G N/A KLIM G KLIM G 0.345 lbs in TORONTO X X X 40-1209-00A -HDWS FILLER TUBE, SHOCK ABSORBER MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 2 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 17/6/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 A .002 A 32 32 .005 A-B 63 3 3 2 2 2 2 .100 .080 .560 .540 INT .989 .988 2x .823 .821 .960 .940 .200 .180 A A SECTION A-A 63 63 32 32 32 2 3 3 2 2 .265 .255 .060 .040 R .760 .740 R .015 .005 R 2x .045 .035 R 2x A 32 63 3 3 2 .100 .080 .560 .540 INT .200 .180 A A 8 7 6 5 4 3 2 1 9 10 11 12 E D C G H J K L A .002 A 32 32 .005 A-B 63 3 3 2 2 2 2 .100 .080 .560 .540 INT .989 .988 2x .823 .821 .960 .940 .200 .180 A A SECTION A-A 63 63 32 32 32 2 3 3 2 2 .265 .255 .060 .040 R .760 .740 R .015 .005 R 2x .045 .035 R 2x A A G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 1 1 30-1200-21A HYPERLOOP 17/6/2016 KLIM G N/A KLIM G KLIM G 0.345 lbs in TORONTO X X X 40-1209-00A -HDWS FILLER TUBE, SHOCK ABSORBER MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 2 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: MATERIAL: BRASS SHIM STOCK. 2 MAKE QTY 20 FROM EACH OF THE FOLLOWING STOCK THICKNESSES: 3. BAG AND TAG EACH STOCK THICKNESS. PART # THICK. -01 .005 -02 .006 -03 .007 -04 .008 -05 .010 -06 .012 -07 .015 -08 .020 -09 .025 -10 .031 NOTES: 1. MATERIAL: BRASS SHIM STOCK. 1. MATERIAL: BRASS SHIM STOCK. 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 REF ONLY SCALE: NONE 3D VIEW 2 MAKE QTY 20 FROM EACH OF THE FOLLOWING STOCK THICKNESSES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 .770 .730 .260 .250 2 .770 .730 .260 .250 G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 1 20-1000-00 HYPERLOOP 1/12/2016 KLIM G - - KLIM G N/A lbs in TORONTO X X X 40-1210-00 UTL JOINT -HDWS SHIM WASHER, MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 6 : 1 2 0 1 6 C I s . 2 MAKE QTY 20 FROM EACH OF THE FOLLOWING STOCK THICKNESSES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 17/6/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 3 2 1 E D C B A G H J K L NOTES: 1. 1. MATERIAL: BRASS SHIM STOCK. E D C B A G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 1 20-1000-00 HYPERLOOP 1/12/2016 KLIM G - - KLIM G N/A lbs in TORONTO X X X 40-1210-00 UTL JOINT -HDWS SHIM WASHER, MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 6 : 1 2 0 1 6 C I s . 1. MATERIAL: BRASS SHIM STOCK. E d . 2 MAKE QTY 20 FROM EACH OF THE FOLLOWING STOCK THICKNESSES: 3. BAG AND TAG EACH STOCK THICKNESS. 2 MAKE QTY 20 FROM EACH OF THE FOLLOWING STOCK THICKNESSES: 8 7 6 5 4 3 2 1 9 10 11 12 E D G H J K L .770 .730 .260 .250 2 E G H J .770 .730 .260 .250 2 8 7 6 5 9 10 11 12 E G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 1 20-1000-00 HYPERLOOP 1/12/2016 KLIM G - - KLIM G N/A lbs in TORONTO X X X 40-1210-00 UTL JOINT -HDWS SHIM WASHER, MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 6 : 1 2 0 1 6 C I s . 2 MAKE QTY 20 FROM EACH OF THE FOLLOWING STOCK THICKNESSES: E d . 2 MAKE QTY 20 FROM EACH OF THE FOLLOWING STOCK THICKNESSES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 12 .010 A-B D-E C H G F E D C B A K L M N P Q R S 61-T6511 (BAR) PER AMS QQ-A-200/8. 2 MAKE QTY 20 FROM EACH OF THE FOLLOWING STOCK THICKNESSES: 05/.015, UNLESS OTHERWISE INDICATED. RIBING OR PUNCH. 2 MAKE QTY 20 FROM EACH OF THE FOLLOWING STOCK THICKNESSES: NING MISMATCH TO BE .030/.000 UNLESS T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R X N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 1 35-1300-02 HYPERLOOP 7/13/2016 KLIM G ELAHI M SKA KLIM G 3.8 lbs in TORONTO X X X -HDWS RETRACT HOUSING MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O M A M E L I O R A T I O N 1 : 1 2 0 1 6 C 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R T I T L E T I T R E S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 2 28/7/2016 KLIM G - 1 7/11/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR .260 .240 R 2x ALL AROUND .280 .220 R 2x ALL AROUND .280 .220 R 2x A A REF ONLY SCALE: NONE 3D VIEW 2 .002 A-B C .280 .220 R .280 .220 R .460 .440 R 2X 1.555 1.545 43 39 .255 .245 2.010 1.990 4x 2.310 2.290 2.760 2.740 .480 .420 R 2x 1.000 2x 2.680 2.660 1.010 .990 2x 1.690 1.670 2x .280 .220 R ALL AROUND .280 .220 R .530 .470 .800 2X .600 2X 3.460 3.440 2x B B D D .010 A-B .020 D .020 E 1.478 .414 .414 1.891 3.255 3.245 2X .291 .279 THRU ALL 7.510 7.490 .291 .279 THRU ALL C C 5.650 2.060 2.040 1.030 1.020 1.005 .995 .910 .890 2x 1.010 .990 3.410 3.390 1.705 1.695 6.310 6.290 .360 .340 R 2x 2.825 3.155 3.145 .460 .440 R 2x .005 A-B C .002 D-E D .005 A-B C .002 D-E E 1.1881 1.1875 46 44 2x .060 .040 2x 1.480 1.460 2X INT 1.370 1.330 2X .110 .090 R 2X 2.010 1.990 INT 1.005 .995 INT 2.290 2.270 .760 .740 R 2X 46 44 2X .685 .675 2X 1.1881 1.1875 1.100 .900 R 2X 46 44 2X REF ONLY SCALE: NONE 3D VIEW 1 SECTION A-A MINOR DIAMETER .005 B .002 A-B 63 B .002 A-B .010 A-B A .002 A-B 4.125 -16 UNJ-3B 4.210 4.190 2.450 2.430 3.3479 3.3465 .260 .240 R .360 .340 R .130 .110 R .020 .010 R .399 .389 1.197 ( ) 30 .063 .043 .813 .793 2.404 2.384 .205 .195 46 44 3.220 3.180 4 46 44 2x 1.510 1.490 1.505 1.495 3.010 2.990 1.253 1.233 .626 .616 .260 .240 R 2x 2.010 1.990 .880 .870 1.760 1.740 2.290 2.270 .050 .000 MISMATCH 1.960 1.940 .260 .240 R 2X ALL AROUND 1.100 .900 R 2X 2.260 2.240 4.510 4.490 PARTIAL SECTION C C 2X .164 -32 UNRC-3B TAP DRILL .560/470 DEEP MIN FULL THREAD .400 DEEP 47 43 2X .040 .020 2X TIAL SECTION D-D THRU ALL, FULL THREAD .010 A-B C 47 43 4X .040 .020 4X .190 4X -32 UNRF-3B 4 46 44 2x 1.510 1.490 1.505 1.495 3.010 2.990 1.253 1.233 .626 .616 .260 .240 R 2x 2.010 1.990 .880 .870 1.760 1.740 2.290 2.270 .050 .000 MISMATCH 1.960 .260 .240 R 2X ALL AROUND 1.100 .900 R 2X 2.260 2.240 4.510 4.490 1. NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - 3 11/5/2016 KLIM G - 2 9/5/2016 KLIM G - 1 7/5/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 D E S I G N E T U D E S .005 C 2.320 2.300 THREAD RELIEF P Q R S T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R E N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 3 30-1300-02 HYPERLOOP 7/5/2016 KLIM G ELAHI M, KLIM G SKA KLIM G, SKA 2.18 lbs in TORONTO X X X 40-1302-02 - HDWS NUT, RETRACT MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O M A M E L I O R A T I O N 2 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - 3 11/5/2016 KLIM G - 2 9/5/2016 KLIM G - 1 7/5/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 D E S I G N E T U D E S .070 .050 0X 3 I s . NOTES: 1 0 0 m m 2.5602 2.5591 2.790 2.770 2.790 2.770 2.5602 2.5591 .660 .640 2.360 2.340 .510 .490 .710 .690 1.860 1.840 .160 .140 R 2X .020 .010 R 2X 63 63 A B .002 A-B .002 A-B .002 B .002 A .002 B .002 A 5 A A 3.922 3.912 MAJO .005 A-B SECTION A-A .005 A-B C .005 C .005 C 2.25-3 ACME-3G INTERNAL THREAD: MAJOR DIAMETER: 2.2900/2.2700in PITCH DIAMETER: 2.0833/2.0956in MINOR DIAMETER: 1.9334/1.9167in T.P.I.: 3 PITCH: .333333 2.320 2.300 THREAD RELIEF .015 R MIN 1.9333 1.9167 THREAD MINOR DIAMETER 2.290 2.270 LEAD-IN CHAMFER 16 14 .460 .440 INT THREAD RELIEF 76 74 .070 .050 90X 46 44 90X 2.5602 2.5591 2.790 2.770 2.790 2.770 2.5602 2.5591 .660 .640 2.360 2.340 .510 .490 .710 .690 1.860 1.840 .160 .140 R 2X .020 .010 R 2X 63 63 A B .002 A-B .002 A-B .002 B .002 A .002 B .002 A 5 A A SECTION A-A .005 A-B C .005 C .005 C 2.25-3 ACME-3G INTERNAL THREAD: MAJOR DIAMETER: 2.2900/2.2700in PITCH DIAMETER: 2.0833/2.0956in MINOR DIAMETER: 1.9334/1.9167in T.P.I.: 3 PITCH: .333333 2.320 2.300 THREAD RELIEF .015 R MIN 1.9333 1.9167 THREAD MINOR DIAMETER 2.290 2.270 LEAD-IN CHAMFER 16 14 .460 .440 INT THREAD RELIEF 76 74 .070 .050 90X 46 44 90X 76 74 16 14 N P Q R S T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R E N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 3 30-1300-02 HYPERLOOP 7/5/2016 KLIM G ELAHI M, KLIM G SKA KLIM G, SKA 2.18 lbs in TORONTO X X X 40-1302-02 - HDWS NUT, RETRACT MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O M A M E L I O R A T I O N 2 : 1 2 0 1 6 C I s . NOTES: E d . 2 MAKE QTY 20 FROM EACH OF THE FOLLOWING STOCK THICKNESSES: MATERIAL: ALUMINUM 6061-T6511 (BAR) PER AMS QQ-A-200/8. 1.760 1.740 REF ONLY SCALE: NONE 3D VIEW 1 1.960 1.940 D 1.197 ( ) 2.404 2.384 .063 .043 .063 .043 A .813 .793 A .010 A-B 7.510 7.490 SECTION A-A .020 E .291 .279 THRU ALL .020 D .291 .279 THRU ALL 1.478 .414 .414 PARTIAL SECTION D-D 35-1300-02 P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E 1 0 0 m m HDWS RETRACT HOUSING PARTIAL SECTION C-C -HDWS 40-1301-02 2 I s . E d . 2 I s . E d . NOTES: E d . 3 I s . E d . NOTES: 1. MATERIAL: ALUMINUM 6061-T6511 (BAR) PER AMS QQ-A-200/8. 2. DEBURR SHARP EDGES .005/.015, UNLESS OTHERWISE INDICATED. 6. ANODIZING ALL OVER. T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g A S M E I S O X 125 3 MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E C L A S S I F I C A T I O N - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 13/9/2016 KLIM G .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1.080 1.040 4.125 -16 UNJ-3A .041 .021 R 40X .830 .810 .002 A MAJOR DIAMETER A A REF ONLY SCALE: NONE 3D VIEW 3.3478 3.3464 3.220 3.180 .020 .010 R 46 44 .060 .040 .456 .436 63 A .002 A .002 A 63 .330 .310 20X .165 .155 20X 18.5 17.5 20X B B 4.039 4.019 .060 .040 R .230 .210 20X 31 29 .065 .035 46 44 46 44 .260 .240 SECTION B-B .005 A B 4.125 -16 UNJ-3A .002 A MAJOR DIAMETER B 46 44 4.039 4.019 .005 A 63 3.220 3.180 .002 A SECTION B-B SECTION A-A 1 I s . E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 13/6/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 REF ONLY SCALE: NONE 3D VIEW E D G H J 4 .202 .192 2X .305 .295 86 85 48 42 4X .120 .080 4x .060 .040 R 2X 5 4 .520 .480 2X .036 .026 INT .595 .575 .270 .230 2x .260 .240 .706 .686 .135 .115 E D G H J K L 4 .202 .192 2X .305 .295 86 85 48 42 4X .120 .080 4x .060 .040 R 2X 5 4 .520 .480 2X .036 .026 INT .595 .575 .270 .230 2x .260 .240 .706 .686 .135 .115 4 .202 .192 2X .305 .295 86 85 48 42 4X .120 .080 4x .060 .040 R 2X 5 4 .520 .480 2X .036 .026 INT .595 .575 .270 .230 2x .260 .240 .706 .686 .135 .115 4 .202 .192 2X .305 .295 86 85 48 42 4X .120 .080 4x .060 .040 R 2X 5 4 .520 .480 2X .036 .026 INT .595 .575 .270 .230 2x .260 .240 .706 .686 H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D X N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 1 30-1300-02 HYPERLOOP 12/6/2016 KLIM G - SKA KLIM G 0.022 lbs in TORONTO X X X 40-1304-00 -HDWS LOCK TAB, RETRACT ASSY MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 4 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 1. MATERIAL: MACHINE FROM AISI 1018 STEEL, COLD ROLLED, BAR STOCK. 2. DEBURR SHARP EDGES .005/.015, UNLESS OTHERWISE INDICATED. 3. MACHINE IN THE FULLY HEAT TREATED CONDITION. 4 PART MARKING USING SCRIBING OR PUNCH. 5. BLACK OXIDE CORROSION PROTECTION ALL OVER. NOTES: E D C B G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D X N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 1 30-1300-02 HYPERLOOP 12/6/2016 KLIM G - SKA KLIM G 0.022 lbs in TORONTO X X X 40-1304-00 -HDWS LOCK TAB, RETRACT ASSY MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 4 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 13/6/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 12 3 14 15 16 E D C B A G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 1 30-1300-02 HYPERLOOP 7/5/2016 KLIM G ELAHI M SKA KLIM G 0.16 lbs in TORONTO X X X 40-1305-01 - HDWS PIN, RETRACTION ASSY MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 2 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: E D C G H J K L 1.710 1.690 .291 .279 THRU ALL .019 A A A SECTION A-A 32 A .005 A .610 .590 .9995 .9985 .760 .740 .060 .040 2X .060 .040 2X 20 10 2X 31 29 A A H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 1 30-1300-02 HYPERLOOP 7/5/2016 KLIM G ELAHI M SKA KLIM G 0.16 lbs in TORONTO X X X 40-1305-01 - HDWS PIN, RETRACTION ASSY MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 2 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 3D VIEW REF ONLY SCALE: NONE NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 SECTION A-A 12 3 14 15 16 E D C B A G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 2 2 30-1300-02 HYPERLOOP 5/20/2016 KLIM G ELAHI M SKA KLIM G 0.093 lbs in TORONTO X X X 40-1308-00 RETRACT ASSEMBLY -HDWS BUSH, PIVOT PIN MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 3 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 2 7/13/2016 KLIM G - 1 5/23/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 REF ONLY SCALE: NONE 3D VIEW 1.750 1.730 1.1909 1.1901 .380 .370 A 63 .010 A .002 A A A 1.0015 1.0005 .030 .020 R .510 .490 1.010 .990 31 29 31 29 16 14 .050 .030 2X .0625 .0575 .065 .055 .030 .010 47 43 SECTION A-A 32 .001 A 63 63 NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 2 7/13/2016 KLIM G - 1 5/23/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 REF ONLY SCALE: NONE 3D VIEW E D C B A G H J K L 5400) PER ASTM B505 OR EQUIVALENT. NOTES: .015, UNLESS OTHERWISE INDICATED. AT TREATED CONDITION. & TAG. NOTES: T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 2 2 30-1300-02 HYPERLOOP 5/20/2016 KLIM G ELAHI M SKA KLIM G 0.093 lbs in TORONTO X X X 40-1308-00 RETRACT ASSEMBLY -HDWS BUSH, PIVOT PIN MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 3 : 1 2 0 1 6 C I s . NOTES: E D C B G H J K L 4. PART MARKING USING BAG & TAG. 1.750 1.730 1.1909 1.1901 .380 .370 A 63 .010 A .002 A A A 1.0015 1.0005 .030 .020 R .510 .490 1.010 .990 31 29 31 29 16 14 .050 .030 2X .0625 .0575 .065 .055 .030 .010 47 43 SECTION A-A 32 .001 A 63 63 8 7 6 5 4 3 2 1 9 10 11 E D C G H J K L 4. PART MARKING USING BAG & TAG. 1.750 1.730 1.1909 1.1901 .380 .370 A 63 .010 A .002 A A A 1.0015 1.0005 .030 .020 R .510 .490 1.010 .990 31 29 31 29 16 14 .050 .030 2X .0625 .0575 .065 .055 .030 .010 47 43 SECTION A-A 32 .001 A 63 63 A 1.0015 1.0005 .030 .020 R .510 .490 1.010 .990 31 29 31 29 16 14 .050 .030 2X .0625 .0575 .065 .055 .030 .010 47 43 SECTION A-A 32 .001 A 63 63 SECTION A-A 12 NOTES: E D C B A G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 1 10-1000-00 HYPERLOOP 10/3/2016 KLIM G - - KLIM G 0 lbs in TORONTO X X X 40-1309-00 -HDWS SHIM STOCK, MOTOR BRACKET MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 3 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: -01 .005 -02 .006 -03 .007 -04 .008 -05 .010 -06 .012 -07 .015 -08 .020 -09 .025 -10 .031 NOTES: 1. MATERIAL: C260 BRASS HALF HARD SHIM STOCK. 2 MAKE QTY 20 FROM EACH OF THE FOLLOWING STOCK THICKNESSES: 3. BAG AND TAG EACH STOCK THICKNESS. 1. MATERIAL: C260 BRASS HALF HARD SHIM STOCK. NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 REF ONLY SCALE: NONE 3D VIEW PART # THICK. NOTES: 1. MATERIAL: ALUMINUM 6061-T6511 (BAR) PER AMS QQ-A-200/8. 8 7 6 5 4 3 2 9 10 11 12 E D C B G H J K L ZING ALL OVER. MARKING USING SCRIBING OR PUNCH. NOTES: T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 1 1 35-1400-01 HYPERLOOP 6/28/2016 KLIM G ELAHI M - KLIM G 0.32 lbs in TORONTO X X X 40-1401-01 -HDWS LOWER TORQUE LINK MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 2 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 4.000 .410 .390 R 2x FULL RAD 4 A A REF ONLY SCALE: NONE 3D VIEW .3753 .3743 IN-LINE .290 .270 4x .4379 .4369 IN-LINE 1.923 1.917 .823 .817 .040 .020 4X .255 .245 R 2X .260 .240 R 2X .810 .790 2X INT 1.320 1.280 2X INT 1.310 1.290 1.420 1.380 2.010 1.990 R 2X 5.010 4.990 R 2X 17 15 2X 17 15 2.020 1.980 R 2x .770 .750 2x INT SECTION A-A A .005 A B .005 B .002 A .002 A C .002 C .002 C 32 32 D C B NG ALL OVER. NOTES: RKING USING SCRIBING OR PUNCH. 4.000 .410 .390 R 2x FULL RAD 4 A A REF ONLY SCALE: NONE 3D VIEW 8 7 6 5 4 3 2 1 9 10 11 12 E D C B G H J K L 3 4 PART MARKING USING SCRIBING OR PUNCH. NOTES: 1 0 0 m m 0 4.000 .410 .390 R 2x FULL RAD 4 A A .3753 .3743 IN-LINE .290 .270 4x .4379 .4369 IN-LINE 1.923 1.917 .823 .817 .040 .020 4X .255 .245 R 2X .260 .240 R 2X .810 .790 2X INT 1.320 1.280 2X INT 1.310 1.290 1.420 1.380 2.010 1.990 R 2X 5.010 4.990 R 2X 17 15 2X 17 15 2.020 1.980 R 2x .770 .750 2x INT SECTION A-A A .005 A B .005 B .002 A .002 A C .002 C .002 C 32 32 4.000 .410 .390 R 2x FULL RAD 4 A A REF ONL SCALE: NO 3D VI REF ONLY SCALE: NONE .3753 .3743 IN-LINE .290 .270 4x .4379 .4369 IN-LINE 1.923 1.917 .823 .817 .040 .020 4X .255 .245 R 2X .260 .240 R 2X .810 .790 2X INT 1.320 1.280 2X INT 1.310 1.290 1.420 1.380 2.010 1.990 R 2X 5.010 4.990 R 2X 17 15 2X 17 15 2.020 1.980 R 2x .770 .750 2x INT A .005 A B .005 B .002 A .002 A C .002 C .002 C 32 32 H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 1 1 35-1400-01 HYPERLOOP 6/28/2016 KLIM G ELAHI M - KLIM G 0.32 lbs in TORONTO X X X 40-1401-01 -HDWS LOWER TORQUE LINK MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 2 : 1 2 0 1 6 C I s . NOTES: E d . 2 MAKE QTY 20 FROM EACH OF THE FOLLOWING STOCK THICKNESSES: 8 7 6 5 4 3 2 1 9 10 11 12 E G H J K L 1.405 1.395 .265 .255 2X R FULL RAD 2X .285 .265 2X 2 1.405 1.395 .265 .255 2X R FULL RAD 2X .285 .265 2X 2 1.405 1.395 .265 .255 2X R FULL RA 2X .285 .265 2X 12 8 7 6 5 4 9 10 11 12 13 14 15 16 D C B A -T6511 (BAR) PER AMS QQ-A-200/8. /.015, UNLESS OTHERWISE INDICATED. BING OR PUNCH. 4.000 .410 .390 R 2x FULL RAD 4 A A REF ONLY SCALE: NONE 3D VIEW NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 REF ONLY SCALE: NONE 3D VIEW NOTES: 1. MATERIAL: ALUMINUM 6061-T6511 (BAR) PER AMS QQ-A-200/8. 4. THREADS PER AS8879 5 PART MARKING USING SCRIBING OR PUNCH. 5 C .385 .365 R 2X FULL RAD A A 2.500 .040 .020 4X .3753 .3743 1.043 1.037 .558 .552 2.010 1.990 R 4X .3753 .3743 .595 .555 2X INT .660 .640 2X INT 11 10 2X 46 44 4x .510 .490 .510 .490 .1380 2X -32 UNJC-3B SECTION A-A A .005 B B .010 A-B C .002 A .002 A BOTH SIDES BOTH SIDES THRU ALL, FULL THREAD .005 A H. NOTES: 5 C .385 .365 R 2X FULL RAD A A A A 2.500 .040 .020 4X .3753 .3743 1.043 1.037 .558 .552 2.010 1.990 R 4X .3753 .3743 .595 .555 2X INT .660 .640 2X INT 11 10 2X 46 44 4x .510 .490 .510 .490 .1380 2X -32 UNJC-3B SECTION A-A A .005 B B .010 A-B C .002 A .002 A BOTH SIDES BOTH SIDES THRU ALL, FULL THREAD .005 A .002 A BOTH SIDES H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D X N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 1 1 35-1402-02 HYPERLOOP 11/22/2016 KLIM G ELAHI M - KLIM G 0.16 lbs in TORONTO X X X 40-1402-02 -HDWS UPPER TORQUE LINK MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 3 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 SECTION A-A 3 14 15 16 E D C B A G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D X N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 1 1 35-1402-02 HYPERLOOP 11/22/2016 KLIM G ELAHI M - KLIM G 0.16 lbs in TORONTO X X X 40-1402-02 -HDWS UPPER TORQUE LINK MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 3 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 2. DEBURR SHARP EDGES .005/.015, UNLESS OTHERWISE INDICATED. NOTES: 11 10 9 8 7 6 12 13 14 15 16 17 18 H G F E K L M N P Q R S T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R E X N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 1 2 30-1501-02 HYPERLOOP 6/10/2016 KLIM G ELAHI M SKA KLIM G 1.25 lbs in TORONTO X X X 40-1501-02 -HDWS AXLE , AXLE ASSEMBLY MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O M A M E L I O R A T I O N 2 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 2 6/28/2016 KLIM G 2 1 13/6/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 D E S I G N E T U D E S 2 I s . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 SECTION A-A 12 NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 2 6/28/2016 KLIM G 2 1 13/6/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 D E S I G N E T U D E S A .010 A 32 5 A MAJOR DIAMETER .002 B .9995 .9990 .270 .260 THRU ALL .051 .031 2X 46 44 4X 75 -16 UN-3A .847 .837 2X A A B B SECTION A-A .005 A .635 .615 .060 .040 R 4X .139 .119 2X INT 4.720 4.710 2X 4.885 4.865 2X 4.225 4.215 2X .035 .025 2X 16 14 2X .210 .150 R 2X .190 .140 2X .020 .010 R 2X .021 .011 R 8X .187 .167 2X .420 .410 2X .094 .084 2X A .010 A 32 .005 A MAJOR DIAMETER B .002 B .9995 .9990 .270 .260 THRU ALL .051 .031 2X 46 44 4X .9375 2X -16 UN-3A .847 .837 2X A A B B .021 .011 R 8X .187 .167 2X .420 .410 2X .094 .084 2X 010 A 165 155 THRU ALL A .010 A 32 .005 A MAJOR DIAMETER B .002 B .9995 .9990 .270 .260 THRU ALL .051 .031 2X 46 44 4X .9375 2X -16 UN-3A .847 .837 2X A A B B SECTION A-A .005 A .635 .615 .060 .040 R 4X .139 .119 2X INT 4.720 4.710 2X 4.885 4.865 2X 4.225 4.215 2X .035 .025 2X 16 14 2X .210 .150 R 2X .190 .140 2X .020 .010 R 2X SECTION B-B 2 PLACES .010 A 61 59 6X .165 .155 6X THRU ALL 1 9 A 31 29 .005 A MAJOR DIAMETER .9375 2X -16 UN-3A B .010 A .165 .155 6X THRU ALL A P Q R S T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R E X N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 1 2 30-1501-02 HYPERLOOP 6/10/2016 KLIM G ELAHI M SKA KLIM G 1.25 lbs in TORONTO X X X 40-1501-02 -HDWS AXLE , AXLE ASSEMBLY MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O M A M E L I O R A T I O N 2 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 6/10/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 REF ONLY SCALE: NONE 3D VIEW E D G H J K L 2 3 1.385 1.365 .156 ( ) 1.010 ( ) .424 ( ) .125 ( ) 2 3 1.385 1.365 .156 ( ) 1.010 ( ) .424 ( ) .125 ( ) G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D X N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 1 30-1500-02 HYPERLOOP 6/10/2016 KLIM G N/A SKA KLIM G 0.025 lbs in TORONTO X X X 40-1503-00 AXLE ASSEMBLY -HDWS WASHER, KEY, RETAINING MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 4 : 1 2 0 1 6 C I s . NOTES: E d . 8 8 7 7 6 6 5 5 4 4 3 3 2 2 1 1 9 10 11 9 10 11 E D C B A G H J K L NOTES: 1. MAKE FROM MS21258C16 (CADMIUM PLATED 1018 STEEL) 2 DIMENSION TO BE MET AFTER MODIFICATION 3 BREAK SHARP EDGES 0.05/0.015 4. PART MARKING USING BAG & TAG OR FIXED TAG. 2 3 1.385 1.365 .156 ( ) 1.010 ( ) .424 ( ) .125 ( ) 16 NOTES: E d . NOTES: E D C B A G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D X N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 3 1 30-1500-02 HYPERLOOP 6/10/2016 KLIM G N/A SKA KLIM G 0.025 lbs in TORONTO X X X 40-1503-00 AXLE ASSEMBLY -HDWS WASHER, KEY, RETAINING MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 4 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 1. MATERIAL: 303 STAINLESS STEEL. 2. DEBURR SHARP EDGES .005/.015, UNLESS OTHERWISE INDICATED. 3. PART MARKING USING BAG & TAG. NOTES: REF ONLY SCALE: NONE 8 7 6 5 4 3 2 9 10 11 12 E D G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 1 1 20-2000-00 HYPERLOOP 1/12/2016 KLIM G - - KLIM G 0.75 lbs in TORONTO X X X 40-2102-00 MOTOR ASSEMBLY - HDWS SPUR GEAR, PINNION MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 2 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 6/10/2016 KLIM G - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 12 NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 .180 .120 R A A .164 -32, THRU ALL, FULL THREAD 1.210 1.190 1.070 1.030 .660 .640 .810 .790 SECTION VIEW A-A .005 A B B 2.172 2.162 MAJOR DIAMETER .5483 .5458 .4735 .4725 .1580 .1570 .0790 .0785 PUR GEAR: IAMETRAL PITCH: 12 o OF TEETH: 24 RESSURE ANGLE: 20 ITCH DIAMETER: 2 in ASE CIRCLE DIAMETER: 1.8794 in IRCULAR PITCH: .2618 in OOTH THICKNESS: .1309 in DDENDUM: .0833 in EDENDUM: .1042 in EAR FILLET RADIUS: .0250 in .002 A A SCALE: NONE A 1.070 1.030 A SECTION VIEW A-A This page was intentionally left blank 166 166 This page was intentionally left blank 167 8 7 6 5 4 3 2 9 10 11 12 13 14 15 16 E D C B A G H J K L S: E FROM DELRIN. URR SHARP EDGES .005/.015, UNLESS OTHERWISE INDICATED. 8 7 6 5 4 3 2 1 9 10 11 12 E D C B A G H J K L 1. MAKE FROM DELRIN. 2. DEBURR SHARP EDGES .005/.015, UNLESS OTHERWISE INDICATED. .770 .730 2.020 1.980 0.010 A A A .927 .925 .180 .120 R .180 .120 R 22 18 .200 .160 SECTION A-A 32 32 32 32 A 2. DEBURR SHARP EDGES .005/.015, UNLESS OTHERWISE INDICATED. 8 7 6 5 4 3 2 1 9 10 11 12 E D C B A G H J K L 1. MAKE FROM DELRIN. 2. DEBURR SHARP EDGES .005/.015, UNLESS OTHERWISE INDICATED. .770 .730 2.020 1.980 0.010 A A A .927 .925 .180 .120 R .180 .120 R 22 18 .200 .160 SECTION A-A 32 32 32 32 A REF ONLY SCALE: NONE REF ONLY SCALE: NONE G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 NA 1 - HYPERLOOP 7/8/2016 KLIM G N/A - KLIM G 0 lbs in TORONTO X X X ST-FT-2 FILLER TUBE -HDWS SEAL COMPRESSION TOOL, MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 3 : 1 2 0 1 6 C I s . 8 7 6 5 4 3 2 1 9 10 11 12 E D C B A G H J K L 1. MAKE FROM DELRIN. 2. DEBURR SHARP EDGES .005/.015, UNLESS OTHERWISE INDICATED. .770 .730 2.020 1.980 0.010 A A A .927 .925 .180 .120 R .180 .120 R 22 18 .200 .160 SECTION A-A 32 32 32 32 A E d . This page was intentionally left blank T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 NA - HYPERLOOP 7/8/2016 KLIM G N/A - KLIM G 0 lbs in TORONTO X X X FILLER TUBE -HDWS SEAL COMPRESSION TOOL, MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 3 : 1 2 0 1 6 C 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR .770 .730 2.020 1.980 0.010 A A A REF ONLY SCALE: NONE 3D VIEW 1 REF ONLY SCALE: NONE 3D VIEW 2 .927 .925 .180 .120 R .180 .120 R 22 18 .200 .160 SECTION A-A 32 32 32 32 A NOTES: FROM DELRIN. R SHARP EDGES .005/.015, UNLESS OTHERWISE INDICATED. .770 .730 2.020 1.980 0.010 A A A REF ONLY SCALE: NONE 3D VIEW 1 REF ONLY SCALE: NONE 3D VIEW 2 E D C B A G H J K L 1. MAKE FROM DELRIN. 2. DEBURR SHARP EDGES .005/.015, UNLESS OTHERWISE INDICATED. Y E S O K E C A R D M A X T G E T O L I S a p p l i c G E O M T O L E R A S I F O R C A B 1 0 0 m m 0 .X .X An Ec .770 .730 2.020 1.980 0.010 A A A REF ONLY SCALE: NONE 3D VIEW .927 .925 .180 .120 R .180 .120 R 22 18 .200 .160 SECTION A-A 32 32 32 32 A 8 7 6 5 4 3 2 1 9 10 11 12 E D C B A G H J K L 1. MAKE FROM DELRIN. 2. DEBURR SHARP EDGES .005/.015, UNLESS OTHERWISE INDICATED. .770 .730 2.020 1.980 0.010 A A A .927 .925 .180 .120 R .180 .120 R 22 18 .200 .160 SECTION A-A 32 32 32 32 A 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 12 NOTES: 2. DEBURR SHARP EDGES .005/.015, UNLESS OTHERWISE INDICATED. A SECTION A-A 10 NOTES: 2. DEBURR SHARP EDGES .005/.015, UNLESS OTHERWISE INDICATED. 3.020 2.980 MACHINE TURNING CENTRE AS REQUIRED. A A 1.060 1.058 1.020 .980 R .145 .105 R .588 .568 1.470 1.430 R FULL RAD .298 .292 .265 .255 R 2X .030 .000 R ALL AROUND 1.670 1.630 SECTION A-A 16 16 32 16 0.001 A A 0.005 A DRILL DEPTH 16 14 0.005 A .801 .781 INT 3.020 2.980 INE TURNING CENTRE AS REQUIRED. A A MACHINE TURNING CENTRE AS REQUIRED. NOTES: A A H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 NA 1 N/A HYPERLOOP 7/8/2016 KLIM G N/A - KLIM G 0 lbs in TORONTO X X X ST-PG-1 PISTON GLAND -HDWS SEAL RESIZING TOOL, MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 2 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 16 R FULL RAD ST-PG-1 8 8 7 7 6 6 5 5 4 4 3 3 2 2 9 10 11 12 13 14 15 16 9 10 11 12 E D C B A G H J K L DELRIN. NOTES: ARP EDGES .005/.015, UNLESS OTHERWISE INDICATED. NOTES: T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 NA 1 N/A HYPERLOOP 7/8/2016 KLIM G N/A - KLIM G 0 lbs in TORONTO X X X ST-PG-2 PISTON GLAND -HDWS SEAL COMPRESSION SLEEVE MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 3 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: E D C B G H J K L 2. DEBURR SHARP EDGES .005/.015, UNLESS OTHERWISE INDICATED. 2.020 1.980 1.020 .980 0.010 A A A 1.493 1.491 22 18 .180 .120 R .180 .120 R .260 .240 SECTION A-A 32 32 32 32 A 2. DEBURR SHARP EDGES .005/.015, UNLESS OTHERWISE INDICATED. 2. DEBURR SHARP EDGES .005/.015, UNLESS OTHERWISE INDICATED. NOTES: REF ONLY SCALE: NONE REF ONLY SCALE: NONE G 1.493 1.491 22 18 .180 .120 R .180 .120 R .260 .240 SECTION A-A 32 32 32 32 A H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 NA 1 N/A HYPERLOOP 7/8/2016 KLIM G N/A - KLIM G 0 lbs in TORONTO X X X ST-PG-2 PISTON GLAND -HDWS SEAL COMPRESSION SLEEVE MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 3 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 2.020 1.980 1.020 .980 0.010 A A A 1.493 1.491 22 18 .180 .120 R .180 .120 R .260 .240 SECTION A-A 32 32 32 32 A REF ONLY SCALE: NONE 3D VIEW 1 REF ONLY SCALE: NONE 3D VIEW 2 NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 SECTION A-A This page was intentionally left blank 172 16 16 15 15 14 14 13 13 12 12 11 11 10 10 9 9 8 8 7 7 6 6 5 5 4 4 3 3 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 17 18 19 20 21 22 23 24 25 26 27 28 ING CONSTRUCTION GRADE STAINLESS STEEL (OR EQUIVALENT) D SCREWS. NTERFACE HOLES ARE APROPRIATELY PRE-DRILLED WHERE TOTAL SCREW DEPTH OR GREATER. 1. FABRICATE USING CONSTRUCTION GRADE STAINLESS STEEL (OR EQUIVALENT) PHILLIPS HEAD SCREWS. This page was intentionally left blank 0 1 0 0 m m P R O D U C T A C C E P T A N C E T E S T S C H E D U L E I N A C C O R D A N C E W I T H P A R T N U M B E R : C O N D I T I O N S D E R E C E P T I O N S U I V A N T R E F E R E N C E : P R O G R A M M E P R O G R A M M E P A R R E F E R E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T S C A L E E C H E L L E S I Z E F O R M A T S H E E T P L A N C H E T I T L E T I T R E P A R T N U M B E R R E F E R E N C E 3 5 9 6 2 C 2 0 1 6 1 : 1 A M E L I O R A T I O N A M E N D M E N T A M E N D E M E N T D E S I G N O F F I C E B U R E A U D ' E T U D E S D R A W N B Y D E S S I N E P A R C H E C K E D B Y V E R I F I E P A R D A T E D E S I G N E T U D E S U N I T U N I T E S T R E S S C A L C U L (sauf indications particulières): (unless otherwise stated): Unit / Unité KLIM G - - KLIM G 18/09/2016 0 lbs TORONTO HYPERLOOP - TRANSPORTATION FIXTU - HDWS TF-1000-00 1 : 1 M A X N O M I N A L C A L C U L A T E D W E I G H T M A S S E C A L C U L E E J D E S I G N E T U D E S S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R - - - 1 in C L A S S I F I C A T I O N NA - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION AMENDMENT AMENDEMENT MODIFICA FICHE D'AP Y E S O U I N O N O N X K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 0 1 0 0 m m P A R T N U M B E R R E F E R E N C E I s s u e E d i t i o n S H E E T P L A N C H E V I E W V U E TF-1000-00 1 1 2 1 2 P/N BASE PLATE, TF-1101-00 8X 2 4.40 ±.50 8.75 ±.50 5.00 ±.25 REF ONLY SCALE: NONE 3D VIEW 2 P/N HOUSING SUPPORT, TF-1102-00 (QTY 2) P/N T-ARM SUPPORT, TF-1103-00 (QTY 2) P/N HORIZONTAL SUPPORT, TF-1104-00 (QTY 4) 8X 2 4.14 ±.01 7.66 ±.01 1.463 2X ( ) REF ONLY SCALE: NONE 3D VIEW 1 1. This page was intentionally left blank FABRICATE USING CONSTRUCTION GRADE STAINLESS STEEL (OR EQUIVALENT) PHILLIPS HEAD SCREWS. 2 ENSURE ALL INTERFACE HOLES ARE APROPRIATELY PRE-DR MARKED, 3/4 TOTAL SCREW DEPTH OR GREATER. P/N HOUSING SUPPORT, TF-1102-00 (QTY 2) REF ONLY SCALE: NONE 3D VIEW 1 P/N BASE PLATE, TF-1101-00 8X 2 4.40 ±.50 8.75 ±.50 5.00 ±.25 1 2X ( 4.40 ±.50 8.75 ±.50 8X 2 NOTES: 1. MAKE FROM 3/4" PLYWOOD, MAPLE OR BALTIC BIRCH. 1. MAKE FROM 3/4" PLYWOOD, MAPLE OR BALTIC BIRCH. 1. MAKE FROM 3/4" PLYWOOD, MAPLE OR BALTIC BIRCH. 2 MAY BE PRE-DRILLED AS SHOWN OR MARKED USING AN AGREED METHOD. NOTES: .709 (18 MM STANDARD PLY) 22.000 ±.500 22.000 ±.500 REF ONLY SCALE: NONE 2 000 12.00 ±.25 3.750 ±.020 4.849 ±.020 5.000 ±.020 8.359 ±.020 3.125 ±.010 3.576 1.755 ±.010 2X .625 ±.010 2X .050 ±.030 8X 2 3.750 ±.020 4.849 ±.020 5.000 ±.020 8.359 ±.020 3.125 ±.010 3.576 1.755 ±.010 2X .625 ±.010 2X .050 ±.030 8X .625 ±.010 2X 12 12 13 14 15 16 12 E D C B A G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X N/A NA 1 TF-1000-00 HYPERLOOP 18/09/16 KLIM G - - KLIM G 0 lbs in TORONTO X X X TF-1102-00 -TRANSPORTATION FIXTURE HOUSING SUPPORT MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 1 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 .709 (18 MM STANDARD PLY) REF ONLY SCALE: NONE 3D VIEW NOTES: 1. MAKE FROM 3/4" PLYWOOD, MAPLE OR BALTIC BIRCH. 2 MAY BE PRE-DRILLED AS SHOWN OR MARKED USING AN AGREED METHOD. 8 7 6 5 4 3 2 9 10 11 5.0 ±1.0 R .500 ±.100 R 3X 5.407 ±.020 1.250 ±.020 3.445 ±.020 57.5 ±1 2.000 1.000 STANDARD TOOLING TOLERANCES ACCEPTED 1.000 ±.020 R 80 ±1 12.050 ±.010 6.291 INT 57.5 ±1 1.250 ±.010 1.000 ±.020 3.000 ±.020 .500 ±.020 1.650 ±.020 2 .050 ±.030 4X 1.250 ±.010 1.000 ±.020 R G .500 ±.100 R 3X 6.291 INT 2.000 2 .050 ±.030 4X 8 10 11 6 12 NOTES: 1. MAKE FROM 3/4" PLYWOOD, MAPLE OR BALTIC BIRCH. 1. MAKE FROM 3/4" PLYWOOD, MAPLE OR BALTIC BIRCH. 2 MAY BE PRE-DRILLED AS SHOWN OR MARKED USING AN AGREED METHOD. NOTES: 1.000 STANDARD TOOLING TOLERANCES ACCEP 1.500 ±.020 1.000 ±.020 3.000 ±.020 .050 ±.020 4X .500 ±.020 1.000 ±.020 R 60 ±0.5 2X 3.300 ±.010 6.120 ±.050 2 1.000 STANDARD TOOLING TOLERANCES ACCEP 1.500 ±.020 1.000 ±.020 3.000 ±.020 .050 ±.020 4X .500 ±.020 1.000 ±.020 R 60 ±0.5 2X 3.300 ±.010 6.120 ±.050 2 1.000 STANDARD TOOLING TOLERANCES ACCEPTED .709 (18 MM STANDARD PLY) H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X N/A NA 1 TF-1000-00 HYPERLOOP 18/09/16 KLIM G - - KLIM G 0 lbs in TORONTO X X X TF-1103-00 -TRANSPORTATION FIXTURE T-ARM LUG MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 1 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 1. MAKE FROM 3/4" PLYWOOD, MAPLE OR BALTIC BIRCH. 2 MARKED USING AN AGREED METHOD, PRE-DRILL NOT REQUIRED. NOTES: 8 7 6 5 4 3 2 1 9 10 11 12 E G H J K L 1.050 ±.020 2.000 ±.020 .709 (18 MM STANDARD PLY) .354 ±.020 1.000 ±.020 .050 4X (BOTH SIDES) .500 ±.020 STANDARD TOOLING TOLERANCES ACCEPTED 2 1.050 ±.020 2.000 ±.020 .709 (18 MM STANDARD PLY) .354 ±.020 1.000 ±.020 .050 4X (BOTH SIDES) .500 ±.020 STANDARD TOOLING TOLERANCES ACCEPTED 2 .050 4X (BOTH SIDES) STANDARD TOOLING TOLERANCES ACCEPTED 2 .050 4X (BOTH SIDES) STANDARD TOOLING TOLERANCES ACCEPTED 2 G .709 (18 MM STANDARD PLY) 1.050 ±.020 2.000 ±.020 H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X N/A NA 1 TF-1000-00 HYPERLOOP 18/09/16 KLIM G - - KLIM G 0 lbs in TORONTO X X X TF-1104-00 -TRANSPORTATION FIXTURE CONNECTOR MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 1 : 1 2 0 1 6 C I s . NOTES: E d . 8 8 9 10 11 12 13 14 15 16 9 10 11 12 E D C B A G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X N/A NA 1 TF-1000-00 HYPERLOOP 18/09/16 KLIM G - - KLIM G 0 lbs in TORONTO X X X TF-1104-00 -TRANSPORTATION FIXTURE CONNECTOR MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 1 : 1 2 0 1 6 C I s . E d . 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 .709 (18 MM STANDARD PLY) .354 ±.020 1.000 ±.020 .500 ±.020 REF ONLY SCALE: NONE 3D VIEW 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 REF ONLY SCALE: NONE 3D VIEW 8 8 9 10 11 12 13 14 15 16 9 10 11 12 E D C B A G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X N/A NA 1 TF-1000-00 HYPERLOOP 18/09/16 KLIM G - - KLIM G 0 lbs in TORONTO X X X TF-1104-00 -TRANSPORTATION FIXTURE CONNECTOR MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 1 : 1 2 0 1 6 C I s . E d . 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 .709 (18 MM STANDARD PLY) .354 ±.020 1.000 ±.020 .500 ±.020 REF ONLY SCALE: NONE 3D VIEW E D C B A G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X N/A NA 1 TF-1000-00 HYPERLOOP 18/09/16 KLIM G - - KLIM G 0 lbs in TORONTO X X X TF-1104-00 -TRANSPORTATION FIXTURE CONNECTOR MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 1 : 1 2 0 1 6 C I s . 8 8 9 10 11 12 13 14 15 16 9 10 11 12 E D C B A G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X N/A NA 1 TF-1000-00 HYPERLOOP 18/09/16 KLIM G - - KLIM G 0 lbs in TORONTO X X X TF-1104-00 -TRANSPORTATION FIXTURE CONNECTOR MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 1 : 1 2 0 1 6 C I s . E d . 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 .709 (18 MM STANDARD PLY) .354 ±.020 1.000 ±.020 .500 ±.020 REF ONLY SCALE: NONE 3D VIEW E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 3.300 ±.010 1.000 ±.020 .500 ±.020 1.500 ±.020 NOTES: C 2 0 1 6 1 : 1 A M E L I O R A T I O N A M E N D M E N T A M E N D E M E N T D E S I G N O F F I C E B U R E A U D ' E T U D E S D R A W N B Y D E S S I N E P A R C H E C K E D B Y V E R I F I E P A R D A T E D E S I G N E T U D E S U N I T U N I T E S T R E S S C A L C U L (sauf indications particulières): (unless otherwise stated): Unit / Unité KLIM G - - KLIM G 8/5/2016 0 lbs TORONTO HYPERLOOP - 1 WHEEL TEST RIG - HDWS WTR-1000-00 1 : 1 N/A M A X N O M I N A L C A L C U L A T E D W E I G H T M A S S E C A L C U L E E J D E S I G N E T U D E S S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R - - - 1 in D A T E C L A S S I F I C A T I O N NA - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION AMENDMENT AMENDEMENT MODIFICATION APPROVAL FORM / EDES FICHE D'APPROBATION DE MODIFICATION Y E S O U I N O N O N X K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 0 1 0 0 m m P A R T N U M B E R R E F E R E N C E I s s u e E d i t i o n S H E E T P L A N C H E V I E W V U E WTR-1000-00 1 1 2 1 2 1.795 2X ( ) 25.000 2X ( ) 13.030 12.970 4.000 ( ) P/N WTR-1102-00, SUPPORT TUBE P/N WTR-1103-00, DUMMY AXLE 5.000 2X ( ) 2.000 2X ( ) P/N WTR-1101-00, FIXTURE MOUNT PLATE REF ONLY SCALE: NONE 3D VIEW 2 3D VIEW 1 REF ONLY SCALE: NONE P A R T N U M B E R R E F E R E N C E I s s u e E d i t i o n S H E E T P L A N C H E V I E W V U E 1 2 1.795 2X ( ) 25.000 2X ( ) 13.030 12.970 .530 .470 .630 .570 4.000 ( ) 2.410 ( ) WELD JOINT AS REQUIRED WELD JOINT AS REQUIRED, BOTH SIDES P/N WTR-1102-00, SUPPORT TUBE P/N WTR-1102-00, SUPPORT TUBE P/N WTR-1103-00, DUMMY AXLE P/N WTR-1105-00, DUMMY AXLE SPACERS (QTY 2) LOCK JOINT, COMBINATION OF (OR EQUIVALENT): P/N NAS6204-29, BOLT P/N NAS1149C0432R, WASHER (QTY 2) P/N MS14145L4, HEX NUT P/N MS246658-151 LOCK FLANGE, MAKE FROM HS STEEL, MATING BORE .252/0.258. NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 .500 ±.020 .354 ±.020 1.050 ±.020 12 16 16 15 15 14 14 13 13 12 12 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 17 18 19 20 21 22 23 24 25 26 27 28 H G F E D C B A K L M N P Q R S 0 1 0 0 m m P R O D U C T A C C E P T A N C E T E S T S C H E D U L E I N A C C O R D A N C E W I T H P A R T N U M B E R : C O N D I T I O N S D E R E C E P T I O N S U I V A N T R E F E R E N C E : P R O G R A M M E P R O G R A M M E P A R T N U M B E R O F U N I T D R A W I N G R E F E R E N C E D U D E S S I N D E D E F I N I T I O N M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T S C A L E E C H E L L E S I Z E F O R M A T S H E E T P L A N C H E T I T L E T I T R E P A R T N U M B E R R E F E R E N C E 3 5 9 6 2 E d . NOTES: I s . NOTES: 2.015 2.005 A A 2X ( 2.0 2X ( SECTION A-A PARTIAL VIEW ONLY 1.030 .970 WELD JOINT AS REQUIRED, BOTH SIDES H-BRIDGE, SUPPORT TUBE, MAKE FROM STOCK HS STEEL, 2"X4"X1/4" TUBE. 2.000 ( ) P/N WTR-1105-00, DUMMY AXLE SPACERS (QTY 2) .530 .470 .630 .570 WELD JOINT AS REQUIRED P/N WTR-1105-00, DUMMY AXLE SPACERS (QTY 2) LOCK JOINT, COMBINATION OF (OR EQUIVALENT): P/N NAS6204-29, BOLT P/N NAS1149C0432R, WASHER (QTY 2) P/N MS14145L4, HEX NUT P/N MS246658-151 LOCK FLANGE, MAKE FROM HS STEEL, MATING BORE .252/0.258. 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 17 18 19 20 21 22 23 24 25 26 H G F E D C B A K L M N P Q R S 0 1 0 0 m m P A R T N U M B E R R E F E R E N C E I s s u e E d i t i o n S H E E T P L A N C H E V I E W V U E WTR-1000-00 1 1 2 1 2 1.795 2X ( ) 25.000 2X ( ) 13.030 12.970 .530 .470 .630 .570 4.000 ( ) 2.410 ( ) WELD JOINT AS REQUIRED WELD JOINT AS REQUIRED, BOTH SIDES P/N WTR-1102-00, SUPPORT TUBE P/N WTR-1102-00, SUPPORT TUBE P/N WTR-1103-00, DUMMY AXLE P/N WTR-1105-00, DUMMY AXLE SPACERS (QTY 2) LOCK JOINT, COMBINATION OF (OR EQUIVALENT): P/N NAS6204-29, BOLT P/N NAS1149C0432R, WASHER (QTY 2) P/N MS14145L4, HEX NUT P/N MS246658-151 LOCK FLANGE, MAKE FROM HS STEEL, MATING BORE .252/0.258. 2.015 2.005 A A 5.000 2X ( ) 2.000 2X ( ) P/N WTR-1101-00, FI 3D VI REF SCALE: SECTION A-A PARTIAL VIEW ONLY 1.030 .970 WELD JOINT AS REQUIRED, BOTH SIDES H-BRIDGE, SUPPORT TUBE, MAKE FROM STOCK HS STEEL, 2"X4"X1/4" TUBE. 2.000 ( ) NOTES: 1. MAKE FROM STOCK 10 X 14 836 HOT ROLLED STEEL. 1. MAKE FROM STOCK 10 X 14 836 HOT ROLLED STEEL. 2. DEBURR SHARP EDGES .005/.015, UNLESS OTHERWISE INDICATED. NOTES: .8175 .8075 4X 1.000 8.000 .005 A B C 1.000 C 12.000 14.000 ( ) B E D C B G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 1 1 WTR-1000-00 HYPERLOOP 8/5/2016 KLIM G - - KLIM G 0 lbs in TORONTO X X X WTR-1101-00 WHEEL TEST RIG -HDWS PLATE, FIXTURE MOUNT MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 1 : 1 2 0 1 6 C I s . NOTES: E d . 16 15 14 13 12 11 10 9 8 7 6 5 17 18 19 20 21 22 23 24 25 26 27 28 H G F E D C B A K L M N P Q R S 0 1 0 0 m m P R O D U C T A C C E P T A N C E T E S T S C H E D U L E I N A C C O R D A N C E W I T H P A R T N U M B E R : C O N D I T I O N S D E R E C E P T I O N S U I V A N T R E F E R E N C E : P R O G R A M M E P R O G R A M M E P A R T N U M B E R O F U N I T D R A W I N G R E F E R E N C E D U D E S S I N D E D E F I N I T I O N M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T S C A L E E C H E L L E S I Z E F O R M A T S H E E T P L A N C H E T I T L E T I T R E P A R T N U M B E R R E F E R E N C E 3 5 9 6 2 E d . I s . C 2 0 1 6 1 : 1 A M E L I O R A T I O N A M E N D M E N T A M E N D E M E N T D E S I G N O F F I C E B U R E A U D ' E T U D E S D R A W N B Y D E S S I N E P A R C H E C K E D B Y V E R I F I E P A R D A T E D E S I G N E T U D E S U N I T U N I T E S T R E S S C A L C U L (sauf indications particulières): (unless otherwise stated): Unit / Unité KLIM G - - KLIM G 8/5/2016 0 lbs TORONTO HYPERLOOP - 1 WHEEL TEST RIG - HDWS WTR-1000-00 1 : 1 N/A M A X N O M I N A L C A L C U L A T E D W E I G H T M A S S E C A L C U L E E J D E S I G N E T U D E S S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R - - - 1 in D A T E C L A S S I F I C A T I O N NA - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION MODIFICATION AMENDMENT AMENDEMENT MODIFICATION APPROVAL FORM / EDES FICHE D'APPROBATION DE MODIFICATION Y E S O U I N O N O N X K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 0 1 0 0 m m P A R T N U M B E R R E F E R E N C E I s s u e E d i t i o n S H E E T P L A N C H E V I E W V U E WTR-1000-00 1 1 2 1 2 1.795 2X ( ) 25.000 2X ( ) 13.030 12.970 .530 .470 .630 .570 4.000 ( ) 2.410 ( ) WELD JOINT AS REQUIRED EQUIRED, BOTH SIDES P/N WTR-1102-00, SUPPORT TUBE R-1102-00, SUPPORT TUBE P/N WTR-1103-00, DUMMY AXLE 0, DUMMY AXLE SPACERS (QTY 2) NT): EEL, 2.015 2.005 A A 5.000 2X ( ) 2.000 2X ( ) P/N WTR-1101-00, FIXTURE MOUNT PLATE REF ONLY SCALE: NONE 3D VIEW 2 3D VIEW 1 REF ONLY SCALE: NONE 1.030 .970 NT AS REQUIRED, BOTH SIDES SUPPORT TUBE, STOCK HS STEEL, 2"X4"X1/4" TUBE. 2.410 ( ) .530 .470 P/N WTR-1103-00, DUMMY AXLE .630 .570 2.015 2.005 P/N WTR-1102-00, SUPPORT TUBE 1.030 .970 WELD JOINT AS REQUIRED, BOTH SIDES H-BRIDGE, SUPPORT TUBE, MAKE FROM STOCK HS STEEL, 2"X4"X1/4" TUBE. 2.000 ( ) A .030 .970 4.000 ( ) 25.000 2X ( ) 25.000 2X ( ) SECTION A-A PARTIAL VIEW ONLY NOTES: 1. MAKE FROM STOCK HS STEEL, 2"X4"X1/4" TUBE. NOTES: 25.530 25.470 2.010 1.990 1.0015 1.0005 THRU ALL 24.030 23.970 1.500 ( ) 25.530 25.470 4.000 ( ) 24.030 23.970 24.030 23.970 N P Q R S T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R E N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 1 1 WTR-1000-00 HYPERLOOP 8/5/2016 KLIM G - - KLIM G 0 lbs in TORONTO X X X WTR-1102-00 WHEEL TEST RIG -HDWS SUPPORT TUBE MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O M A M E L I O R A T I O N 1 : 1 2 0 1 6 C I s . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 .8175 .8075 4X 10.000 ( ) 1.000 8.000 .005 A B C 1.000 12.000 14.000 ( ) B A 1.000 ( ) REF ONLY SCALE: NONE 3D VIEW 1.000 ( ) REF ONLY SCALE: NONE 11 12 NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 P Q R S T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R E N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 1 1 WTR-1000-00 HYPERLOOP 8/5/2016 KLIM G - - KLIM G 0 lbs in TORONTO X X X WTR-1102-00 WHEEL TEST RIG -HDWS SUPPORT TUBE MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O M A M E L I O R A T I O N 1 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 WTR-1102-00 1 I s . NOTES: E d . 8 7 6 5 4 3 2 9 10 11 12 13 14 15 16 E D C B A G H J K S: E FROM O1 TOOL STEEL, BAR STOCK, PER ASTM A681 OR EQUIVALENT. URR SHARP EDGES .005/.015, UNLESS OTHERWISE INDICATED. NOTES: T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S S T R E S S - C H E C K E D B Y V E R I F I E P A R Y E S O U I A S M E A S M E I S O I S O A S M E X 125 1 WTR-1000-00 HYPERLOOP - KLIM G TORONTO X X X MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N S T R E S S C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R C A L C U L A T E D W E I G H T D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR .9995 .9985 .115 .085 2X 16 14 2X .130 .070 R 2X 32 A A .258 .252 THRU ALL 8.030 7.970 .393 .373 SECTION A-A .002 A OPTIONAL SECOND BORE, NOT REQUIRED. NOTES: REF ONLY SCALE: NONE 3D VIEW NOTES: E D C B A G H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 1 1 WTR-1000-00 HYPERLOOP KLIM G - - KLIM G 0 lbs in TORONTO X X X WTR-1105-00 WHEEL TEST RIG -HDWS SPACER, DUMMY AXLE MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 4 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 REF ONLY SCALE: NONE 3D VIEW 8 7 6 5 4 3 2 1 9 10 E D C B A G H J K L 1. NOTES: MAKE FROM DELRIN. 2. DEBURR SHARP EDGES .005/.015, UNLESS OTHERWISE INDICATED. NOTES: 47 43 1.520 1.480 .135 .115 A A SECTION A-A .205 .195 .040 .020 2X 32 28 2X 1.0015 1.0005 47 43 1.520 1.480 .135 .115 A A H J K L T r a c e a b i l i t y a n d m a r k i n g f o l l o w i n g X N O N O N Y E S O U I K E Y C H A R A C T E R I S T I C S C A R A C T E R I S T I Q U E S C L E S 1 - D E S I G N E T U D E S - S T R E S S C A L C U L - C H E C K E D B Y V E R I F I E P A R D N O M I N A L M A X Y E S O U I A S M E A S M E I S O I S O A S M E X 125 1 1 WTR-1000-00 HYPERLOOP KLIM G - - KLIM G 0 lbs in TORONTO X X X WTR-1105-00 WHEEL TEST RIG -HDWS SPACER, DUMMY AXLE MAXIMUM AND LEAST MATERIAL PRINCIPLE PRINCIPE DU MAXIMUM ET MINIMUM MATIERE U N I T U n l e s s o t h e r w i s e i n d i c a t e d , R a : S a u f i n d i c a t i o n s c o n t r a i r e s , R a : Unit / Unité G E N E R A L T O L E R A N C E S T O L E R A N C E S G E N E R A L E S a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : S U R F A C E T E X T U R E E T A T D E S U R F A C E I S O a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : a p p l i c a b l e w i t h s t a n d a r d / a v e c n o r m e : D E S I G N E T U D E S G E O M E T R I C A L T O L E R A N C I N G T O L E R A N C E M E N T G E O M E T R I Q U E M A M E L I O R A T I O N 4 : 1 2 0 1 6 C I s . NOTES: E d . NOTES: 1. MAKE FROM O1 TOOL STEEL, BAR STOCK, PER ASTM A681 OR EQUIVALENT. 2. DEBURR SHARP EDGES .005/.015, UNLESS OTHERWISE INDICATED. 1. MAKE FROM O1 TOOL STEEL, BAR STOCK, PER ASTM A681 OR EQUIVALENT. 1. MAKE FROM O1 TOOL STEEL, BAR STOCK, PER ASTM A681 OR EQUIVALENT. 2. DEBURR SHARP EDGES .005/.015, UNLESS OTHERWISE INDICATED. A A A A 32 8.030 7.970 SECTION A-A WTR-1103-00 NOTES: 3 5 9 6 2 D A T E D A T E S T R E S S C A L C U L C H E C K E D B Y V E R I F I E P A R D R A W N B Y D E S S I N E P A R P A R T N U M B E R R E F E R E N C E T I T L E T I T R E S H E E T P L A N C H E S I Z E F O R M A T S C A L E E C H E L L E M A N U F A C T U R E R C O D E - C O D E F A B R I C A N T C A L C U L A T E D W E I G H T M A S S E C A L C U L E E D E S I G N O F F I C E B U R E A U D ' E T U D E S P A R T N U M B E R O F A S S E M B L Y R E F E R E N C E D E L ' E N S E M B L E P R O G R A M M E P R O G R A M M E N O N O N A R T I C L E O F A P P R O V E D M A N U F A C T U R E A R T I C L E A F A B R I C A T I O N A P P R O U V E E C L A S S I F I C A T I O N 1 0 0 m m 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 - - - ISSUE EDITION DATE NAME / NOM ISUM FICHETTE D'EVOLUTION .XX = .030 .XXX = .010 Angle = .5 Eccen = .002 TIR 1 SECTION A-A 12 Appendix D: Gas Spring Test Result Data The gas spring test data as recorded during testing is presented here, as recorded during the test. Two data samples are provided as recorded for HDWS #4 and HDWS#5 here below. For reference, the gas spring data, as calculated by Dynatool© [60], is presented here with the data collected during test shown below marked in pen under the “static load column,” measured at pre- determined intervals as shown in the figure below. The first page for test is shown, and then only relevant pages corresponding to values recorded in the scan below are shown thereafter. 183 Gas spring data recorded for HDWS #4: Pg 1 of 7 Gas spring data recorded for HDWS #4: Pg 1 of 7 Gas spring data recorded for HDWS #4: Pg 1 of 7 184 Gas spring data recorded for HDWS #4: Pg 3 of 7 Gas spring data recorded for HDWS #4: Pg 3 of 7 185 Gas spring data recorded for HDWS #4: Pg 5 of 7 Gas spring data recorded for HDWS #4: Pg 5 of 7 186 Gas spring data recorded for HDWS #4: Pg 6 of 7 187 Gas spring data recorded for HDWS #4: Pg 7 of 7 End of gas spring test data, HDWS #4. End of gas spring test data, HDWS #4. 188 188 Appendix E: Retractable Shock Strut Concept Art 189 190 190 191 Appendix F: Galvanic Chart Metals compatibility chart as found in the Unbrako catalogue [32] Metals compatibility chart as found in the Unbrako catalogue [32] 192 References [1] E. Musk, "Hyperloop Alpha," Space Exploration Technologies Corp., 2013. [2] Hyperloop One, "Fact Sheet And FAQ," [Online]. Available: https://hyperloop- one.com/fact-sheet-and-faq. [Accessed 28 8 2017]. [3] SpaceX, "Hyperloop," Space Exploration Technologies Corp., [Online]. Available: http://www.spacex.com/hyperloop. [Accessed 8 8 2017]. [4] SpaceX, "SpaceX Hyperloop Test-Track Specification," Space Exploration Technologies Corp., Hawthorne, 2016. [5] N. S. Currey, Aircraft Landing Gear Design: Principles and Practices, Washington DC: AIAA, 1988. [6] H. G. Conway, Landing Gear Design, London: Chapman & Hall, 1958. [7] R. C. Churchill and W. E. 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https://openalex.org/W4391929176	https://link.springer.com/content/pdf/10.1007/s10346-024-02216-w.pdf	English	null	Geomorphological assessment as basic complement of InSAR analysis for landslide processes understanding	Landslides	2,024	cc-by	16,908	Davide Torre · Jorge Pedro Galve · Cristina Reyes‑Carmona · David Alfonso‑Jorde · Daniel Ballesteros · Marco Menichetti · Daniela Piacentini · Francesco Troiani · José Miguel Azañón Geomorphological assessment as basic complement of InSAR analysis for landslide processes understanding Davide Torre · Jorge Pedro Galve · Cristina Reyes‑Carmona · David Alfonso‑Jorde · Daniel Ballesteros · Marco Menichetti · Daniela Piacentini · Francesco Troiani · José Miguel Azañón Geomorphological assessment as basic complement of InSAR analysis for landslide processes understanding Abstract Landslide research has benefited greatly from advances in remote sensing techniques. However, the recent increase in available data on land surface movement provided by InSAR tech- niques can lead to identifying only those areas that were active during data acquisition as hazardous, overlooking other potentially unsafe areas or neglecting landslide-specific geological settings in hazard assessments. Here, we present a case study that serves as a reminder for landslide researchers to carefully consider the geology and geomorphology of study areas where complex active move- ments are detected using InSAR technology. In an area extensively studied using InSAR and UAV-related techniques, we provide new insights by applying classical approaches. The area is the coastal stretch of La Herradura, and its importance lies in the fact that it has served as an illustrative example in the Product User Manual of the European Ground Motion Service, a platform that provides ground motion data on a European scale. Our approach is to revisit the area and carry out qualitative geological and geomorphological assessments supported by UAV surveys and GIS spatial analysis on a broader scale than previously published investigations. Our clas- sical approach has yielded the following new observations, crucial for risk assessment and land management: active landslides identi- fied by InSAR techniques since 2015 are bodies nested within large mass movements that affect entire slopes. A variety of processes contribute to slope dynamics, such as large slumps, marble rock spreading and block sliding, and surface rock falls and topples. The revised delineation of the landslide bodies reveals an area almost five times larger than previously mapped. These new findings in a well-known area highlight (1) the importance of updating and downscaling previous maps and (2) the ongoing importance of classical fieldwork and desk studies as basic complements to mod- ern InSAR analyses. Synthetic Aperture Radar Interferometry (InSAR) data (e.g., Cigna et al. 2011; Bianchini et al. 2013; Barra et al. 2022; Reyes-Carmona et al. 2023) and photogrammetric low-altitude aerial photos acquired from Unmanned Aerial Vehicles (UAVs) (e.g., Hackney and Clayton 2015; Tziavou et al. 2018; Giordan et al. 2020) have emerged as common tools for geological and geomorphological mapping and ground characterization. Davide Torre · Jorge Pedro Galve · Cristina Reyes‑Carmona · David Alfonso‑Jorde · Daniel Ballesteros · Marco Menichetti · Daniela Piacentini · Francesco Troiani · José Miguel Azañón Geomorphological assessment as basic complement of InSAR analysis for landslide processes understanding pp g g The increasing accessibility of remote data, now readily avail- able, may lead to base landslide research mainly on remote sensing information alone. However, it is crucial always to consider the spe- cific geological and geomorphological conditions, which can only be known by developing comprehensive studies integrating classi- cal field surveys and desk studies with remote sensing analyses as Griffiths (2019) or Hearn (2019) point out. In the landslide research environment, fieldwork and desk study are considered essential for obtaining sound results but, as methodological procedures, these classical techniques nowadays have been relegated to a secondary role. In some cases, field work is perceived laboriousness or difficult to develop in the time frames within which contemporary science operates. This particularly occurs under challenging conditions due to field accessibility problems of diverse nature and, in this case, fieldwork is reduced and left out of the picture to only underscore the technologies or methods applied to analyze data acquired by remote sensors. In our experience in other professional spheres, classical approaches are sometimes considered obsolete if not cou- pled with new technology or dismissed when there is existing data in the study areas. In the latter scenario, problems may arise as the prior information may be outdated or not refined to the necessary detail required by the research scale. Although classical approaches remain theoretically fundamental for the surface dynamics scien- tific community, there is a growing trend in practice to decrease the focus on qualitative information acquired by classical techniques such as geological and geomorphological observations, although, it is widely accepted that this information is a basic complement to remote sensing analysis in order to interpret results, and it should be given its due importance. Here, we present a case study that reflects the above described, in which some authors of the study themselves have realized that they overlooked a more general research based on classical surveys in an area extensively studied using InSAR and UAV technologies. Observations obtained through traditional approaches, which aim for a thorough understanding of the terrain of the study area, have yielded a more detailed model of the geological context that helps to interpret the ground motion detected by InSAR techniques. Through this study, we can highlight Keywords Geomorphological mapping · Rocky coast · InSAR · Landslides · Block sliding Original Paper Landslides (2024) 21:1273–1292 DOI 10.1007/s10346-024-02216-w Received: 21 July 2023 Accepted: 11 January 2024 Published online: 19 February 2024 The Author(s) 2024 © Original Paper Original Paper Study area The coastal section of La Herradura (36°44′ N, 3°44′ W; Fig. 1) is a 3 km long bay located in the province of Granada (Southern Spain). The bay is bounded by two promontories, Cerro Gordo (ca. 343 m a.s.l.) and Punta de la Mona (ca. 126 m a.s.l.), which are included in two Special Areas of Conservation (SAC): Maro-Cerro Gordo Cliff Natural Site (MCGCNS) and Cliffs and Seabed of La Punta de la Mona (CSPM), stated within the framework of the European Union Habitats Directive (9242/EEC). The area is heavily anthropized with agricultural terraces, housing, resorts, and hotels, which contribute to the regional economic vitality. The inner part of La Herradura bay is the most densely urbanized sector of the study area, while both promontories also feature urban develop- ments. Notably, the resorts of Cármenes del Mar (on the Cerro Gordo promontory) and Marina del Este (on the Punta La Mona promontory) were built on the eastern slopes of the capes, which are affected by active slope movements. p The present study returns to the abovementioned stretch of the Granada’s coast, albeit with a complete approach that under- lines the importance of classical geomorphological assessments as an important asset for remote sensing studies. For that, in-depth geological-geomorphological surveys have been conducted in the La Herradura section revealing meaningful changes in the previ- ous interpretations of the ground movements detected by InSAR and photogrammetry (Notti et al. 2015; Galve et al. 2017; Mateos et al. 2017; Barra et al. 2022). This contribution includes a wide range analysis of the landforms, as well as a comprehensive geo- morphological map, which serves as a basis for the subsequent interpretation of the movements detected by the aforementioned techniques. In that sense, we have (i) enlarged landslide extensions compared to the previously mapped landslide area and, amongst other observations, (ii) determined the process underlying a defor- mation of unknown origin detected by Notti et al. (2015). These two points have important implications for land-use planning, urban development, the design of structural engineering meas- ures as well as superficial and groundwater management, especially considering the occurrence of local aquifers within marble units (Andreo et al. 2018; Montiel et al. 2018) affected by slope instabili- ties. Introduction The knowledge of the geomorphological processes of an area is fundamental to understand changes in the territory for risk iden- tification and mitigation as well as urban development (Howard 2013; Sreenivasan and Jha 2022). In the present day, geomorpho- logical assessments are assuming an increasingly important role in supporting engineering geology (Hearn 2019; Laimer 2021). Contemporary remote sensing methodologies are facilitating the acquisition and analysis of spatio-temporal data. In this sense, Landslides 21 • (2024) 1273 Original Paper interpretations derived from EGMS, which will also help to design effective landslide mitigation measures there and in other coastal areas around the Globe. the importance of considering the “big picture” and updating geo- logical and geomorphological data for an accurate diagnosis of an area affected by complex landslides. The results of the work hold particularly significant, especially considering that previous research, including contributions from some authors of this paper, extensively examined the study area in recent years, leading to the perception that the area was fully understood from a geological/geomorphological point of view. It appeared that existing knowledge and evidence regarding the land- slides in La Herradura were comprehensive and conclusive, leaving little expectation for significant new discoveries. However, it is cru- cial to note that previous research has relied on prior information based on 1:50,000-scale geological maps and site-scale geomorpho- logical assessments that did not allow the geomorphic “big picture” to be seen. This is the first medium-scale geomorphological assess- ment of the area that brings to light new aspects of the slopes. The new perspective gained from this assessment has the potential to reshape the interpretation of monitoring data in an area of high interest for InSAR studies, particularly within the EGMS. Background of the case study Remote sensing techniques play a crucial role in assessing coastal areas (Sreenivasan and Jha 2022). These regions, characterized by high population density, cultural significance, valuable ecosys- tems, and critical freshwater resources face substantial impacts from many interactions between natural and human processes. Global Change factors, including sea-level rise, escalating aridity, and intense and sudden rainfall (López-Fernández et al. 2022), fur- ther contribute to the challenges. In this context, one of the most human-altered areas globally is the Mediterranean shoreline (Fal- cucci et al. 2007), with half of it composed of rocky coasts (Furlani et al. 2014), where unstable slopes are common (e.g., Mantovani et al. 2016; Polcari et al. 2018; Svigkas et al. 2020). Identification and monitoring of coastal landslides are a priority in this setting, and the research using remote sensing techniques focused on the coast of Granada province in the Mediterranean shoreline of South- ern Spain has attracted international attention on this topic (Notti et al. 2015; Galve et al. 2017; Mateos et al. 2017; Barra et al. 2022). So much so that the European Ground Motion Service (EGMS), a Europe-wide project on InSAR applications, has taken this stretch of coastline as one of its example cases to show the results of the service (Crosetto et al. 2020; Kotzerke et al. 2022). The EGMS is an ambitious initiative of the Copernicus Land Monitoring Service (CLMS) that provides information on ground motion occurring in the majority of European countries. The European Space Agency (ESA), the European Union, and other national organizations and academic institutions are working together on it to identify and track ground motion events including earthquakes, landslides, and subsidence. This initiative is focused on providing ground motion data, but the interpretation of the results is the concern of those who use the platform. Study area The main purpose of this work was to refine the conceptual model of landslides along the coastline of La Herradura to validate The coastal sector under study shows a NW-SE orientation, with local N-S variations due to the promontories morphology (Fig. 1). It is characterized by a Mediterranean climate, a humid temperate climate with dry and hot summer (Csa, according to the Köppen- Geiger classification) (Cunha et al. 2011), with a mean annual pre- cipitation of about 430 mm (Chacón et al. 2019). Rainfall in this coastal area above the yearly average occurred in the wet seasons of 1995–1997, 2000–2003, and 2009–2010 (Irigaray et al. 2000; Vicente- Serrano et al. 2011) breaking all the previous records in 2010, with an annual average value of about 857 mm (Chacón et al. 2019); the greatest amount was reported in 1973 with 350 mm in 24 h (Notti et al. 2015). Due to the latitude, the coastal orientation, and the Betic Cordillera, which protects the coastline from the Northern winds (Mooser et al. 2021), temperatures are mild both during winter and summer with a mean annual range of 18–20 °C (Chica Ruiz and Barragán Muñoz 2011; Consejería de Medio Ambiente y Ordenación del Territorio Junta de Andalucía 2015). Dominant winds approach from E-SE with a speed up to 9 m/s (Manno et al. 2016; Molina et al. 2019) causing storm waves. The coast is dominated by E-SE high-frequency waves (significant wave height 1 m) (Guisado and Malvárez 2009) that generate a littoral drift towards W (Fig. 1); Landslides 21 • (2024) 1274 1275 Fig. 1 La Herradura coastal section, showing major urban areas, main roads (A-7 highway, N-340 national road), and special conservation areas. The map of Spain with zones of Betic-Rif orogen is after Moragues et al. (2021). The geological map is after Marín-Lechado et al. (2009). La Herradura fault is after Ruano (2003) and Ruano et al. (2004). Preferred wave movement direction in percent and wave height by colour range in the range 2011–2021 (is from State Port, Ministry of Transport, Mobility and Urban Agenda of Spanish on https://www. puertos.es/es-es) Fig. 1 La Herradura coastal section, showing major urban areas, main roads (A-7 highway, N-340 national road), and special conservation areas. The map of Spain with zones of Betic-Rif orogen is after Moragues et al. (2021). The geological map is after Marín-Lechado et al. (2009). Study area La Herradura fault is after Ruano (2003) and Ruano et al. (2004). Preferred wave movement direction in percent and wave height by colour range in the range 2011–2021 (is from State Port, Ministry of Transport, Mobility and Urban Agenda of Spanish on https://www. puertos.es/es-es) the average tidal range is less than 20 cm (Manno et al. 2016), and the ongoing local sea-level rising is estimated in 2.65 mm per year (Serrano et al. 2020). exhibits a km-scale recumbent syncline affected by SW-dipping low-angle normal faults and other Alpine structures (Simancas and Campos 1993). The normal fault displaced the Marble Fm. belong- ing to the hanging wall towards the S-SW. Marble Fm. is classically considered a karst aquifer with high fis- suration and a low degree of karstification, while the remaining rocks, such as schists, are aquitards (Andreo et al. 2018; Calvache et al. 2020). The main recharge of the aquifer occurs by rainfall infiltration inland and the discharge takes place via submarine outlets or through karst springs located at sea level (Montiel et al. 2018). Both promontories are associated with Triassic marble that exhibits karst features often related to fractures and faults, especially in Cerro Gordo cape, where 11 caves totalling 330 m in length have been reported by speleological teams. Previous landslides studies (2022) confirmed the activity of the Marina del Este and Punta de la Mona landslides from November 2015 to May 2020, using InSAR techniques and without delimiting land- slide extension, both with LOS average displacement rates ranging from − 10 to − 20 mm/yr. The ground displacement was inferred to be around 10 mm/yr affecting the Marina del Este resort. y The approach used for this study involved consulting the litera- ture, geotechnical reports, and historical pre-urbanization aerial images. Geological and geomorphological terrain models were developed based on various in situ and remote sensing surveys. Moreover, a comparison was made with studies that investigated the same areas using InSAR data from the EGMS Explorer (Euro- pean Environment Agency 2023). The calibrated displacement data in ascending and descending orbits from the period January 2018–September 2022 were downloaded by using the EGMStream app (Festa and Del Soldato 2023), which facilitated the data man- agement. For both geometries, the stability range was established as two times the standard deviation of the data (Barra et al. 2017), and satellite Line-Of-Sight (LOS) velocity maps were displayed. Moreover, the average time series of accumulated displacement of the main unstable areas was extracted and analyzed. In the laboratory, aerial photography, land, and subma- rine Digital Elevation Models (DEM), and previous geological maps were performed and analyzed using ArcGIS 10.8.2 (ESRI®, 2022/2023-licensed “DISPEA University of Urbino”). Aerial photos were downloaded by the National Geographic Institute (IGN) of Spain (https://centrodedescargas.cnig.es/; https://fototeca.cnig.es/ fototeca/) resulting from photogrammetric flights conducted by the USA over the years 1945–1946 and 1956–1957 and by Spanish administrations since 1973; these were used to visualize the area previously to its heavy urbanization and to define the recent pre- urbanization landslide activity. The images were interpreted using a stereoscope for a 3D interpretation of landforms in combination with Google Earth optical satellite images providing a multitempo- ral analysis. The IGN also provided base maps and the 3D LiDAR point cloud in LAZ format; this zip file was decompressed into LAS archives and filtered with LAStools (Isenburg 2014), generating a DEM with a 1 m cell-size resolution. Data for bathymetric recon- struction were downloaded from the Ministry for the Ecologic Transition and Demographic Challenge of Spain (https://www. miteco.gob.es/es/costas/temas/proteccion-costa/ecocartografias/ ecocartografia-granada.aspx), generating a 5 m-resolution bathy- metric DEM. Previous geological cartography is from Avidad et al. Previous landslides studies The terrain instability studies of the Cerro Gordo and Punta de la Mona promontories started with their urban development of Cármenes del Mar and Marina del Este resorts. From 1977 onwards, the first buildings were constructed, with urbanization increas- ing very rapidly from 1997 to the 2000s (Notti et al. 2015; Mateos et al. 2017). Signs of coastal instability in this area were firstly described in 1988. From 2005 onwards, unpublished technical reports, described in Chacón et al. (2019), analyzing landslide areas were carried out, focusing on the urbanization of Cármenes del Mar. After those reports centred on geotechnical aspects, scientific research was developed focused on remote sensing techniques. Thus, the recent activity of landslides in both the Cerro Gordo and Punta de la Mona promontories have been largely confirmed by applying Differential Interferometric Synthetic Aperture Radar (DInSAR) (Notti et al. 2015; Mateos et al. 2017; Chacón et al. 2019; Barra et al. 2022). Regarding the Cerro Gordo promontory, the most studied is Calaiza landslide previously reported by several unpublished geotechnic studies, by Landslide Database of the Spanish Geological Survey (CN IGME 2016), the inventory of the Granada Province (Chacón et al. 2007) and by Azañón et al. (2016), Mateos et al. (2017), Chacón et al. (2019), and Barra et al. (2022). A Line-Of-Sight (LOS) displacement rate of up to 2.5 mm/yr was obtained in the period 1997–2000, related to the Calaiza land- slide activity (Chacón et al. 2019). Mateos et al. (2017) estimated a maximum LOS displacement rate of − 10 mm/yr for this landslide from May 2003 to December 2009, a period in which the first dam- ages of the Cármenes del Mar resort appeared. These authors also estimated displacements of up to 1.98 m in 8 years through UAV digital photogrammetry in relation to the intense rainfall period during 2009–2010. Regarding the Punta de la Mona promontory, Marina del Este and Peñón del Lobo landslides were reported by unpublished geotechnic studies, Chacón et al. (2007), CN IGME (2016), Azañón et al. (2016) and Notti et al. (2015); an average LOS displacement rate of − 11 mm/yr was registered on the land- slide affecting the Marina del Este resort (Notti et al. 2015). More recently, Barra et al. Geological and geomorphological setting La Herradura coastal sector is settled in the Alpujárride Complex, formed by deformed metamorphic rocks belonging to the Alboran Domain of the Betic Cordillera (Azañón et al. 1994; Azañón and Crespo-Blanc 2000). The main structures of the Alpujárride Complex are Alpine regional-scale anticline folds, with a mean E-W trend and NW vergence (Simancas and Campos 1993; Azañón et al. 1997; Williams and Platt 2017). These compressive structures are cut by top-to-the-SW low-angle normal faults and later high-angle transtensional faults and other structures, formed since the Miocene (Azañón et al. 1997; Ruano 2003; Ruano et al. 2004; Simancas 2018). However, a NE-SW extensional phase characterizes the whole region at present (Azañón et al. 2015; Galindo-Zaldívar et al. 2015). g g p y p g The plunging cliff (Sunamura 2015), which reaches a depth of over 40 m below the sea level (Consejería de Medio Ambiente y Ordenación del Territorio Junta de Andalucía 2015), is the main coastal morphology along the shoreline. Alternating with the plunging cliff, there are small coves with beach deposits and larger pebbly and sandy beaches such as La Herradura bay. The seabed in front of the coast consists of a continental shelf that slopes dip- ping 1.4° to the continental escarpment zone located at about 4 km from the coast. The promontories are dominated by small torren- tial streams within short and narrow valleys and slope/run-off processes in slopes. Slope processes involve prominent landslides impacting Cármenes del Mar and Marina del Este resorts (Fig. 1). Chacón et al. (2014) reported the occurrence of landslides with slide kinematics mostly at the contact between marble and schist or phyllite units. The activity of these instabilities was directly The study area is settled in the tectonic unit of La Herradura, formed by the following lithostratigraphic sequence, from bottom to top (Azañón and Crespo-Blanc 2000): (i) Dark Schists Formation (Fm.), dark-coloured schists and graphitic micaschists (Paleozoic), with Migmatite Gneisses at the bottom; (ii) Light Schists Fm., light- coloured fine-grained schists with calcschists in the uppermost part (Permian-Triassic); (iii) Marble Fm., calcareous and dolomitic marbles with interbedded phyllites and schists (Middle-Upper Tri- assic). The geological structure of the coast stretch is complex and Landslides 21 • (2024) 1275 Original Paper correlated with extreme rainfall events (Notti et al. 2015; Mateos et al. 2017; Chacón et al. 2019). Geological and geomorphological setting the rocky coastline, and to establish a relationship between slope processes and bedrock lithology and structure. The cartography allowed to know the landslide extent and dynamics and to elaborate cross-sections along the main slope instabilities, as the basis for performing a conceptual model of the slope processes previously detected by DInSAR studies. Previous landslides studies (1973), and the Continuous Digital Geological Map of Spain per- formed at a 1:50,000 by Marín-Lechado et al. (2009). Lineaments (related to landslide failure surfaces, faults, and/ or fractures) and landforms were identified by analyzing also the hillshade model derived by the DEM and were later verified by fieldwork carried out from September to December 2022. The landslide mechanisms were classified according to Cruden and Varnes (1996) and Hungr et al. (2014). The field surveys were novelty supported by UAV flights to visualize hard-to-reach spots such as the rocky cliffs along the coast. The drone model used was a DJI Mavic 2 pro equipped with a 20 MP Hasselblad camera with a 1’’ CMOS sensor size; the camera is fastened on a 3-axis gimbal for image stabilization and a 28 mm lens, with a FOV of ca. 77° and an aperture of f/2.8-f/11, allowing a 20 Mpixel photo resolution. Furthermore, this drone is equipped with a Finally, landslides have resulted in a dramatic situation. The authorities have currently proclaimed an emergency state after ground movement damage forced the evacuation of 42 buildings in the resort of Cármenes del Mar (Chacón et al. 2019). For these reasons, all the investigations bring the La Herradura coastal sec- tion to be one of the selected illustrative cases of the Product User Manual of EGMS (Kotzerke et al. 2022; Crosetto and Solari 2023). Landslides in the Cerro Gordo promontory: reconfiguring the conceptual model of slope failures Landslides in the Cerro Gordo promontory: reconfiguring the conceptual model of slope failures Landslides in the Cerro Gordo promontory: reconfiguring the conceptual model of slope failures GPS/Glonass system for image georeferencing. Mobile device applications (e.g., FieldMove Clino) were also utilized to collect data directly in digital form. GPS/Glonass system for image georeferencing. Mobile device applications (e.g., FieldMove Clino) were also utilized to collect data directly in digital form. Cerro Gordo promontory (343 m altitude) is mainly composed of Marble Fm. exhibiting the typical massive morphology influenced by the occurrence of a recumbent syncline (Simancas and Campos 1993), with a NW-SE to E-W trend. Here, bedding dips 15° to the SE at the summit, while it is subvertical in the southern plunging cliff of the cape. The fold shows an axial plane tectonic foliation towards S-SE dipping 30–40°. Both Marble and Schists Fms. are partially weathered, although the schists show a more extreme alteration. The deformation that the Dark Schists Fm. has undergone, and the high degree of fracturing, due to the tectonics present in the area, led to a more ductile than brittle rheology. Materials and methods The geomorphological and geological map of La Herradura coastal section was carried out at a scale of 1:20,000 to identify gravity-induced deposits, to recognize surface processes acting on Landslides 21 • (2024) 1276 Results a Cross-section along the active Calaiza Cármenes del Mar resort was built on the eastern slope of Cerro Gordo promontory. An inferred shear zone associated w tational slope deformation is also shown. b Cross-section of Las Palomas landslide that showcases the foundations of Las th i i it f C G d hill Whil th j it f thi l d lid i tl i ti th i ti it b d i it l 278 Original Paper eomorphological cross-sections along La Herradura coastal section. The legend and position of the s om position of the aerial image in profile b can be seen in Fig. 4a. a Cross-section along the active Cala t b ilt th t l f C G d t A i f d h i t d Fig. 3 Geological-geomorphological cross-sections along La Herradura coastal section. The legend and position of the sections are depicted in Fig. 2 and the zoom position of the aerial image in profile b can be seen in Fig. 4a. a Cross-section along the active Calaiza landslides, where Cármenes del Mar resort was built on the eastern slope of Cerro Gordo promontory. An inferred shear zone associated with a larger gravi- tational slope deformation is also shown. b Cross-section of Las Palomas landslide that showcases the foundations of Las Palomas resort, at the vicinity of Cerro Gordo hill. While the majority of this landslide is currently inactive, there is activity observed in its lower part, where Mar- ble blocks are sliding at the present day. Aerial image of the 1957 flight displays trenches revealing the occurrence of large, slipped marble blocks (highlighted by red ellipses). Vertical scale is ca. two times exaggerated. c Cross-section along Punta de la Mona promontory exhibit- ing active Punta de la Mona and Marina del Este landslides Fig. 3 Geological-geomorphological cross-sections along La Herradura coastal section. The legend and position of the sections are depicted in Fig. 2 and the zoom position of the aerial image in profile b can be seen in Fig. 4a. a Cross-section along the active Calaiza landslides, where Cármenes del Mar resort was built on the eastern slope of Cerro Gordo promontory. An inferred shear zone associated with a larger gravi- tational slope deformation is also shown. b Cross-section of Las Palomas landslide that showcases the foundations of Las Palomas resort, at the vicinity of Cerro Gordo hill. Results Desk study and field investigations provided the first compre- hensive geological-geomorphological map of La Herradura coastal section at a scale of 1:20,000 (Fig. 2). Three landslides (named Cantarriján, Calaiza, and Las Palomas) have been iden- tified, surveyed, and/or studied in Cerro Gordo promontory. Additionally, four major instabilities (namely Playa, Punta de la Mona, Marina del Este, and Peñón del Lobo landslides) have been delimited and studied in the surroundings of Punta de la Monta promontory. In our work, we examined Calaiza, Las Palo- mas, Punta de la Mona, and Marina del Este landslides in detail by means of three geological cross-sections to synthesize all newly gathered observations in the vertical dimension (Fig. 3). The studied instabilities are affecting or could impact resorts, infrastructures (highway, national road) and other urban areas. Subsequently, we compared the results with previous works that have analyzed only some of these landslides, particularly through InSAR data. The eastern side of the Cerro Gordo promontory before the urban development is shown in Fig. 4a, and the corresponding improved geological-geomorphological map (Fig. 4b) shows a zoom of Fig. 2. Here, Dark Schists Fm. has been identified in areas previously mapped as Marble Fm. in previous works. Moreover, we observed that Paleozoic Dark Schist Fm. lies over the Marble Fm. through a contact of tectonic origin. This evidence and the absence of the Permian-Lower Triassic Light Schists Fm. (which stratigraphically appears between the Marbles and the Dark Schists Fig. 2 Detailed geological and geomorphological map of La Herradura coastal section. Bedrock geology is after Avidad et al. (1973) and Marín-Lechado et al. (2009). In the legend and in the map light-coloured features remain inactive/quiescent, dashed-line features are sup- posed/uncertain, and barbs (in normal fault) and triangles (in thrusts) are in the hanging wall Fig. 2 Detailed geological and geomorphological map of La Herradura coastal section. Bedrock geology is after Avidad et al. (1973) and Marín-Lechado et al. (2009). In the legend and in the map light-coloured features remain inactive/quiescent, dashed-line features are sup- posed/uncertain, and barbs (in normal fault) and triangles (in thrusts) are in the hanging wall Landslides 21 • (2024) 1277 1278 Original Paper Fig. 3 Geological-geomorphological cross-sections along La Herradura coastal section. The legend and position of the sect in Fig. 2 and the zoom position of the aerial image in profile b can be seen in Fig. 4a. Results While the majority of this landslide is currently inactive, there is activity observed in its lower part, where Mar- ble blocks are sliding at the present day. Aerial image of the 1957 flight displays trenches revealing the occurrence of large, slipped marble blocks (highlighted by red ellipses). Vertical scale is ca. two times exaggerated. c Cross-section along Punta de la Mona promontory exhibit- ing active Punta de la Mona and Marina del Este landslides Landslides 21 • (2024) 1278 Fig. 4 a Aerial image of 1957 exhibiting landslides and their detachment areas, where the landslides are clearly visible without anthropiza- tion. Continuous red lines indicate landslide bodies, orange lines indicate the detachment areas, black rectangle indicates zoom in Fig. 3b; b Zoom of the geological and geomorphological map (Fig. 2) on the Cerro Gordo promontory. The survey details each single slipped block of marble. The legend for b is in Fig. 2; c Panoramic view of the eastern slope of Cerro Gordo promontory with the villages built on Calaiza and Las Palomas landslides. The point of view of c is shown in a Fig. 4 a Aerial image of 1957 exhibiting landslides and their detachment areas, where the landslides are clearly visible without anthropiza- tion. Continuous red lines indicate landslide bodies, orange lines indicate the detachment areas, black rectangle indicates zoom in Fig. 3b; b Zoom of the geological and geomorphological map (Fig. 2) on the Cerro Gordo promontory. The survey details each single slipped block of marble. The legend for b is in Fig. 2; c Panoramic view of the eastern slope of Cerro Gordo promontory with the villages built on Calaiza and Las Palomas landslides. The point of view of c is shown in a Fms.; Azañón and Crespo-Blanc 2000) suggest that the contact between Dark Schists and Marble Fms. is a thrust in line with observation carried out by Sanz de Galdeano and López-Garrido (2003) in Betic Cordillera. (2017). Just below the ridge, two escarpments have been discovered using the hillshade model of 1 m resolution and later confirmed by fieldwork (Fig. 5d). The uppermost, with an approximately 10 m height and a N30° E mean direction can be recognized along the entire slope (Fig. 4b). The second escarpment at lower altitudes is definitely shorter and discontinuous. The origin of this landform will be explored in the discussion section. Results Cantarriján landslide stands out in the western slope of the Cerro Gordo promontory (Fig. 4). This inactive landslide is a rock avalanche, has a minimum volume of 3 × 106 m3, and produces a significant effect on the littoral relief and the bathymetry until the depth of − 50 m visible in the Visor de Cartografiado Marino (https://sig.mapama.gob.es/marino/). The landslide deposit is quiescent/inactive, except for sporadic rock falls along the land- slide scarp and along the cliff of the landslide deposit generated by marine erosion. Las Palomas landslide has been discovered towards the NE of the Calaiza landslide. It is almost masked by the urban area, and the observation of geological and geomorphological features here is really difficult. Specifically, the northern limit of the landslide is now largely obscured from view, making it challenging to delimit. Fortunately, historical aerial photographs taken in 1957 (Fig. 4a) display key morphological features to map the sliding mass. In addition, the sea cliff face provides a clear view of the landslide deposit, allowing for an accurate understanding of its structure and composition. Thanks to low-altitude aerial photos captured using a UAV, the inaccessible cliff has been examined revealing (i) the fallen marble rocks from the landslide deposit into the sea (Fig. 5e), which are not found anywhere else on the promontory where landslides were not reported; (ii) the alternation of massive marble outcrops (huge blocks on Las Palomas landslide) to the landslide deposit. These observations were crucial for developing The eastern slope of the Cerro Gordo promontory is affected by the Calaiza landslide, which has impacted on Cármenes del Mar resort up to the present (Chacón et al. 2014, 2016, 2019; Notti et al. 2015; Azañon et al. 2016; Mateos et al. 2017). The landslide volume was estimated by Chacón et al. (2019) in ca. 8 × 105 m3, and its cartography has been updated in our work (Figs. 2, 3, and 4). Lateral spread and block sliding of marble blocks with volumes over 300 m3 (within and above the landslide) have been detailed in the southernmost sector, detached from the 25 m height escarp- ment (Fig. 5a–c), some of them already mapped by Mateos et al. Landslides 21 • (2024) 1279 1280 Original Paper Fig. 5 a Contact between a detached marble block and the Dark Schists Fm. marked by a shear zone made of a sandy gouge. Results In the insert frame are visible drag folds in the foot of the deformation zone; b Marble block detached from the escarpment c and slipped above the Cal- aiza landslide deposit in the southern part; c Panoramic view of the Calaiza landslide (dashed red area) in the Cerro Gordo promontory. The right flank escarpment of the Calaiza landslide is also visible; d Main upper escarpment (ca. 10 m height) above the Calaiza landslide; e Las Palomas landslide body involving up to metric marble blocks, falling into the sea through marine erosive action; f Detail on the Las Palomas landslide body, with medium-fine matrix and up to metric marble blocks Fig. 5 a Contact between a detached marble block and the Dark Schists Fm. marked by a shear zone made of a sandy gouge. In the insert frame are visible drag folds in the foot of the deformation zone; b Marble block detached from the escarpment c and slipped above the Cal- aiza landslide deposit in the southern part; c Panoramic view of the Calaiza landslide (dashed red area) in the Cerro Gordo promontory. The right flank escarpment of the Calaiza landslide is also visible; d Main upper escarpment (ca. 10 m height) above the Calaiza landslide; e Las Palomas landslide body involving up to metric marble blocks, falling into the sea through marine erosive action; f Detail on the Las Palomas landslide body, with medium-fine matrix and up to metric marble blocks Fig. 5 a Contact between a detached marble block and the Dark Schists Fm. marked by a shear zone made of a sandy gouge. In the insert frame are visible drag folds in the foot of the deformation zone; b Marble block detached from the escarpment c and slipped above the Cal- aiza landslide deposit in the southern part; c Panoramic view of the Calaiza landslide (dashed red area) in the Cerro Gordo promontory. The right flank escarpment of the Calaiza landslide is also visible; d Main upper escarpment (ca. 10 m height) above the Calaiza landslide; e Las Palomas landslide body involving up to metric marble blocks, falling into the sea through marine erosive action; f Detail on the Las Palomas landslide body, with medium-fine matrix and up to metric marble blocks the weathering schists and phyllites (Fig. 5f) does not fit with the current source area of the landslide (i.e., crown area) composed of schists. Results Two landslides are now being analyzed that had so far not been catalogued, neither in the Land- slide Database of the Spanish Geological Survey (CN IGME 2016) nor in the inventory of the Granada Province (Chacón et al. 2007). Punta de la Mona landslide, with composite rock slump kinemat- ics, affects the entire western slope and is characterized by active superficial reactivations in its central part, exhibiting a minimum volume of 1.5 × 106 m3. The landslide scarp is not evident, but it would be recognizable S of the landslide body and continues along the southern flank, coinciding with a large open fracture on the plunging cliff (Fig. 7b). In the northern part of the western flank, Playa landslide was recognized, which caused some damages to the overlying buildings and roads. This active landslide has a slow- moving flow kinematic and developed entirely on Dark Schists Fm. In the eastern sector of the promontory, the Marina del Este landslide shows a minimum volume of about 2.5 × 106 m3 and has been much studied in geotechnics reports and through InSAR analysis (Notti et al. 2015 and reference therein). The landslide escarpment follows the entire slope showing heights of up to 30 m (Fig. 7c), vertical fractures, and detached large marble blocks up p Landslides involve both sides of the Punta de la Mona prom- ontory, which is heavily urbanized. Two landslides are now being analyzed that had so far not been catalogued, neither in the Land- slide Database of the Spanish Geological Survey (CN IGME 2016) nor in the inventory of the Granada Province (Chacón et al. 2007). Punta de la Mona landslide, with composite rock slump kinemat- ics, affects the entire western slope and is characterized by active superficial reactivations in its central part, exhibiting a minimum volume of 1.5 × 106 m3. The landslide scarp is not evident, but it would be recognizable S of the landslide body and continues along the southern flank, coinciding with a large open fracture on the plunging cliff (Fig. 7b). In the northern part of the western flank, Playa landslide was recognized, which caused some damages to the overlying buildings and roads. This active landslide has a slow- moving flow kinematic and developed entirely on Dark Schists Fm. Results Particularly, it is clear how the Las Palomas landslide changed the previous coastline (as for the Cantarriján landslide), which was about 150 m further back. a more comprehensive understanding of the processes that con- tribute to landslides in this area. Las Palomas landslide, had a short runout, with a general composite rock slump kinematic and nested movements of marble block local lateral spreads and block slides (Figs. 3b and 4b). With an estimated minimum volume of about 6 × 106 m3, the main body involves a large part of the slope reaching the N-340 national road. The crown, which is not very discernible within the landscape, encompasses the two different types of nested bodies, with the largest gravitational process that involves the entire slopes with a very large and deep sliding surface (Fig. 3b). The structure of the landslide with up to metric-scale marble blocks embedded in a medium-fine matrix derived from In some cases, a reddish-brown sandy-rich sediment consisting of fine matrix and angular centimetric marble clasts, not previously described in the area, has been found at the contact between marble blocks and schists (Fig. 5a). Field evidence observed in an excavated talus reports that this sediment is probably a gouge as a result of large marble blocks sliding process on schists (Fig. 5a). The out- crop exhibits a marble block situated above the layer in question, Landslides 21 • (2024) 1280 related to the lower altitude of the promontory itself, which reaches about 126 m in contrast with over 300 m of the Cerro Gordo cape. as well as significantly deformed schist displaying a drag folding zone associated with the movement of the block (zoom in Fig. 5a). According to this, marble blocks are comparable to ploughing blocks commonly observed in periglacial environments (Goudie 2004). These blocks exhibit faster downslope movement compared to the underlying sediment due to solifluction processes, which produce inner deformation within the sediment. as well as significantly deformed schist displaying a drag folding zone associated with the movement of the block (zoom in Fig. 5a). According to this, marble blocks are comparable to ploughing blocks commonly observed in periglacial environments (Goudie 2004). These blocks exhibit faster downslope movement compared to the underlying sediment due to solifluction processes, which produce inner deformation within the sediment. 3 p Landslides involve both sides of the Punta de la Mona prom- ontory, which is heavily urbanized. Gravitational collapse of the Punta de la Mona promontory: redefining the landslide areas Punta de la Mona promontory (Fig. 6) shows similar lithologies as the previous promontory, with the additional presence of spo- radic outcrops of highly altered and fractured Migmatite Gneisses in the eastern slope. Here too, the strata dip towards the S, and the recumbent synclinal axis turns in an E-W direction following the coastline. One of the regional extensional faults, with a NW-SE direction, here cutting across the promontory and generating a par- tially visible tectonic contact in the ridge between Marble and Dark Schists Fms. (Fig. 7a). The main difference from the Cerro Gordo is the scarcity of Marble Fm., which crops out in situ only in the southernmost promontory and at the ridge (Fig. 6a). This is also Fig. 6 a Aerial image taken in 1957 exhibiting landslides and their detachment areas, which are easily recognizable in the pre-urbanized Punta de la Mona promontory. Continuous red lines indicate landslide bodies, orange lines indicate the detachment areas, the green arrow indicates the part of the body landslide flattened for the parking; b Zoom of the geological and geomorphological map (Fig. 2) on the Punta de la Mona promontory. It can be seen the detail with which the landslide deposits and their shapes have been mapped. The legend of b is shown in Fig. 2; c Panoramic view of the eastern slope of the Punta de la Mona promontory with Marina del Este resort, after which the land- slide was named. The rock fall deposit with the up to 30 m height escarpment in the southeastern part of the promontory is clearly visible. The point of view of c is shown in a Fig. 6 a Aerial image taken in 1957 exhibiting landslides and their detachment areas, which are easily recognizable in the pre-urbanized Punta de la Mona promontory. Continuous red lines indicate landslide bodies, orange lines indicate the detachment areas, the green arrow indicates the part of the body landslide flattened for the parking; b Zoom of the geological and geomorphological map (Fig. 2) on the Punta de la Mona promontory. It can be seen the detail with which the landslide deposits and their shapes have been mapped. The legend of b is shown in Fig. 2; c Panoramic view of the eastern slope of the Punta de la Mona promontory with Marina del Este resort, after which the land- slide was named. Gravitational collapse of the Punta de la Mona promontory: redefining the landslide areas The rock fall deposit with the up to 30 m height escarpment in the southeastern part of the promontory is clearly visible. The point of view of c is shown in a Fig. 6 a Aerial image taken in 1957 exhibiting landslides and their detachment areas, which are easily recognizable in the pre-urbanized Punta de la Mona promontory. Continuous red lines indicate landslide bodies, orange lines indicate the detachment areas, the green arrow indicates the part of the body landslide flattened for the parking; b Zoom of the geological and geomorphological map (Fig. 2) on the Punta de la Mona promontory. It can be seen the detail with which the landslide deposits and their shapes have been mapped. The legend of b is shown in Fig. 2; c Panoramic view of the eastern slope of the Punta de la Mona promontory with Marina del Este resort, after which the land- slide was named. The rock fall deposit with the up to 30 m height escarpment in the southeastern part of the promontory is clearly visible. The point of view of c is shown in a Landslides 21 • (2024) 1281 1282 Original Paper Original Paper Fig. 7 a Contact between Marble and Dark Schists Fms. that is associated with a tectonic shear zone composed by brecciated and well-con- solidated material. Here, one of the extensional faults cuts across the promontory, exhibiting this tectonic contact in the ridge; b Plunging cliffs of Punta de la Mona promontory, obtained from a drone video of the Inmobiliaria de La Cuesta. Indicated by a red arrow is an open fracture that coincides with the escarpment (dashed orange line) of the landslide affecting the western slope; c The escarpment (30 m height) of Marina del Este landslide showing vertical open fractures (red arrow) related to the detachment of marble blocks; d Peñón de las Caballas rock, a huge marble block slipped into the sea, where the harbour of Marina del Este was built Fig. 7 a Contact between Marble and Dark Schists Fms. that is associated with a tectonic shear zone composed by brecciated and well-con- solidated material. Here, one of the extensional faults cuts across the promontory, exhibiting this tectonic contact in the ridge; b Plunging cliffs of Punta de la Mona promontory, obtained from a drone video of the Inmobiliaria de La Cuesta. Gravitational collapse of the Punta de la Mona promontory: redefining the landslide areas Indicated by a red arrow is an open fracture that coincides with the escarpment (dashed orange line) of the landslide affecting the western slope; c The escarpment (30 m height) of Marina del Este landslide showing vertical open fractures (red arrow) related to the detachment of marble blocks; d Peñón de las Caballas rock, a huge marble block slipped into the sea, where the harbour of Marina del Este was built EGMS InSAR data in La Herradura coastal section to 100 m in diameter such as the Peñón de las Caballas block in the marina (Fig. 7d).l Looking at the aerial images of the 1957 flight, it is clear how the promontory was modified when the resorts were constructed (Fig. 6a). For instance, the bottom of the Marina del Este landslide was flattened for the construction of the harbour car park (green arrow in Fig. 6a) or the innumerable gullies engraving the landslide body. Furthermore, as in the case of Las Palomas landslide, huge slipped marble blocks were found along the coastline and on the landslide deposit along the slope, mapped in detail in the geological- geomorphological map (Fig. 6b). In this context, it is noteworthy that an accumulation of marble rocks can be observed at the bottom of the southeastern slope of the promontory, just situated beneath the crown scarp. Based on their distribution pattern, it is possible to infer that the rocks have been dislodged and fallen into the sea as a result of a topple or rockfall event, rather than through sliding over the underlying schist. This inference is supported by the occurrence here of marble outcrops without preserved marble blocks inland. Figure 8 shows the InSAR calibrated data in ascending and descending orbit, extracted from the EGMS, at the Cerro Gordo (Fig. 8a, b) and Punta de la Mona (Fig. 8c, d) promontories. The stability range was settled from 2.5 to − 2.5 mm/yr for both geom- etries. All the landslides exhibit ground motion through at least one geometry, with the exception of Cantarriján, Las Palomas, and Peñón del Lobo landslides where clear ground displacement was not captured. The LOS maximum velocity registered in ascend- ing orbit is 14 mm/yr in the Calaiza landslide (Fig. 8a), while in descending geometry, LOS velocities reach 15.3 mm/yr in the Punta de la Mona landslide (Fig. 8d). The average velocities of the Calaiza, Punta de la Mona, and Marina del Este landslides range from 3.5 to 8.1 mm/yr (absolute values), while the Playa landslide shows a lower mean LOS velocity of − 2.8 mm/yr. Moreover, the displace- ment pattern provided by EGMS in these four landslides follows a linear and constant trend during the measured period of time Landslides 21 • (2024) 1282 1283 Fig. EGMS InSAR data in La Herradura coastal section 10a) although this is not evident in Punta de la Mona promontory. According to this, the contact here is represented by a vertical fault in Fig. 10b. In general, the geological structure of the bedrock favoured the primary development of landslides on the eastern hillsides, creat- ing an apparent suitable smooth area for building. The landslides are really complex and involve three main clear movements: (i) a deepest movement characterized by rock slump kinematics at a 40 m depth in Cerro Gordo promontory (Mateos et al. 2017) and 20 to 25 m depth in Punta la Mona Promontory (Notti et al. 2015); (ii) superficial movements composed of a slow-moving earthflow in combination with lateral spreads and sliding of large marble blocks, and (iii) local toppling and rockfall processes on scarps and detached marble blocks, confined in areas proximal to escarp- ments and cliffs. Another potential main movement is related to the escarpments recognized at the top of Cerro Gordo promontory and a hypothetic shear zone in depth (Fig. 10a). The scarps would be formed by a gravitational process, and it is related to one of the most persistent joint family with a NE-SW orientation (Mateos et al. 2017). This joint family is also correlated with the NNE-SSW exten- sional tectonic structures that characterize the Alpujarride complex (Azañón et al. 1997; Simancas 2018). The presence of enormous, detached marble blocks and the mentioned escarpment suggests that the entire eastern slope of Cerro Gordo promontory is affected by a complex and old gravitational movement. The morphological features resulting from this gravitational process appear to have been largely obliterated by watershed erosion, which implies that the development of such phenomena has been notably slow and over a long period of time. Marine erosive action may have played a key role in the main land- slide activities. In fact, the highest concentration of landslide phenom- ena is found on the eastern slopes, where the prevailing waves and storm waves with the highest frequency are active (Fig. 1). In relation to the existing mapping, that is the BD-MOVES cata- logue of the Geological and Mining Institute of Spain (CN IGME 2016), the total landslide areas in the La Herradura coastal stretch would be 2 × 105 m2. EGMS InSAR data in La Herradura coastal section Again, the landslides that are highlighted in the literature are only the active bodies. From the morphological evidence, the eastern side, where Marina del Este is located, was completely involved in a very extensive mass movement, with only the central portion currently active, above which the Marina del Este resort was built. From the bathymetric trend, it is visible that the seabed slopes more gently than on the western side of the promontory, most likely due to the transport of sediments from the landslide deposit to the seabed close to the shoreline. As a further indication of the above, the − 50 m isobath also has an extension in the area below the slope (Fig. 6b). The west- ern side is also entirely affected by gravitational processes, which activity was recently displayed by Barra et al. (2022) and mapped in our research for the first time. As can be seen, the evolution of both promontories is very simi- lar, with equal landslide kinematics and quite similar sliding sur- face depths. Surface movements, detected by InSAR, are nothing more than reactivations of previous landslide deposits, caused by a combination of exceptional rainfall events and rapid and mas- sive urban development. Another important common feature is the presence of marble blocks of varying size (from metric to decamet- ric scale) that lie along the slope or on the coastline (Figs. 4 and 6). Due to the development of major discontinuities (Fig. 7c) within the marbles, with an approximately NNE-SSW trend, the detachment of blocks, even large ones, occurs through a process similar to lateral spreading. The weathering of the schists and the landslide depos- its means that the block can slide, thus highlighting the evolution from lateral spreading to block sliding (Fig. 10). Examples of this type of kinematics have already been studied on Mediterranean coasts made of sedimentary rocks (Mantovani et al. 2013; Devoto et al. 2021). In the La Herradura coastal section, we present one of the few comprehensible examples of an Alpine metamorphic set- ting, highlighted just by few contributions until now in other areas (Poisel et al. 2009). The new landslide conceptual model of Cerro Gordo and Punta de la Mona promontories is shown in Fig. 10. In both cases, the gen- eral attitude of the strata dips to the S-SE. The contact between the geological formations would be an Alpine thrust (Fig. EGMS InSAR data in La Herradura coastal section 8 Line-Of-Sight (LOS) velocity maps in ascending (a) and descending (b) orbits in the Cerro Gordo promontory; and in ascending (c) and descending (d) orbits in the Punta de la Mona promontory (data downloaded from https://egms.land.copernicus.eu/). The main landslide boundaries delimited in this research (Fig. 2) are also indicated in order to compare current active areas according to EGMS with the entire area of landslides Fig. 8 Line-Of-Sight (LOS) velocity maps in ascending (a) and descending (b) orbits in the Cerro Gordo promontory; and in ascending (c) and descending (d) orbits in the Punta de la Mona promontory (data downloaded from https://egms.land.copernicus.eu/). The main landslide boundaries delimited in this research (Fig. 2) are also indicated in order to compare current active areas according to EGMS with the entire area of landslides LOS accumulated displacement is up to − 30 mm in the Calaiza (Fig. 9a) and Punta de la Mona (Fig. 9b) landslides, in ascending and descending orbit, respectively. (from January 2018 to September 2022), as evidenced by the time series of accumulated displacement (Fig. 9). There is no significant acceleration of the movement in any of the landslides, and the total Fig. 9 Times series of accumulated displacement, measured along the satellite Line-Of-Sight (LOS), of the Calaiza and Marina del Este land- slides in ascending geometry (a) and Punta de la Mona and Playa landslides in descending orbit (b). In both graphs, daily precipitation recorded by the closer meteorological station of the Spanish State Meteorological Agency (Station Salobreña 6267X, 36°44′54″ N 3°34′43″ W) to the study area is represented. No clear accelerations of landslide displacements are observed in relation to daily rainfall peaks or periods of prolonged rainfall in the timeframe analyzed Fig. 9 Times series of accumulated displacement, measured along the satellite Line-Of-Sight (LOS), of the Calaiza and Marina del Este land- slides in ascending geometry (a) and Punta de la Mona and Playa landslides in descending orbit (b). In both graphs, daily precipitation recorded by the closer meteorological station of the Spanish State Meteorological Agency (Station Salobreña 6267X, 36°44′54″ N 3°34′43″ W) to the study area is represented. No clear accelerations of landslide displacements are observed in relation to daily rainfall peaks or periods of prolonged rainfall in the timeframe analyzed Landslides 21 • (2024) 1283 Original Paper Internal discontinuity planes of the marble bedrock manifested as bedding, tectonic foliation, and joint families, constrained the mobilized blocks. EGMS InSAR data in La Herradura coastal section would act as a sliding layer for marble blocks, leading to their eventual mingling with the underlying weathered and slid-down Dark Schists mass. In addition, the Dark Schists Fm. is of low permeability and concentrated groundwater on its top, favouring landslide movements. This agrees with the InSAR moni- toring that reordered higher ground movement velocities during wet periods (Notti et al. 2015; Mateos et al. 2017; Chacón et al. 2019). Landslides affected both sides of the Punta de la Mona promon- tory (Fig. 10b) according to Notti et al. (2015) and Barra et al. (2022), showing detached large marble boulders. Again, the landslides that are highlighted in the literature are only the active bodies. From the morphological evidence, the eastern side, where Marina del Este is located, was completely involved in a very extensive mass movement, with only the central portion currently active, above New landslide conceptual model of La Herradura coastal section The field survey developed in the present investigation has cor- roborated that the evolution of the eastern slope of the Cerro Gordo promontory was undoubtedly influenced by the outcrop- ping Dark Schists Fm. as previous investigations had highlighted. Notwithstanding, the new geological-geomorphological map (Fig. 2) and cross-sections (Fig. 3a, b) have yielded dissimilar results compared to earlier works, which were based on the geo- logical map of Avidad et al. (1973). This map was created prior to significant advancements in tectonics during the 1980s (a period when, for instance, the conceptual understanding of thrust sys- tems underwent significant development) and suggested that the promontory has a core of dark schists covered by the marble unit. Our investigation conducted at a scale of 1:20,000 (utilizing high-resolution aerial images, LiDAR DEM, and an extensive field survey supported by UAV technology) unveiled the occurrence of a NW-direct thrust dipping to the SE. This geological structure is parallel to the eastern slope of Cerro Gordo promontory and, therefore, would influence widely the slope processes here. These novel insights and new geomorphological evidence allowed us to redefine the geological and geomorphological conception of the study area and subsequently to revise the local landslide concep- tualization made by Notti et al. (2015), Mateos et al. (2017), and Chacón et al. (2019). Landslides affected both sides of the Punta de la Mona promon- tory (Fig. 10b) according to Notti et al. (2015) and Barra et al. (2022), showing detached large marble boulders. EGMS InSAR data in La Herradura coastal section Meanwhile, the presence of schist interbedded into the Marble Fm. would act as a sliding layer for marble blocks, leading to their eventual mingling with the underlying weathered and slid-down Dark Schists mass. In addition, the Dark Schists Fm. is of low permeability and concentrated groundwater on its top, favouring landslide movements. This agrees with the InSAR moni- toring that reordered higher ground movement velocities during wet periods (Notti et al. 2015; Mateos et al. 2017; Chacón et al. 2019). Internal discontinuity planes of the marble bedrock manifested as bedding, tectonic foliation, and joint families, constrained the mobilized blocks. Meanwhile, the presence of schist interbedded into the Marble Fm. would act as a sliding layer for marble blocks, leading to their eventual mingling with the underlying weathered and slid-down Dark Schists mass. In addition, the Dark Schists Fm. is of low permeability and concentrated groundwater on its top, favouring landslide movements. This agrees with the InSAR moni- toring that reordered higher ground movement velocities during wet periods (Notti et al. 2015; Mateos et al. 2017; Chacón et al. 2019). Landslides affected both sides of the Punta de la Mona promon- tory (Fig. 10b) according to Notti et al. (2015) and Barra et al. (2022), showing detached large marble boulders. Again, the landslides that are highlighted in the literature are only the active bodies. From the morphological evidence, the eastern side, where Marina del Este is located, was completely involved in a very extensive mass movement, with only the central portion currently active, above which the Marina del Este resort was built. From the bathymetric trend, it is visible that the seabed slopes more gently than on the western side of the promontory, most likely due to the transport of sediments from the landslide deposit to the seabed close to the shoreline. As a further indication of the above, the − 50 m isobath also has an extension in the area below the slope (Fig. 6b). The west- ern side is also entirely affected by gravitational processes, which activity was recently displayed by Barra et al. (2022) and mapped in our research for the first time. Internal discontinuity planes of the marble bedrock manifested as bedding, tectonic foliation, and joint families, constrained the mobilized blocks. Meanwhile, the presence of schist interbedded into the Marble Fm. EGMS InSAR data in La Herradura coastal section According to our surveys and to the new land- slide mapping, the landslide area would amount to about 1 × 106 m2, five times more extension than the previously mapped landslides (Fig. 11). It is important to clarify that not all the areas affected by landslides are presently exhibiting clear signs of instability. Landslides 21 • (2024) 1284 10 Conceptual landscape model. 3D illustrations show the morphodynamic style that characterizes the promontories of Cerro Go Punta de la Mona b. In addition to the large landslides present, the process of lateral spreading and block sliding is highlighted. For ures (such as marble blocks or detachment areas) vertical scale is exaggerated more or less two times for a better view of the single b Fig. 10 Conceptual landscape model. 3D illustrations show the morphodynamic style that characterizes the promontories of Cerro Gordo and Punta de la Mona b. In addition to the large landslides present, the process of lateral spreading and block sliding is highlighted. For som features (such as marble blocks or detachment areas) vertical scale is exaggerated more or less two times for a better view of the single block Fig. 10 Conceptual landscape model. 3D illustrations show the morphodynamic style that characterizes the promontories of Cerro Gordo a and Punta de la Mona b. In addition to the large landslides present, the process of lateral spreading and block sliding is highlighted. For some features (such as marble blocks or detachment areas) vertical scale is exaggerated more or less two times for a better view of the single block Classical methods offer fresh insights in areas extensively explored by advanced technologies This observation can be attributed to the fact that all investigations thus far have been limited to the damaged area of Cármenes del Mar resort, without any broader research being undertaken in the region. Furthermore, in the historical images, it can be recognized that the lower part of the landslide shows two large marble blocks affected by an internal process of lateral spread and block sliding provoked by the coastal erosion (zoom of the aerial image in Fig. 3b). Nowadays, this zone is covered by urban areas which prevents us from observing these fea- tures. These features suggest an active movement in the date when those photographs were taken. Currently, the landslide seems to be inactive, which is also indicated by the calibrated EGMS InSAR data (Fig. 8), as movement has not been detected in that area up to now. However, this should be treated with caution because the InSAR data cover a limited range of time and ground movements (few millimetres), and the technique could be blind for movements with N-S orientations. Another feature worth mentioning in the area of the Las Palomas landslide is the perturbation of the drainage network caused by this landslide. The path of the main impluvium of the slope is deflected showing a sharp bend. In the longitudinal profile of the ravine, it is clear how there is an anomaly, in part certainly due to the anthropic component, but with a change in convexity downstream of the anomaly (Fig. 12). This could indicate a diversion of the ravine due to lithological contrast between schist and marbles, due to the existence of a slipped marble block, or the recent advance of the landslide body, in a centennial temporal scale. The impluvium, in the section previous to the anomaly, engraved the main rock slump landslide deposit by crossing it and is diverted due to the subsequent reactivation of the deposit and lithological change of the marble block. Thanks to the geological and geomorphological field survey, the use of multi-temporal aerial images and InSAR data, it was possible to give a complete evolutionary picture of the La Herradura coastal section. In Table 1, the present work is compared with the previous scientific papers, which describe researches in this coastal section apply advanced technologies, through the methods used, work products, and results obtained. Notti et al. Classical methods offer fresh insights in areas extensively explored by advanced technologies Nevertheless, it should be noted that landslide deposits typically lack consolidation, and as such, an area currently deemed stable, such as those discernible by InSAR, may undergo reactivation in the future. This is particularly true during periods of exceptional rain- fall events or earthquakes, which are common in the region. These extraordinary events could also alter the linear constant trend of the active landslides (Fig. 9) and trigger catastrophic accelerations of these movements as it has already occurred in the extraordinary wet period between 2009 and 2010 (Notti et al. 2015; Chacón et al 2019). Calaiza landslide is one of the most studied landslides in Spain because of the countless significant damages to the overlying build- ings and the forced evacuation of 42 dwellings (Chacón et al. 2019). This led to analyzing the activity, speed, and extent of landslide displacement with all the latest techniques, particularly remotely and from the geotechnical point of view. By concentrating only on Landslides 21 • (2024) 1285 1286 Original Paper Fig. 11 Comparison of landslide boundaries defined by this study (red lines) and previous works (blue lines Notti et al. 2015; orange lines Mateos et al. 2017; green lines Chacón et al. 2019) in both Cerro Gordo (a) and Punta de la Mona (b) promontories. It is clearly visible how the use of different techniques leads to a much more defined picture of an area, mapping not only the active landslide portions but also those that could reactivate Fig. 11 Comparison of landslide boundaries defined by this study (red lines) and previous works (blue lines Notti et al. 2015; orange lines Mateos et al. 2017; green lines Chacón et al. 2019) in both Cerro Gordo (a) and Punta de la Mona (b) promontories. It is clearly visible how the use of different techniques leads to a much more defined picture of an area, mapping not only the active landslide portions but also those that could reactivate the analysis of the abovementioned things and not carrying out a detailed geological and geomorphological survey at the scale of the entire slope, the basis on which all the following analyses can then be carried out has been lacking. This is also partly demonstrated by the Las Palomas landslide. Classical methods offer fresh insights in areas extensively explored by advanced technologies It is noteworthy that photointer- pretation of historical images allowed the novel identification of the latter landslide, which with a short runout, a large part of the landslide body is mainly lying on the slope. This observation can be attributed to the fact that all investigations thus far have been limited to the damaged area of Cármenes del Mar resort, without any broader research being undertaken in the region. Furthermore, in the historical images, it can be recognized that the lower part of the landslide shows two large marble blocks affected by an internal process of lateral spread and block sliding provoked by the coastal erosion (zoom of the aerial image in Fig. 3b). Nowadays, this zone is covered by urban areas which prevents us from observing these fea- tures. These features suggest an active movement in the date when those photographs were taken. Currently, the landslide seems to be inactive, which is also indicated by the calibrated EGMS InSAR data (Fig. 8), as movement has not been detected in that area up to now. However, this should be treated with caution because the InSAR data cover a limited range of time and ground movements (few millimetres), and the technique could be blind for movements with N-S orientations. Another feature worth mentioning in the area of the Las Palomas landslide is the perturbation of the drainage network caused by this landslide. The path of the main impluvium of the slope is deflected showing a sharp bend. In the longitudinal profile of the ravine, it is clear how there is an anomaly, in part certainly due to the anthropic component, but with a change in convexity downstream of the anomaly (Fig. 12). This could indicate a diversion of the ravine due to lithological contrast between schist the analysis of the abovementioned things and not carrying out a detailed geological and geomorphological survey at the scale of the entire slope, the basis on which all the following analyses can then be carried out has been lacking. This is also partly demonstrated by the Las Palomas landslide. It is noteworthy that photointer- pretation of historical images allowed the novel identification of the latter landslide, which with a short runout, a large part of the landslide body is mainly lying on the slope. Classical methods offer fresh insights in areas extensively explored by advanced technologies (2015) have analyzed landslides in Punta de la Mona promontory using Permanent Scat- terers (PS)InSAR technique, mapping all the active processes and made a building damages assessment; furthermore, they compared recent photos with historical pre-urbanization images but focaliz- ing only on the active portion of Marina del Este landslide. Mateos et al. (2017) have examined the Calaiza landslide on Cerro Gordo promontory combining PSInSAR and UAV photogrammetry; they mapped the active landslide deposits and smaller, shallower mar- ble blocks, studied the recent reactivations of the landslide body in relation to exceptional rainfall events, and compiled a build- ing damages inventory. Chacón et al. (2019) focused on the Calaiza landslide, meticulously describing all the interventions carried out to stabilize the slope over the years and compiling an inven- tory of damages. Barra et al. (2022) chose the two promontories as study areas, starting from the PSInSAR data and providing a map of damages for the Calaiza, Punta de la Mona, and Marina del Este landslides while always considering recent activity. The application of a large variety of remote sensing techniques but Landslides 21 • (2024) 1286 1287 Fig. 12 Longitudinal profile of a ravine perturbated by Las Palomas landslide. The red arrow shows a relief anomaly (sharp slope) defined by a profile-changing curvature. Other relief anomalies are related to anthropic works. In the aerial image, the diverted path of the impluvium is clearly visible (red circle) Fig. 12 Longitudinal profile of a ravine perturbated by Las Palomas landslide. The red arrow shows a relief anomaly (sharp slope) defined by a profile-changing curvature. Other relief anomalies are related to anthropic works. In the aerial image, the diverted path of the impluvium is clearly visible (red circle) problems, and the presence of the sea are some of the aspects that make classical field surveys difficult on rocky coasts, as in the case study described. For this reason, remote surveys are widely used in these environments, both for their convenience and their lower cost. Furthermore, they have, for example, taken advantage of UAVs and Land Surface Quantitative (LSQ) analysis based on high-resolution DEMs, in particular when studying gravitational processes (Devoto et al. 2020; Troiani et al. 2020; Piacentini et al. 2021). In our case, the coastal stretch of La Herradura revealed the important role of UAVs in the exploration of cliff areas, as well as in providing additional perspectives on the ground. Classical methods offer fresh insights in areas extensively explored by advanced technologies In addition, with the help of high-resolution DEM, we were able to discern geological and geomorphological features and understand the landscape from dif- ferent points of view. Thanks to InSAR techniques, researchers in previous works were able to identify active zones where impercep- tible movements developed. In this sense, active landslides can be remotely analyzed, monitored, and mapped using InSAR data and nowadays, more easily through web-based digital platforms that provide already processed data (Galve et al. 2017; EGMS 2023). By exploiting the InSAR data from the EGMS Explorer (Fig. 8), it can be seen that ground displacement is registered in the most impor- tant landslides described in this work: Calaiza, Playa, Punta de la Mona, and Marina del Este. Notice that the detection sensitivity varies between the ascending and descending orbit data, and dis- placement is not equally captured in both geometries for all land- slides. In this regard, the availability of InSAR data in the two geom- etries of the EGMS was truly valuable, preventing the oversight of active landslides that could be undetected using a single geometry. However, as demonstrated by this study, all the aforementioned techniques are complementary. Each one provides information at without the knowledge provided by a more extensive survey (Notti et al. 2015; Mateos et al. 2017; Chacón et al. 2019; Barra et al. 2022) allowed to describe in detail only 40% of the existing landslides in La Herradura coastal section (Fig. 11). All the landslides previ- ously reported correspond to active instabilities, which occurrence is also evidenced by damages observed in urban areas. However, the geological and geomorphological mapping at the 1:20,000-scale conducted in our work has enabled the identification of additional landslide bodies in the same area. It is crucial to consider the occurrence of a priori inactive landslides in land management as they have the potential to be reactivated by human activities (e.g., excavations, new constructions, drainage network modifications). Consequently, their reactivation could significantly impact urban areas and infrastructures. In addition, the meticulous field survey- ing has led to the recognition of novel instability processes, includ- ing the lateral-spreading and sliding of marble blocks. The presence of these boulders has been previously documented by Notti et al. (2015) and Mateos et al. (2017), but these researches do not link these blocks with the landslides’ dynamics. Classical methods offer fresh insights in areas extensively explored by advanced technologies Two escarpments have been also discovered in Cerro Gordo promontory through field- work, suggesting the development of a major potential instability that affects the entire slope. New technologies supporting classical methods and their synergies In the previous section, the advantages of carrying out geomor- phological and geological surveys as a complement to remote sensing surveys have been outlined. However, it is also important to underline the importance of remote sensing techniques to sup- port classical methods. For example, site accessibility, logistical Landslides 21 • (2024) 1287 Table 1 Comparison table between the present work and previous works in the La Herradura coastal section. Only 40% of the landslides evidence and processes were detected and mapped by previous works, even those that had not been identified because they showed in the present day no activity a InSAR data produced by the authors b Peñón del Lobo landslide was already mapped by Chacón et al. (2007) in their inventory of the Province of Granada Notti et al. (2015) Mateos et al. (2017) Chacón et al. (2019) Barra et al. (2022) This work Methods Interferometry InSARa x x x Photointerpretation Recent aerial or satellite images x x x x Historical images x x x x UAV (drone) images x x Field surveying Geological mapping x x x x Geomorphological mapping x x Damages inspection x x x Landslides activity Active processes x x x x x No evidence of active processes x Products Products Geomorphological assessment x Geological map x x Geomorphological map x x Building damages inventory x x x x Results Landforms Landslides Cerro Gordo promontory Cantarriján x x x Calaiza x x x x Las Palomas x Punta de la Mona promontory Playa x Punta de la Mona x x Marina del Este x x x Peñón del Lobob x Detached large marble blocks x x x Upper scarps of Cerro Gordo promontory x Types of instability processes Rock slump x x x x Superficial slow- moving earthflow x x x x Superficial lateral- spreading and block-sliding x Rock falls x Table 1 Comparison table between the present work and previous works in the La Herradura coastal section. Only 40% of the landslides evidence and processes were detected and mapped by previous works, even those that had not been identified because they showed in the present day no activity evidence and processes were detected and mapped by previous works, even those that had not been identified because they showed in the present day no activity a InSAR data produced by the authors b Peñón del Lobo landslide was already mapped by Chacón et al. New technologies supporting classical methods and their synergies (2007) in their inventory of the Province of Granada Notti et al. (2015) Mateos et al. (2017) Chacón et al. (2019) Barra et al. (2022) This work Methods Interferometry InSARa x x x Photointerpretation Recent aerial or satellite images x x x x Historical images x x x x UAV (drone) images x x Field surveying Geological mapping x x x x Geomorphological mapping x x Damages inspection x x x Landslides activity Active processes x x x x x No evidence of active processes x Products Products Geomorphological assessment x Geological map x x Geomorphological map x x Building damages inventory x x x x Results Landforms Landslides Cerro Gordo promontory Cantarriján x x x Calaiza x x x x Las Palomas x Punta de la Mona promontory Playa x Punta de la Mona x x Marina del Este x x x Peñón del Lobob x Detached large marble blocks x x x Upper scarps of Cerro Gordo promontory x Types of instability processes Rock slump x x x x Superficial slow- moving earthflow x x x x Superficial lateral- spreading and block-sliding x Rock falls x a InSAR data produced by the authors AR data produced by the authors ón del Lobo landslide was already mapped by Chacón et al. (2007) in their inventory of the Province of Granada InSAR data produced by the authors b Peñón del Lobo landslide was already mapped by Chacón et al. (2007) in their inventory of the Province of Granada Peñón del Lobo landslide was already mapped by Chacón et al. (2007) in their inventory of the Province of Granada Landslides 21 • (2024) 1288 varying spatial and temporal scales with precisions that collectively enable a more certain diagnosis of slope conditions. urbanized. The occurrence of these inactive or quiescent landslides must be considered in land management due to the potential for reactivation resulting from anthropic activities or extreme rainfalls. In conclusion, the present work shows the immense value of traditional geomorphological and geological survey approaches that complement novel techniques and facilitate multidisciplinary investigations, as well as the great advances made in mapping tech- niques assisted by remote sensing technologies. The accessibility of InSAR data through EGMS has provided widespread access to this valuable information. However, it is essential to emphasize that detailed geological and geomorphological investigations combin- ing fieldwork and remote sensors are crucial to avoid misinterpre- tations. New technologies supporting classical methods and their synergies These investigations play a critical role in understanding ground motions detected by InSAR techniques and developing accurate conceptual models of slope instabilities. Furthermore, geological maps typically prioritize tectonics and stratigraphy and may not specifically address slope instability investigations. Therefore, it is necessary to conduct a thorough review of previous geological data at the local scale to check them as the first step to gain a comprehensive understanding of slope processes and enable effective risk assessment. By incorporating these investigations, we can ensure a more robust and reliable analysis of slope instabilities. In summary, we illustrate the importance of thorough geological and geomorphological studies focused on understanding the con- text of landslides in ensuring an accurate interpretation of remotely sensed information. This work serves as a call to action for geolo- gists and geomorphologists to meticulously review existing map- ping and update it as needed, thereby enhancing the reliability of terrain information and instilling greater confidence in the inter- pretation of remotely sensed data. It also highlights the growing reliance on InSAR and other advanced technologies, which may lead to a focus solely on areas in motion during data acquisition, potentially underestimating the true extent of landslides as exem- plified in the described case study. In this regard, the incorporation of qualitative geological and geomorphological information can prevent misinterpretations, ensuring the effectiveness of landslide risk assessment. Acknowledgements We would like to thank Inmobiliaria de La Cuesta for providing a UAV video of a part of Punta de La Mona’s rocky coastline. Con- sejería de Sostenibilidad, Medio Ambiente y Economía Azul (Junta de Andalucía) authorized the research within the Special Area of Conservation: Natural Site of Maro-Cerro Gordo Cliffs (ES6170002) and Cliff and Seabed of Punta de la Mona cape (ES6140016). Conclusion A new comprehensive conceptual model has been developed for the La Herradura coastal section, which serves as an example case in the landslide paragraph of the Product User Manual of the EGMS. The model incorporates instability mechanisms and their extension in relation to the lithology and structure of the bedrock. The conceptual model identifies three main types of instabilities: (i) a deep rock slump movement occurring along a failure surface at a depth of 20–40 m, (ii) surface movements resulting from slow-moving earth flows, lateral spreads, and sliding of large marble blocks resembling ploughing boulders found in periglacial environments, and (iii) local toppling and rockfall pro- cesses involving scarps and detached marble blocks. Furthermore, a potential major movement affecting the entire slope has also been deducted. All these landslides are associated with the tectonic contact between Triassic marbles and Permian-Triassic schists. Intensive schist weathering has resulted in a surface deposit with a more plastic behaviour, facilitating the sliding of marble blocks. These blocks were detached following the natural bedding, tec- tonic foliation, and fissures of the marble, which derived from an intense tectonic deformation. Data availability The data employed in this work are available on request from the corresponding authors. The development of a new comprehensive model was made possible through an in-depth geological and geomorphological investigation supported by fieldwork, historical and recent aer- ial photography (1956–2022), a LiDAR 1-m resolution DEM, and advanced technologies such as unmanned aerial vehicle (UAV) imagery and InSAR. The geological map was meticulously revised with the study objective in mind, and the geomorphological map- ping focused specifically on slope processes. These techniques have led to the discovery of previously unknown landslides along the La Herradura coast, which were not detected using remote sens- ing alone over the past two decades. Remote sensing methods only identified approximately 40% of the existing landslides, whereas their actual extent is five times greater than previously known. The detailed geological and geomorphological survey also revealed the presence of apparently inactive landslides, some of which have been Funding Funding for open access publishing: Universidad de Granada/ CBUA. Funding for open access publishing: Universidad de Gra- nada/CBUA. The research was supported by the following funds: the “Ramón y Cajal” Programme (RYC-2017-23335) of the Spanish Min- istry of Science; the project “MORPHOMED” (PID2019-107138RB- I00) funded by MCIN/SRA (State Research Agency/https://doi. org/10.13039/501100011033); and FEDER/Junta de Andalucía-Conseje- ría de Transformación Económica, Industria, Conocimiento y Univer- sidades/Projects (A-RNM-508-UGR20 and P18-RT-3632). The work of DB and DA-J is funded byPlan Andaluz de Investigación,Desarrollo e Innovación 2020(Junta de Andalucía), and by the programmeGarantía Juvenilof the Spanish Government, respectively. 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Tectonics 36:756–786. https://doi.org/10.1002/2016TC004358 Daniela Piacentini · Francesco Troiani Department of Earth Sciences, Sapienza University of Rome, P.le Aldo Moro, 00185 Rome, Italy Daniela Piacentini · Francesco Troiani Department of Earth Sciences, Sapienza University of Rome, P.le Aldo Moro, 00185 Rome, Italy 1292 Landslides 21 • (2024) 1292
https://openalex.org/W3099926419	https://edoc.hu-berlin.de/bitstream/18452/26178/1/s41467-020-19375-8.pdf	English	null	NeoR, a near-infrared absorbing rhodopsin	Nature communications	2,020	cc-by	12,485	ARTICLE 1 Institute for Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, 10115 Berlin, Germany. 2 Department of Physics and Astronomy, Faculty of Science, Vrije Universiteit Amsterdam, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands. 3 Fritz Haber Center for Molecular Dynamics, Institute of Chemistry, The Hebrew University of Jerusalem, 9190401 Jerusalem, Israel. 4 Molecular Sensory Systems, Center of Advanced European Studies and Research (caesar), Ludwig-Erhard-Allee 2, 53175 Bonn, Germany. ✉email: matthias.broser@hu-berlin.de NeoR, a near-infrared absorbing rhodopsin Matthias Broser 1✉, Anika Spreen1, Patrick E. Konold2, Enrico Peter 1, Suliman Adam Igor Schapiro 3, Reinhard Seifert4, John T. M. Kennis 2, Yinth Andrea Bernal Sierra Matthias Broser 1✉, Anika Spreen1, Patrick E. Konold2, Enrico Peter 1, Suliman Adam 3, Veniamin Borin 3, Igor Schapiro 3, Reinhard Seifert4, John T. M. Kennis 2, Yinth Andrea Bernal Sierra 1 & Peter Hegemann 1 The Rhizoclosmatium globosum genome encodes three rhodopsin-guanylyl cyclases (RGCs), which are predicted to facilitate visual orientation of the fungal zoospores. Here, we show that RGC1 and RGC2 function as light-activated cyclases only upon heterodimerization with RGC3 (NeoR). RGC1/2 utilize conventional green or blue-light-sensitive rhodopsins (λmax = 550 and 480 nm, respectively), with short-lived signaling states, responsible for light- activation of the enzyme. The bistable NeoR is photoswitchable between a near-infrared- sensitive (NIR, λmax = 690 nm) highly ﬂuorescent state (QF = 0.2) and a UV-sensitive non- ﬂuorescent state, thereby modulating the activity by NIR pre-illumination. No other rhodopsin has been reported so far to be functional as a heterooligomer, or as having such a long wavelength absorption or high ﬂuorescence yield. Site-speciﬁc mutagenesis and hybrid quantum mechanics/molecular mechanics simulations support the idea that the unusual photochemical properties result from the rigidity of the retinal chromophore and a unique counterion triad composed of two glutamic and one aspartic acids. These ﬁndings substantially expand our understanding of the natural potential and limitations of spectral tuning in rhodopsin photoreceptors. 1 Institute for Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, 10115 Berlin, Germany. 2 Department of Physics and Astronomy, Faculty of Science, Vrije Universiteit Amsterdam, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands. 3 Fritz Haber Center for Molecular Dynamics, Institute of Chemistry, The Hebrew University of Jerusalem, 9190401 Jerusalem, Israel. 4 Molecular Sensory Systems, Center of Advanced European Studies and Research (caesar), Ludwig-Erhard-Allee 2, 53175 Bonn, Germany. ✉email: matthias.broser@hu-berlin.de 1 NATURE COMMUNICATIONS \| (2020) 11:5682 \| https://doi.org/10.1038/s41467-020-19375-8 \| www.nature.com/naturecommunicati TURE COMMUNICATIONS \| (2020) 11:5682 \| https://doi.org/10.1038 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19375-8 R h t v from distinct families revealed that RGC1/2 and RGC3(NeoR) from R. globosum separate as two distinct phylogenetic branches (Fig. 1a and Supplementary Fig. 1). In contrast the coiled-coil linker and the cyclase domain are highly conserved among all three proteins (Supplementary Fig. 2). R hodopsins are widely distributed sensory photoreceptors that, among other functions, promote different levels of vision in prokaryotes, algae, and animals1,2. Results F i Functional characterization of R. globosum RGCs. Phylogenetic analysis and multiple sequence alignment of the three R. globo- sum RGC rhodopsin modules with other microbial rhodopsins Normalized Photoresponse 0 0.5 1 λ (nm) 400 500 600 700 Absorbance (RGC2) Actionspectrum (NeoR/RGC2) 0 0.5 1 Normalized absorbance 10 s 200 nA RGC1 + NeoR RGC2 + NeoR NeoR RGC1 + RGC2 0 –0.3 –0.6 –0.9 –1.2 Initial slope nA/s RGC1 + NeoR RGC2 + NeoR RGC1 + RGC2 RGC1 RGC2 NeoR N=8 N=15 N=23 N=20 N=10 N=18 a b Tree scale: 1 Enzymerhodopsins RhoPDE RGC HKR HR PR BR NaR ACR MerMAIDs CCR Ion-pumping rhodopsins Channelrhodopsins RGC2 (R. globosum) RGC1 (R. globosum) NeoR ( NeoR (R. globosum R. globosum) c d e 1 10 0 200 400 600 800 1000 1200 1400 Photocurrent (pA) 660 nm cwi (N=8) Light intensity (mW·mm–2) EC50 = 4.60 mW·mm-2 100 pA 10 s Dark background (N=7) EC50 = 4.64 mW·mm-2 NeoR RGC1/2 GTP cGMP Fig. 1 Phylogeny, model, and activity of the Rhizoclosmatium globosum rhodopsin-guanylyl cyclases (RGCs). a Unrooted phylogenetic tree of microbial hodopsins, with red circles indicating bootstrap values >90%; the scale bar represents average amino acid substitutions per site. b Model of R. globosum heterodimeric RGCs: rhodopsins, magenta (RGC1/2) and cyan (NeoR); cyclase, blue; and linker, yellow. c Representative currents from Xenopus oocytes expressing RGC1/2 and NeoR individually or in combination, in response to a 2 s light pulse (yellow box); initial photocurrent slopes are plotted as mean ± standard error (s.e.m.); N = number of biological replicates. d Photocurrent action spectrum (red squares; means ± standard deviation (s.d.), derived from hree biological replicates) of RGC2/NeoR in ND7/23 cells, normalized to the maximum current underlayed by the RGC2 absorption. e Photocurrents means ± s.e.m., with N = number of biological replicates) of RGC2/NeoR heterodimers generated by 10 ms blue-light (490 nm) ﬂashes of different ntensities (as visualized in the inset) in the absence (blue cycles) or presence (red cycles) of 660 nm background light (continuous wave illumination cwi)). 10 s 200 nA RGC1 + NeoR RGC2 + NeoR NeoR RGC1 + RGC2 0 –0.3 –0.6 –0.9 –1.2 Initial slope nA/s RGC1 + NeoR RGC2 + NeoR RGC1 + RGC2 RGC1 RGC2 NeoR N=8 N=15 N=23 N=20 N=10 N=18 c a Tree scale: 1 Enzymerhodopsins RhoPDE RGC HKR HR PR BR NaR ACR MerMAIDs CCR Ion-pumping rhodopsins Channelrhodopsins RGC2 (R. globosum) RGC1 (R. NeoR, a near-infrared absorbing rhodopsin Although rhodopsins have been color tuned over a wide spectral range during evolution, as well as by molecular engineering3, the limits of detection towards both ends of the spectrum into the ultra- violet (UV) and far-red ranges have not been systematically explored experimentally or by theoretical approaches4. To characterize their function, we ﬁrst expressed the three putative RGCs of R. globosum in Xenopus laevis oocytes, in conjunction with the cGMP-activated cyclic nucleotide-gated (CNG)-A2 ion channel from rat olfactory neurons7. Photocur- rents were exclusively observed when RGC1 or RGC2 were co- expressed with NeoR and CNG-A2, while neither the combina- tion of RGC1 with RGC2 nor individually expressed constructs showed any light response (Fig. 1c). This suggested that both RGC1 and RGC2 are only functional after forming heterodimers with NeoR (RGC1/NeoR and RGC2/NeoR; Fig. 1b). To our knowledge, heterodimerization has not been reported for any rhodopsin, although it does occur with the rhodopsin-related odorant receptors (ORs) in Drosophila olfactory sensory neurons8. Next, we expressed RGC1, RGC2, and NeoR individually and in different combinations together with an engineered cGMP-gated potassium-selective channel in mamma- lian hybrid cells (ND7/23). Once again, neither RGC1, RGC2, nor NeoR alone were able to produce any photocurrents when co-expressed with the channel in ND7/23 cells. In contrast, In several ﬂagellated fungal zoospores, photoorientation is mediated by homodimeric rhodopsin cyclases (termed RhGCs or simply RGCs; Fig. 1a), in which rhodopsins are directly linked to type III guanylyl cyclases for cyclic GMP (cGMP) upregulation upon illumination (cartoon in Fig. 1b)5. Here, we describe two RGCs (RGC1 and RGC2) of the fungus Rhizoclosmatium globo- sum6, from the phylum Chytridiomycota (Fig. 1a and Supple- mentary Fig. 1), each containing a classic blue-green absorbing rhodopsin, which heterodimerize with another uncharacterized rhodopsin, RGC3 or neorhodopsin (NeoR), that we show has sensitivity in the near-infrared (NIR) spectrum. Results F i NATURE COMMUNICATIONS \| (2020) 11:5682 \| https://doi.org/10.1038/s41467-020-19375-8 \| www.nature.com/naturecommunicatio 2 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19375-8 RGC2/NeoR displayed photocurrents with maximal sensitivity in the blue, that were graded with the light-intensity with half- saturation EC50 = 4.6 mW mm−2 (Fig. 1d, e). Although the action spectrum shows no photoresponse in the red spectral range (Fig. 1d), continuous background-illumination with 660 nm light resulted in signiﬁcantly increased photocurrent amplitudes evoked by the application of 490 nm light ﬂashes in a light titration experiment, while the light saturation behavior of the signal remained unaffected (Fig. 1e red dots, Supplementary Fig. 3a). Enlarged photocurrent amplitudes were also observed in single cell experiments upon pre-illumination with a 660 nm light pulse for 15 s (Supplementary Fig. 3b, c). RGC1/NeoR tended to precipitate in large intracellular clusters and were only poorly targeted to the plasma membrane (Supplementary Fig. 4a), with the consequence that no photoresponses were observed in ND7/ 23 cells. known animal vision11. However, these photoreceptor proteins remain uncharacterized, and their light-sensitive cofactors are unknown. Notably, the used R. globosum strain JEL800 was ori- ginally isolated from puriﬁed shrimp chitin, although the sig- niﬁcance of this correlation is unclear12. NeoR is a photochromic bistable rhodopsin with a highly ﬂuorescent NIR-state. We further found that the NeoR main absorption band disappears upon illumination with far-red light and gives rise to short-wavelength absorption, with a maximum at 367 nm (Fig. 2b). This UV-absorbing state is also thermally stable but can be completely reconverted into the NIR state by illumination with UV light (Fig. 2c, d), thereby disclosing that NeoR is a photochromic bistable rhodopsin, i.e., a rhodopsin with two thermally stable states that are spectrally distinct2,13. The spectral properties are similar for the NeoR full-length protein and the rhodopsin fragment in detergent and protein/lipid nanodiscs (Supplementary Fig. 5a–c). According to these results, the increased photocurrents observed for RGC2/NeoR hetero- dimers in ND7/23 cells by illumination with far-red light may be assigned to NeoR that is switched between the two states NeoR690 and NeoR367. However, the application of UV light (365 nm) required for reconversion to RGC2/NeoR690 leads to strong enzyme activation and reduced patch quality, which hindered us from further investigating the reversibility of photocurrent modulation. Spectral properties of the rhodopsin domains. To better understand the spectral properties of the R. Results F i globosum RGCs, we expressed the rhodopsin fragments of all three proteins in human embryonic kidney cells (HEK-T) and insect cells (Sf21). The recombinant RGC1 (from HEK-T) and RGC2 (from Sf21) frag- ments were puriﬁed with low yield as red and yellow proteins with typical rhodopsin spectra showing maxima at 550 and 480 nm, respectively, the latter corresponding to the RGC2/NeoR- dimer action spectra measured in ND7/23 cells (Figs. 1e and 2a). NeoR expressed well in both systems and puriﬁed recombinant NeoR appeared turquoise or cyan (Inset Fig. 2a), with the main narrow band displaying an absorption maximum at 690 nm, a shoulder at 640 nm, and a second lower visible absorption around 400 nm. No other naturally occurring microbial rhodopsin ever showed a peak absorption beyond 610 nm9, and the spectra from only a few animal rhodopsins have been predicted as such, including the goldﬁsh and salamander red cones (λmax = 617 nm and 615 nm, respectively), both incorporating 3,4-dehydroretinal (A2-retinal) as chromophore10. Some of the many rhodopsins from crustacean shrimps, however, may have indeed maxima near 700 nm, a range that is far beyond human and any other Both, the NIR absorption and the spectral band shape resemble phytochromes that use linear tetrapyrroles as the chromophore (Fig. 2e). However, in the absence of retinal no functional rhodopsin was produced when recombinant NeoR was expressed in insect cells, whereas supplementation of the growth media with 3,4-dehydroretinal (A2-retinal) resulted a NeoR with an absorp- tion band even further bathochromic shifted to 759 nm (A2- NeoR; Fig. 2e) and well-preserved bimodal photoswitching. Such a large shift of 70 nm (1317 cm−1) for A2 compared to A1 has never been observed for a rhodopsin to our knowledge14,15. Results F i Similar to other retinylidene proteins, denaturation of NeoR leads to the appearance of a 440 nm absorption band (Supplementary Normalized absorbance 0 0.5 1 λ (nm) λ (nm) λ (nm) λ (nm) λ (nm) λ (nm) 400 500 600 700 800 690 nm 550 nm 480 nm 367 nm Normalized absorbance 0 0.5 1 500 600 700 800 900 NeoR (A2) NeoR (A1) iRFP (BV) A1-retinal A2-retinal BV NH NH HN N H S-Cys COOH COOH O O NH+ Lys NH+ Lys RGC2 RGC1 NeoR690 NeoR367 Normalized Fluorescence 0 0.5 1 600 700 800 Em@720 nm NeoR690 Ex@650 nm NeoR690 Ex@650 nm NeoR367 690 nm 707 nm Absorbance NeoR690 Bleaching 640 nm Recovery 405 nm i) ii) iii) a b c d 400 500 600 700 800 -0.3 -0.2 -0.1 0 0.1 ΔAbs (OD) NeoR690 EADS 1.1 ns 465 nm excited state absorption Stimulated emission 708 nm 762 nm e f 0 1 2 3 Delay / ns -0.2 0 ΔAbs / OD 708 nm fit Absorbance at 690 nm 0.3 0.4 0.5 Time (s) 0 1.000 2.000 3.000 680 nm 375 nm Absorbance 0 0.1 0.2 0.3 300 400 500 600 700 800 NeoR690 NeoR690 60 s NeoR690 120 s NeoR690 240 s NeoR690 420 s 367 nm 690 nm Absorbance 0 0.1 0.2 0.3 300 400 500 600 700 800 NeoR367 NeoR367 10 s NeoR367 20 s NeoR367 60 s NeoR367 180 s 367 nm 690 nm Illumination with 680 nm Illumination with 375 nm g h Fig. 2 Spectral properties of puriﬁed rhodopsin domains from the R. globosum RGCs. a Normalized absorption spectra of the puriﬁed recombinant rhodopsin domains from RGC1, RGC2, and NeoR (red and UV-form); picture of the puriﬁed NeoR sample, inset. b Reversible bleaching of NeoR690 with 680 nm light. c Reversible bleaching of NeoR367 with 375 nm light. d Kinetic traces of NeoR bleaching with 680 nm light and subsequent recovery by 375 nm illumination. e Absorption spectra of puriﬁed NeoR expressed in media supplemented with A1-retinal or 3,4-dihydro-retinal (A2), as compared to those from biliverdin (BV)-containing iRFP50. f Fluorescence spectra of NeoR690: excitation (dots) overlaid with the absorbance (red) and emission (green). g Evolution-associated difference spectra (EADS) of fs–ns transient absorption spectra derived from pump-probe experiments, time trace at 708 nm, inset h Representative confocal images of NeoR ﬂuorescence (red) and the C-terminal eGFP tag (green) in ND7/23 cells. Results F i globosum) NeoR ( NeoR (R. globosum R. globosum) a c Normalized Photoresponse 0 0.5 1 λ (nm) 400 500 600 700 Absorbance (RGC2) Actionspectrum (NeoR/RGC2) 0 0.5 1 Normalized absorbance NeoR NeoR RGC2 b d e 1 10 0 200 400 600 800 1000 1200 1400 Photocurrent (pA) 660 nm cwi (N=8) Light intensity (mW·mm–2) EC50 = 4.60 mW·mm-2 100 pA 10 s Dark background (N=7) EC50 = 4.64 mW·mm-2 NeoR RGC1/2 GTP cGMP Normalized Photoresponse 0 0.5 1 λ (nm) 400 500 600 700 Absorbance (RGC2) Actionspectrum (NeoR/RGC2) 0 0.5 1 Normalized absorbance d e 1 10 0 200 400 600 800 1000 1200 1400 Photocurrent (pA) 660 nm cwi (N=8) Light intensity (mW·mm–2) EC50 = 4.60 mW·mm-2 100 pA 10 s Dark background (N=7) EC50 = 4.64 mW·mm-2 P Normalized Photoresponse 0 0.5 1 λ (nm) 400 500 600 700 Absorbance (RGC2) Actionspectrum (NeoR/RGC2) 0 0.5 1 Normalized absorbance d b NeoR RGC1/2 GTP cGMP e 1 10 0 200 400 600 800 1000 1200 1400 Photocurrent (pA) 660 nm cwi (N=8) Light intensity (mW·mm–2) EC50 = 4.60 mW·mm-2 100 pA 10 s Dark background (N=7) EC50 = 4.64 mW·mm-2 d b e e Light intensity (mW·mm–2) Fig. 1 Phylogeny, model, and activity of the Rhizoclosmatium globosum rhodopsin-guanylyl cyclases (RGCs). a Unrooted phylogenetic tree of microbial rhodopsins, with red circles indicating bootstrap values >90%; the scale bar represents average amino acid substitutions per site. b Model of R. globosum heterodimeric RGCs: rhodopsins, magenta (RGC1/2) and cyan (NeoR); cyclase, blue; and linker, yellow. c Representative currents from Xenopus oocytes expressing RGC1/2 and NeoR individually or in combination, in response to a 2 s light pulse (yellow box); initial photocurrent slopes are plotted as mean ± standard error (s.e.m.); N = number of biological replicates. d Photocurrent action spectrum (red squares; means ± standard deviation (s.d.), derived from three biological replicates) of RGC2/NeoR in ND7/23 cells, normalized to the maximum current underlayed by the RGC2 absorption. e Photocurrents (means ± s.e.m., with N = number of biological replicates) of RGC2/NeoR heterodimers generated by 10 ms blue-light (490 nm) ﬂashes of different intensities (as visualized in the inset) in the absence (blue cycles) or presence (red cycles) of 660 nm background light (continuous wave illumination (cwi)). Results F i NeoR is reversibly bleached by illumination with 640- and 405 nm light. The reversible bleaching of NeoR ﬂuorescence in single cells was repeated in three independent experiments using ﬁve ﬁelds of view each. Scale bar, 100 µm. d Absorbance at 690 nm 0.3 0.4 0.5 Time (s) 0 1.000 2.000 3.000 680 nm 375 nm λ (nm) b Absorbance 0 0.1 0.2 0.3 300 400 500 600 700 800 NeoR690 NeoR690 60 s NeoR690 120 s NeoR690 240 s NeoR690 420 s 367 nm 690 nm Illumination with 680 nm Normalized absorbance 0 0.5 1 λ (nm) 400 500 600 700 800 690 nm 550 nm 480 nm 367 nm RGC2 RGC1 NeoR690 NeoR367 a λ (nm) c Absorbance 0 0.1 0.2 0.3 300 400 500 600 700 800 NeoR367 NeoR367 10 s NeoR367 20 s NeoR367 60 s NeoR367 180 s 367 nm 690 nm Illumination with 375 nm b c a d λ (nm) Normalized Fluorescence 0 0.5 1 600 700 800 Em@720 nm NeoR690 Ex@650 nm NeoR690 Ex@650 nm NeoR367 690 nm 707 nm Absorbance NeoR690 f λ (nm) 400 500 600 700 800 -0.3 -0.2 -0.1 0 0.1 ΔAbs (OD) NeoR690 EADS 1.1 ns 465 nm excited state absorption Stimulated emission 708 nm 762 nm 0 1 2 3 Delay / ns -0.2 0 ΔAbs / OD 708 nm fit g λ (nm) Normalized absorbance 0 0.5 1 500 600 700 800 900 NeoR (A2) NeoR (A1) iRFP (BV) A1-retinal A2-retinal BV NH NH HN N H S-Cys COOH COOH O O NH+ Lys NH+ Lys e h f g Bleaching 640 nm Recovery 405 nm i) ii) iii) h e Fig. 2 Spectral properties of puriﬁed rhodopsin domains from the R. globosum RGCs. a Normalized absorption spectra of the puriﬁed recombinant rhodopsin domains from RGC1, RGC2, and NeoR (red and UV-form); picture of the puriﬁed NeoR sample, inset. b Reversible bleaching of NeoR690 with 680 nm light. c Reversible bleaching of NeoR367 with 375 nm light. d Kinetic traces of NeoR bleaching with 680 nm light and subsequent recovery by 375 nm illumination. e Absorption spectra of puriﬁed NeoR expressed in media supplemented with A1-retinal or 3,4-dihydro-retinal (A2), as compared to those from biliverdin (BV)-containing iRFP50. f Fluorescence spectra of NeoR690: excitation (dots) overlaid with the absorbance (red) and emission (green). ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19375-8 Fig. 6a–d), resembling the absorption of the protonated retinal Schiff base (RSBH+) in water. RSBH+) from 440 nm in solution to 690 nm, corresponding to an opsin shift of 250 nm (8235 cm−1). Shortly after discovery of the light-driven proton pump bacteriorhodopsin as the ﬁrst microbial rhodopsin (BR, λmax = 568 nm), Warshel as well as Honig and coworkers proposed a point charge model to explain the 128 nm (5121 cm−1) BR opsin shift, which was considered large at that time. This model involves two negative charges, one of which operates as a counterion near the RSBH+ and the other one near the β-ionone ring24–26. Upon excitation an intramolecular charge transfer occurs between the Schiff base nitrogen and the β-ionone ring. Translocation of the charge has differential effects on the electronic ground- and excited-state energy levels, which pro- vided a qualitative explanation for the origin of the opsin shift. While the proposed negatively charged residue close to the β- ionone ring was not found in any microbial rhodopsin, it is widely accepted that increasing the polarity near the ring still promotes opsin shifts4,27,28. ( ) A further atypical property for a rhodopsin is the strong ﬂuorescence of NeoR690 with an emission maximum at 707 nm, a quantum yield of ΦF = 0.2 (20%) and a small Stokes shift of only 350 cm−1 (17 nm, Fig. 2f and Supplementary Fig. 7). The small Stokes shift as well as the pronounced mirroring vibrational structure of the emission and absorption spectra indicate a close proximity between the Frank-Condon region and the minimum of the S1 potential energy surface. This proximity implies that the chromophore is undergoing only a small geometrical or structural relaxation. Hence, we conclude that the presence of a barrier in the S1 excited state prevents the photoisomerisation and promotes the excited-state lifetime to the nanosecond range16. We further investigated the excited-state dynamics of NeoR using pump-probe experiments and conﬁrmed a long excited-state lifetime of τ = 1.1 ns (Fig. 2g). In contrast, all other known rhodopsins have a negligible ﬂuorescence, with ΦF values in the order of 10−5 for bovine rhodopsin and HKR117,18 up to 9 × 10−4 for the Arch19 and proteorhodopsin20 proton pumps correspond- ing to excited-state lifetimes in the range of 100 fs to a few ps. ARTICLE Additionally, substantial bioengineering efforts to increase the ﬂuorescence of microbial rhodopsins as voltage sensors or incorporation of retinal analogs have yielded maximal ΦF values of 3.3%21–23. Here, we note that although we have not yet been able to observe a voltage dependency of NeoR ﬂuorescence in mammalian cells, the NIR ﬂuorescence can be utilized for monitoring RGC/NeoR expression in host cells, and this ﬂuorescence may be switched on and off by alternating UVA and far-red light (Fig. 2h and Supplementary Fig. 4a, b). The great ﬂuorescence brightness, which is the product of the large extinction coefﬁcient, ε690 = 129000 M−1 cm−1 (Supplementary Fig. 6a), and high quantum yield ΦF (0.2) suggests NeoR to be suitable for ﬂuorescence-imaging in the NIR spectral range. p p In order to understand how NeoR enables NIR-light absorp- tion, we generated a homology model of the protein. Since no structure with more than 17% sequence identity was available, we combined multiple crystal structures of various microbial rhodopsins matching different regions of NeoR (see SI for details). Despite the low-overall sequence similarity, many of the conserved residues that constitute the chromophore binding pocket in other microbial rhodopsins (e.g., BR) were also present in NeoR. Thus, we did not detect any obvious clues to immediately explain the NIR absorption of NeoR. Therefore, to further elucidate the molecular basis for the large NeoR opsin shift, we mutated the amino acid residues within a radius of ~5 Å from the retinal (Fig. 3a) by changing the polarity with only modest alteration of the structure and referring to analogous mutations carried out in BR (Supplementary Table 1). We found that mutation of residues along the polyene chain that are predicted to increase the polarizability of the NeoR retinal-binding pocket, namely H134Y, E141C/Q, and M192L, induce only small spectral shifts of <10 nm (Supplementary Table 1). More signiﬁcant spectral shifts to the blue (up to ~30 nm) and reduction of ﬂuorescence are achieved by mutation of S191A and T238P/A, which are located near the β- ionone ring (Fig. 3b–e). These effects are additive, with a 65 nm blue shift observed in the double mutant (S191A, T238A). The molecular basis of NeoR NIR absorption maximum. ARTICLE The unique photochemical properties raise the question of how the protein environment in NeoR can shift absorption of the proto- nated retinyl imine (also named protonated retinal Schiff base, NeoR mutations max / nm max / nm sw WT 690 + 20 W110Y 677 -13 + E136Q 420 -270 W137F 677 -13 + D140N 686 -4 + D140C 676 -14 + 13 D140T 630 -60 + E141C 697 +7 + S191A 675 -15 + 15 W234F 693 +3 + T238P 662 -28 + T238A 664 -26 + 10 W241H 686 -4 + E262Q 394 -296 dp D140C. S191A 643 -47 + 5 T238A. S191A 625 -65 +* F / % dp 5 a b c d e W241 W137 W234 E141 T238 I237 H134 S191 M192 D140 E136 E262 R133 Y195 (K265) W110 Normalized absorbance 0 1 2 3 4 5 (nm) 500 600 700 800 NeoR NeoR T238A NeoR D140T Normalized fluorescence 0 0.5 1 600 650 700 Normalized fluorescence 0 0.5 1 (nm) (nm) 700 750 800 NeoR D140C S191A T238A D140T Fig. 3 Molecular model and spectral features of NeoR mutants. a Homology model of the NeoR retinal-binding pocket, with the amino acids targeted by mutagenesis drawn as sticks. b Absorption spectra of NeoR mutants; spectra are normalized to the respective UV-form. c Spectral features of NeoR mutants; sw, bimodal switchable; dp, depronotated chromophore; ФF, ﬂuorescence quantum yield (in %). d Emission spectra (at 650 nm), and e excitation spectra (at 720 nm) of wild-type (WT) NeoR and NeoR mutants. 4 NATURE COMMUNICATIONS \| (2020)11 5682 \| htt //d i /10 1038/ 41467 020 19375 8 \| t / t i ti NeoR mutations max / nm max / nm sw WT 690 + 20 W110Y 677 -13 + E136Q 420 -270 W137F 677 -13 + D140N 686 -4 + D140C 676 -14 + 13 D140T 630 -60 + E141C 697 +7 + S191A 675 -15 + 15 W234F 693 +3 + T238P 662 -28 + T238A 664 -26 + 10 W241H 686 -4 + E262Q 394 -296 dp D140C. S191A 643 -47 + 5 T238A. Results F i g Evolution-associated difference spectra (EADS) of fs–ns transient absorption spectra derived from pump-probe experiments, time trace at 708 nm, inset. h Representative confocal images of NeoR ﬂuorescence (red) and the C-terminal eGFP tag (green) in ND7/23 cells. NeoR is reversibly bleached by illumination with 640- and 405 nm light. The reversible bleaching of NeoR ﬂuorescence in single cells was repeated in three independent experiments using ﬁve ﬁelds of view each. Scale bar, 100 µm. NATURE COMMUNICATIONS \| (2020) 11:5682 \| https://doi.org/10.1038/s41467-020-19375-8 \| www.nature.com/naturecommunications 3 3 NATURE COMMUNICATIONS \| (2020) 11:5682 \| https://doi.org/10.1038/s41467-020-19375-8 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19375-8 Nevertheless, the spectral changes are in the range of analogous mutations in many other microbial rhodopsins or even smaller and do not explain the NIR absorption of NeoR3,22,27–32. QM region. The treatment of the carboxylates by a quantum chemical method allowed to include polarization effects which is an improvement compared to the static point charges typically chosen in the MM region. Further, it provides a ﬂexibility with respect to the protonation state because a proton can be trans- ferred among the titratable residues in the QM region. Since the protonation states of these three titratable residues could be decisive for the spectral tuning, we systematically studied four scenarios where the three potential counterions have a total charge of 0, −1, −2, and −3 (Fig. 4a). Irrespective of the pro- tonation patterns, decreasing the number of charged carboxylates systematically red-shifted the spectrum (Fig. 4a). Notably, the excited-state energy decreased substantially upon reducing the number of negatively charged carboxylates from 2 to 1, while only a small further red shift was observed for the protonation of all three carboxylates (Fig. 4a). The latter, fully protonated conﬁg- uration, however, turned out to be unstable during molecular dynamics (MD) simulations of the entire protein embedded in the membrane. In contrast, the models derived from QM/MM with only one carboxylate, either E136 or E262 deprotonated (systems 5 and 6/7, Fig. 4) were stable during long MD runs of 300 ns. Hence, the strong red shift obtained from QM/MM simulations as well as the stability during a classical MD simu- lation points towards a single deprotonated glutamate. We found the active site, deﬁned by the RSBH+ and its direct protein- and possibly water-environment to be by far the most critical region for the far-red shifted absorption in NeoR. The two RSBH+ aspartate counterions, D85 and D212, in BR are replaced by glutamates, E136 and E262, in NeoR. Mutation of either one of these to Gln results in chromophore deprotonation (Fig. 3c), whereas Asp substitution completely prevents retinal binding in both cases (Supplementary Table 1). In contrast, when the unique third Asp, D140, which is predicted to interact with E136, is replaced by Thr (T89 in BR), the spectrum becomes broad and strongly blue shifted by 60 nm (Fig. 3b), while the photoreceptor retains its bistability. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19375-8 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19375-8 Protonation nm 1 2 x 3 x 4 x 5 x x 6 x* x 7 x x 8 x x x 3.21 2.33 2.08 2.36 1.79 1.82 1.82 1.74 eV 386 533 597 524 693 682 681 712 E136 D140 E262 -3 -2 -2 -2 -1 -1 -1 0 # Charge MAX QM/MM optimization moves the proton to D140 System 5 System 6/7 D140 D140 E136 E136 E262 E262 D140 E136 E262 System 6/7 D140 E136 E262 System 5 a b c Fig. 4 QM/MM Modeling of NeoR. a Overview of QM/MM excitation energy calculations of eight systems (#1–8) with varying protonation of E136, D140 and E262 as indicated. The total charge of the three carboxylates in each system is given. λMAX refers to the absorption maxima as derived from excited- state energy calculation. b Active site from QM/MM-optimized models, with hydrogen bonds represented as dashed lines. c Water intrusion into NeoR as observed during subsequent 300 ns MD simulations with water densities visualized as red meshes. The retinal and amino acid sidechains are taken from the initial structure of the MD simulations. Protonation nm 1 2 x 3 x 4 x 5 x x 6 x x 7 x x 8 x x x 3.21 2.33 2.08 2.36 1.79 1.82 1.82 1.74 eV 386 533 597 524 693 682 681 712 E136 D140 E262 -3 -2 -2 -2 -1 -1 -1 0 # Charge MAX QM/MM optimization moves the proton to D140 a b System 5 D140 E136 E262 b a System 5 System 6/7 D140 D140 E136 E136 E262 E262 D140 E136 E262 System 6/7 D140 E136 E262 System 5 c c D140 E136 E262 System 5 c Fig. 4 QM/MM Modeling of NeoR. a Overview of QM/MM excitation energy calculations of eight systems (#1–8) with varying protonation of E136, D140 and E262 as indicated. The total charge of the three carboxylates in each system is given. λMAX refers to the absorption maxima as derived from excited- state energy calculation. b Active site from QM/MM-optimized models, with hydrogen bonds represented as dashed lines. c Water intrusion into NeoR as observed during subsequent 300 ns MD simulations with water densities visualized as red meshes. The retinal and amino acid sidechains are taken from the initial structure of the MD simulations. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19375-8 y The D140 homolog position occupied by Thr in many microbial rhodopsins has been widely disregarded by experimentalists but was suggested by theoreticians to contribute to the proton transfer from RSBH+ to the acceptor Asp or Glu in rhodopsin pumps and channels33–35. Moreover, replacement of this Thr in the proton pump Arch to Cys, T99C, in combination with the D95E mutation of the primary proton acceptor shifted the absorption from 556 nm to 626 nm thus creating the largest bathochromic color shift and the most red-shifted microbial rhodopsin which, however, remained unexplained21. Previous studies on the Chrimson channelrhodopsin have also demonstrated the importance of residues at the D140 homologous position by inducing color shifts beyond 600 nm upon modiﬁcation of this location31. The most extended color tuning study on a retinal protein was carried on the human cellular retinol binding protein II (hCRBPII) that was converted into a retinal binder36. Mutagenesis of the retinal- binding pocket lead to a protein with absorption at 644 nm37,38, a strong bathochromic shift that depends on the even distribution of the electrostatic potential across the entire polyene. However, one has to point out that hCRBPII is missing any RSBH+ counterion and our data demonstrate that the situation is clearly different in NeoR with low importance of the binding pocket and a main contribution of the active site. Analysis of the QM/MM optimized geometries shows that the negative charge is located relatively far from the RSBH+ (Fig. 4b). Hence, the ground state energy is less stabilized with respect to the electronically excited state, in accordance with the point charge model. All active-site models (system 1–8) have been subjected to extended MD simulations with the protein embedded in the membrane. Interestingly, low water density was found close to the RSBH+ for models with one deprotonated counterion (system 5−7, Fig. 4c), but signiﬁcant water content was present in models of higher total charge (system 1–4). As water promotes extensive ground state dynamics in other microbial rhodopsins39, its absence would explain the narrow spectrum observed for NeoR and thus support an active site model with only one charged carboxylate. Hybrid quantum mechanics/molecular mechanics simulations of NeoR. The contribution of the critical E136, D140, and E262 to the spectral properties of NeoR was further investigated by a hybrid quantum mechanics/molecular mechanics (QM/MM) approach, with the retinal and all three sidechains included in the ARTICLE S191A 625 -65 + F / % dp 5 c d e Normalized fluorescence 0 0.5 1 600 6 Normalized fluorescence 0 0.5 1 (nm) 700 750 NeoR mutations max / nm max / nm sw WT 690 + 20 W110Y 677 -13 + E136Q 420 -270 W137F 677 -13 + D140N 686 -4 + D140C 676 -14 + 13 D140T 630 -60 + E141C 697 +7 + S191A 675 -15 + 15 W234F 693 +3 + T238P 662 -28 + T238A 664 -26 + 10 W241H 686 -4 + E262Q 394 -296 dp D140C. S191A 643 -47 + 5 T238A. S191A 625 -65 +* F / % dp 5 c a b W241 W137 W234 E141 T238 I237 H134 S191 M192 D140 E136 E262 R133 Y195 (K265) W110 Normalized absorbance 0 1 2 3 4 5 (nm) 500 600 700 800 NeoR NeoR T238A NeoR D140T d Normalized fluorescence 0 0.5 1 (nm) 700 750 800 NeoR D140C S191A T238A D140T d a c b e Normalized fluorescence 0 0.5 1 600 650 700 (nm) e Fig. 3 Molecular model and spectral features of NeoR mutants. a Homology model of the NeoR retinal-binding pocket, with the amino acids targeted by mutagenesis drawn as sticks. b Absorption spectra of NeoR mutants; spectra are normalized to the respective UV-form. c Spectral features of NeoR mutants; sw, bimodal switchable; dp, depronotated chromophore; ФF, ﬂuorescence quantum yield (in %). d Emission spectra (at 650 nm), and e excitation spectra (at 720 nm) of wild-type (WT) NeoR and NeoR mutants. 4 4 ARTICLE Discussion Crude membranes of NeoR-rhodopsin fragment were solubilized at room temperature for 16 h using 2% SMA (w/w) in HEPES buffer with 1 mM PMSF. After ultra- zentrifugation at 146,550 x g for 30 min at 4 °C using a Beckman 70.1 Ti rotor, the supernatend was applied to a Strep-Tactin®XT 4Flow® gravity ﬂow column (IBA GmbH, Göttingen, Germany), washed with 50 mM HEPES pH 7.4, 150 mM NaCl and eluted with 2xBTX buffer (IBA GmbH). The formation of SMALPS was proofed by dynamic light scattering (DynaPro NanoStar, Wyatt Technology Cor- poration, Santa Barbara, CA 93117, USA) conﬁrming the expected particle size of ~11 nm. Mutant screening in human embryonic kidney (HEK)-T cells. For expression in mammalian cells, the rhodopsin domains of NeoR (UniProtKB A0A1Y2CSJ0; aa: 1-279) and RGC1/2 (UniProtKB A0A1Y2CSF8; aa: 1-320/UniProtKB A0A1Y2CSL9; aa: 1-319, respectively), as well as the full-length proteins from Chytriomyces confervae (NeoR1: UniProt A0A507FHL0 and RGC1: UniProt A0A507F303) were cloned into the EGFP-C1 vector (Clonetech, Takara Bio, Kusatsu, Shiga Prefecture, Japan) with C-terminal 1D4-tags (TETSQVAPA). Site- directed mutagenesis was performed according to the QuikChange protocol from Agilent, using Pfu polymerase (Agilent Technologies, Santa Clara, CA, USA) using the primers listed in the Supplementary Data 2. For each construct, seven petri dishes (10-cm diameter) of HEK-T (ECACC 12022001, grown in Dulbecco’s Modiﬁed Eagle’s Medium supplemented with 1% fetal bovine serum and Penicillin–Streptomycin (Gibco, Thermo Fisher Scientiﬁc)) cells were transfected using TurboFect (Thermo Fisher Scientiﬁc), according to the manufacturer’s protocol. At 2 days post transfection, cells reached >90 % conﬂuence and were harvested and washed with Dulbecco’s phosphate-buffered saline (DPBS; Gibco, Thermo Fisher Scientiﬁc), containing 2X complete protease inhibitor (Merck, Darmstadt, Germany). The cell pellets were resuspended in 1-ml binding buffer (see above), and membrane proteins were solubilized with 1.5% DDM/0.3% CHS for 4 h at 4 °C. Insoluble material and cell debris were separated by centrifugation at 21,000 x g for 20 min at 4 °C; 150 µl 1D4 afﬁnity beads (CUBE Biotech GmbH, Monheim, Germany) were added to the supernatant, and this was incubated at 4 °C overnight. Beads were washed three times with 1-ml washing buffer (see above), and bound proteins were eluted in two steps with washing buffer, supplemented ﬁrst with 0.3- and then with 1 mg/ml 1D4 peptide. Discussion The supernatant of the solubilized NeoR-rhodopsin fragment was applied to a 5 ml Strep-Tactin®XT Superﬂow® column (IBA GmbH, Göttingen, Germany) and washed with ten column volumes of washing buffer. Protein was eluted with 2X BTX-buffer, containing 100 mM biotin (IBA GmbH), supplemented with 0.02% DDM/0.004% CHS, and colored fractions were pooled and concentrated in washing buffer (Amicon Ultra Centrifugal Filter, molecular weight cutoff (MWCO) 100 kDa, Merck Millipore, Burlington, MA, USA). g ( pp y g ) Admittedly, although we observed a promotion of photo- currents upon pre-illumination with NIR-light, the mechanism of how NeoR is modifying the photoreceptor response remains unclear. We note that the NeoR690 absorption overlaps strongly with the expected broad ﬂuorescence spectrum of RGC1 and RGC2 (Supplementary Fig. 10) thereby potentially allowing Förster resonant energy transfer (FRET) from RGC1/2 to NeoR690 within the RGC heterodimer. Such energy transfer would reduce the photoresponse in the NeoR690 state, but would disappear after NIR illumination and conversion of NeoR690 to NeoR367. A FRET calculation (as detailed in the SI) indicated energy transfer with expected lifetimes in the order of 1–10 ps competing with the sub-ps to ps isomerization of RGC1/2. As an alternative we could imagine an allosteric down-modulation of the activity by NeoR690, which is relieved by conversion to NeoR367. It is known from other heterodimeric type III cyclases that enzyme activation can follow a complex mechanism inﬂu- enced by multiple stimuli, as the synergistic activation of G- protein and Ca2+/calmodulin-binding described for mammalian pseudo-heterodimeric membrane-bound adenylyl cyclase43. Chytridiomycota fungi are ubiquitous microbes present in var- ious aquatic habitats and show a high diversity of saprobiotic and parasitic lifestyles44. Thus, a more complex photo-perceptive system might be requested, which includes sensing of far-red over UVA, whereas direct light-activation of the cyclase can be swit- ched between green (RGC1) and blue-light (RGC2) by differential expression. The three proteins—RGC1, RGC2, and NeoR— together form a photosensory system that covers the entire visible spectrum, expanding into both directions, namely UVA and NIR (Fig. 2a) and thereby blurring the spectral boundaries between rhodopsins and phytochromes. Protein puriﬁcation in SMALPS. Styrene-maleic acid copolymer (SMA 3:1; 20% (w/w) Xiran sl25010 s25, Polyscope Polymers B.V., 6161 CZ Geleen,The Nether- lands) was extensivly dialyzed against 50 mM HEPES pH 7.4, 150 mM NaCl. Discussion During the last ﬁfteen years microbial rhodopsins have attained much attention due to their employment in optogenetic experi- ments especially in the neurosciences for the modulation cellular activities with light. Especially Channelrhodopsins are widely used for neuronal stimulation with unprecedented spatial and temporal precision. Despite the enormous impact of this opto- genetic approach, one of the major limitations of this technique is the low penetration depth of visual light in biological tissues. In contrast to all known rhodopsins, both absorption and ﬂuores- cence of NeoR are within the NIR window (Supplementary Fig. 11), marking this photoreceptor an excellent starting point for developing optogenetic tools that are excited by NIR-light with as-yet-unreached penetration depths, which will enhance our capability for non-invasive in vivo experiments. TEVC measurements in Xenopus oocytes. For two-electrode voltage clamp (TEVC) experiments, cRNAs coding for NeoR and RGC1/2 were synthesized from linearized DNA (NheI), using the mMESSAGE mMACHINE TM T7 Transcription Kit, according to the manufacturer instructions (Invitrogen). Between 2 and 15 ng of total cRNA from either a single construct or a mixture of two were then injected into Xenopus laevis oocytes, together with 5 ng cRNA coding for the cGMP- sensitive cyclic nucleotide-gated (CNG)A2 ion channel from rat olfactory neurons (gb: 6978671, NP_037060.1; K1/2cAMP = 36 μM, K1/2 cGMP = 1.3 μM)7. Oocytes were incubated at 18 °C in Ringer’s solution, containing 96 mM NaCl, 5 mM KCl, Discussion Our data indicate that the speciﬁc spatial organization of the RSBH+ environment, which enables an unusual delocalization of a single negative charge, is responsible for the narrow red-shifted TURE COMMUNICATIONS \| (2020) 11:5682 \| https://doi.org/10.1038/s41467-020-19375-8 \| www.nature.com/naturecommunications 5 5 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19375-8 absorption. We note, however, that structural data will be needed to obtain a more detailed understanding of the chromophore- protein interaction. The polarizability of the retinal-binding pocket determines most of the color shift in the retinal-binding protein hCRBPII40, but only ﬁne tunes the spectral properties of the chromophore in NeoR, similarly as reported from QM/MM calculations on animal rhodopsins41. We predict that similarly rigid chromophore constellations will be found in other enzyme or sensory rhodopsins, but not in ion-transporting rhodopsins— pumps or channels—for which access of water to the active site is essential for function. Indeed, we found a similar active site constellation in an RGC from the related fungus Chytriomyces confervae42, which shares sequence features with the R. globosum NeoR. This CcNeoR1 also exhibits a bistable photochromic chromophore with an absorption maxima at 677 nm and 362 nm. It forms functional heterodimeric photorecptors with CcRGC1 from the same organism (Supplementary Fig. 9) (UniProtKB A0A1Y2CSJ0; aa: 1–279) and rhodopsin-guanylyl-cyclase (RGC)1 and 2 (UniProtKB A0A1Y2CSF8; aa: 1-320 and UniProtKB A0A1Y2CSL9; 1-319, respectively) from Rhizoclosmatium globosum were cloned into the pFastBac1 vector (Invitrogen, Thermo Fisher Scientiﬁc, Waltham, MA, USA), with a C- terminal strepII-tag for NeoR and His(8)-tags for RGC1/2. Further NeoR full- length sequence (UniProtKB A0A1Y2CSJ0; aa: 1-509) was cloned into the pFast- Bac1 vector with C-terminal His(8)-tag. Bacmid preparation, transfection, virus production, and expression were performed following the instruction of the manufacturer (Invitrogen)45. Crude membranes were prepared according to a protocol developed for G-protein-coupled receptors (GPCRs)46. Membranes were solubilized at 4 °C for at least 4 h in binding buffer, containing 50 mM HEPES (pH 7.4), 200 mM NaCl, 1 mM phenylmethylsulfonyl ﬂuoride (PMSF), and 5 µM all- trans retinal, with 1.5% n-dodecyl β-D-maltoside (DDM)/0.3% cholesteryl hemi- succinate (CHS), followed by centrifugation at 30,000 x g for 15 min at 4 °C. Full- length NeoR protein was solubilized overnight at 4 °C in a mixture of DDM/CHS/ 1,2-Dimirystoyl-sn-glycerol-3-phosphocholine(DMPC)/N,N-dimethyl-n-dodecy- lamine N-oxide (LDAO) in a ﬁnal concentration of 2%/0.5%/0.01%/0.25% (w/v)45, but otherwise handled similar as RGC1/2. Discussion For His-tagged proteins, solubilization buffer was supplemented with 30 mM imidazole, and the proteins were puriﬁed using 5 ml HisTrap columns (GE Healthcare, Chicago, IL, USA). Supernatants were applied to the columns, washed with washing buffer (50 mM HEPES (pH 7.4), 200 mM NaCl, 0.02% DDM/0.004% CHS) supplemented with 50 mM Imidazole, eluted with washing buffer with 500 mM imidazole, and concentrated in washing buffer. The supernatant of the solubilized NeoR-rhodopsin fragment was applied to a 5 ml Strep-Tactin®XT Superﬂow® column (IBA GmbH, Göttingen, Germany) and washed with ten column volumes of washing buffer. Protein was eluted with 2X BTX-buffer, containing 100 mM biotin (IBA GmbH), supplemented with 0.02% DDM/0.004% CHS, and colored fractions were pooled and concentrated in washing buffer (Amicon Ultra Centrifugal Filter, molecular weight cutoff (MWCO) 100 kDa, Merck Millipore, Burlington, MA, USA). (UniProtKB A0A1Y2CSJ0; aa: 1–279) and rhodopsin-guanylyl-cyclase (RGC)1 and 2 (UniProtKB A0A1Y2CSF8; aa: 1-320 and UniProtKB A0A1Y2CSL9; 1-319, respectively) from Rhizoclosmatium globosum were cloned into the pFastBac1 vector (Invitrogen, Thermo Fisher Scientiﬁc, Waltham, MA, USA), with a C- terminal strepII-tag for NeoR and His(8)-tags for RGC1/2. Further NeoR full- length sequence (UniProtKB A0A1Y2CSJ0; aa: 1-509) was cloned into the pFast- Bac1 vector with C-terminal His(8)-tag. Bacmid preparation, transfection, virus production, and expression were performed following the instruction of the manufacturer (Invitrogen)45. Crude membranes were prepared according to a protocol developed for G-protein-coupled receptors (GPCRs)46. Membranes were solubilized at 4 °C for at least 4 h in binding buffer, containing 50 mM HEPES (pH 7.4), 200 mM NaCl, 1 mM phenylmethylsulfonyl ﬂuoride (PMSF), and 5 µM all- trans retinal, with 1.5% n-dodecyl β-D-maltoside (DDM)/0.3% cholesteryl hemi- succinate (CHS), followed by centrifugation at 30,000 x g for 15 min at 4 °C. Full- length NeoR protein was solubilized overnight at 4 °C in a mixture of DDM/CHS/ 1,2-Dimirystoyl-sn-glycerol-3-phosphocholine(DMPC)/N,N-dimethyl-n-dodecy- lamine N-oxide (LDAO) in a ﬁnal concentration of 2%/0.5%/0.01%/0.25% (w/v)45, but otherwise handled similar as RGC1/2. For His-tagged proteins, solubilization buffer was supplemented with 30 mM imidazole, and the proteins were puriﬁed using 5 ml HisTrap columns (GE Healthcare, Chicago, IL, USA). Supernatants were applied to the columns, washed with washing buffer (50 mM HEPES (pH 7.4), 200 mM NaCl, 0.02% DDM/0.004% CHS) supplemented with 50 mM Imidazole, eluted with washing buffer with 500 mM imidazole, and concentrated in washing buffer. Protein expression and puriﬁcation. For insect cell expression (Sf21 cells (Gibco, Thermo Fisher Scientiﬁc, Waltham, MA, USA)), the rhodopsin domains of NeoR ARTICLE 10.7 (Molecular Devices). A similar experimental approach was used to investigate the functioning of CcNeoR1 and CcRGC1 from Chytriomyces confervae. The protocols for animal maintenance and oocyte harvesting were approved by the Federation of European Laboratory Animal Science Associations (Berlin, Germany). UV–Vis absorption and ﬂuorescence spectroscopy of puriﬁed protein. Absorption spectra of puriﬁed proteins (expressed either in insect cells or in HEK- T cells) were obtained in buffer containing 50 mM HEPES (pH 7.4), 200 mM NaCl, and 0.02% DDM/0.004% CHS, with 2% (w/v) (ﬁnal concentration f.c.) sodium dodecyl sulfate (SDS) or 100 mM HCl (f.c.) added for protein denaturation, using a Shimadzu UV-2000 photospectrometer with UVProbe v2.34 (Shimadzu Cor- poration, Kyoto, Japan). Transient UV–Vis absorption measurements were obtained with a femtosecond pump–probe setup utilizing a Ti:sapphire ampliﬁed laser system47,48. A CaF2 plate on a homemade moving stage was used for supercontinuum white light generation, which was focused and overlapped with the pump beam in the sample. The probe beam was then spectrally dispersed and detected in a multichannel detection system (Entwicklungsbüro Stresing, Berlin, Germany), comprises a prism spectrograph and a 1024-pixel back-thinned full- frame transfer (FFT)–charge-coupled device (CCD) detector (S7030-1006, Hamamatsu Photonics, Hamamatsu City, Shizuoka Prefecture, Japan). The time delay was varied up to 3.5 ns, and the central wavelength and power of the pump beam were set at 650 nm and 250 nJ, respectively. The instrument response function was ~100 fs. Global analysis ﬁtting was performed for the transient absorption spectra using the Glotaran 1.5.1. program49. Fluorescence spectra were recorded on a Horiba FluoroMax 4 spectrometer with FluorEssence™2.5.2 (HORIBA Instruments Inc., NJ, USA). To determine ﬂuorescence quantum yields (QY), the integrated ﬂuorescence intensity of the sample was plotted over the absorbance at the excitation wavelength (650 nm); iRFP, QY 5.9%50 and Cy 5.5, QY 20% (Molecular Probes, Eugene, OR, USA) were used as references. Phylogenetic analysis. The amino acid sequences of various microbial rhodopsins representing distinct families were obtained, including cation-conducting chan- nelrhodopsin (CCR), metagenomically discovered anion-conducting channelrho- dopsin (MerMAIDs51), proteorhodopsin (PR), sodium-pumping rhodopsin (NaR), bacteriorhodopsin (BR), halorhodopsin (HR), histidine-kinase rhodopsin (HKR), rhodopsin phosphodiesterase (RhoPDE), and rhodopsin guanylyl-cyclase (RGC) (for sequence information see Supplementary Data 1). These were aligned using ClustalO (1.2.4_1) and trimmed with TrimAl (1.4.1), and the phylogenetic tree was constructed with PhyML (3.1_1) in NGPhylogeny.fr (https://ngphylogeny.fr)52 and visualized by iTOL (5.6.3)53. Confocal microscopy. ARTICLE When expressed heterologously in Chinese Hamster Ovary (CHO) cells the generated channel mutant produces large whole-cell cur- rents in the presence of 10 µM cGMP (Supplementary Fig. 12). CHO cells were transfected with using Lipofectamine 2000 (Thermo Fisher Scientiﬁc). Recordings were performed 1–2 days post transfection. Cells were bathed in a solution con- taining (in mM) 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 10 Glucose, 5 HEPES, pH 7.4 (NaOH). The pipette solution contained (in mM) 130 KAsp, 10 NaCl, 2 MgCl2, 1 EGTA, 2 Na2ATP, 10 HEPES, pH 7,4 (KOH). Computation of excitation energies. The eight protonation states of the NeoR homology model were minimized with a hybrid quantum mechanics/molecular mechanics (QM/MM) approach in the ChemShell 3.7.0 software package (https:// www.chemshell.org)69,70. The MM region was described by the CHARMM36 protein force ﬁeld71,72 and the TIP3P water model73. To examine all possible starting locations of the counterion protons, i.e., on either of the carboxylate oxygens and in cis/trans conformation), we used a smaller QM region consisting only of the retinal chromophore and the side chain of K266 (see Supplementary Methods for details). The QM region of the ﬁnal models consisted of the retinal chromophore and the sidechains of E136, D140, E262, and K266. The QM part of the minimization was done at the B3LYP/cc-pVDZ level of theory74–76. Excitation energies were computed in the framework of QM/MM using the algebraic dia- grammatic construction scheme to second order with the resolution-of-identity (RI) approximation (RI-ADC(2))77–80 and cc-pVDZ with the default auxiliary basis set for RI81. The Turbomole (version 7.3) program was used for the RI-ADC (2) method (http://www.turbomole.com). Whole-cell voltage clamp recordings in ND7/23 cells. Patch pipettes with a resistance of 2–3 MΩ were prepared from borosilicate capillaries (GB150P-9P; Science Products GmbH, Hofheim am Taunus, Germany), using the P1000 Micropipette Puller (Sutter Instrument Co., Novato, CA, USA). ND7/23 cells were transfected with RGC2/NeoR and the mutated cGMP-gated potassium channel described above using FuGENE HD Transfection Reagent (Promega). Recordings were made at room temperature in the whole-cell conﬁguration, using an Axon MultiClamp 700B Ampliﬁer. The signals were digitized with the Axon Digidata 1550 A Low-Noise Data Acquisition system (Molecular Devices) and recorded with pCLAMP v10.4 software. The whole-cell recordings had a minimum membrane resistance of 500 MΩ (usual > 1 GΩ) and an access resistance below 10 MΩ. Data were acquired at 5 kHz and ﬁltered at 2 kHz. ARTICLE The full-length NeoR gene sequence with a C-terminal eGFP tag was transfected into ND7/23 (ECACC 92090903) cells using FuGENE HD Transfection Reagent (Promega, Madison, WI, USA), according to the man- ufacture instructions. For coexpression of NeoR and RGC1/2 in ND7/23-cells full- length NeoR was cotransfected together with either full-length RGC1 or RGC2 C- terminally tagged with YFP. Images were recorded by spinning disc confocal microscopy, using an Olympus IX83 Inverted Microscope (Shinjuku, Tokyo, Japan) with a Yokogawa CSU-W1 Confocal Scanner Unit (Musashino, Tokyo, Japan) and an EMCCD Camera (Andor iXon Ultra 888) with a 60X/1.2 NA water immersion objective. Four-hundred eighty-eight nanometer laser light was used for the detection of GFP and YFP ﬂuorescence, while intrinsic ﬂuorescence of NeoR was excited with 640 nm laser light, and ﬂuorescence recovery was achieved by 405 nm laser illumination. Homology modeling. Classical molecular dynamics (MD) simulations and com- putations of excitation energies were based on a homology model of NeoR (rho- dopsin residues: A106–S287). The model was generated using the MODELLER (version 9.20) program (https://salilab.org)54. Multiple retinal proteins have served as a basis for the model generation: Haloquadratum walsbyi bacteriorhodopsin (HwBR, PDB ID: 4QI1)55, Halobacterium salinarum bacteriorhodopsin (HsBR, PDB ID: 5ZIM)56, Archærhodopsin-3 (AR3, PDB ID: 6GUX), Sensory Rhodopsin II (SRII, PDB ID: 5JJE)57, Acetabularia Rhodopsin I (ARI, PDB ID: 5AX0)58, Archærhodopsin-2 (AR2, PDB ID: 3WQJ)59, Blue-light-absorbing proteorhodopsin (BPR, PDB ID: 4JQ6)60, Exiguobacterium sibiricum rhodopsin (ESR, PDB ID: 4HYJ)60, Channelrhodopsin Chrimson, PDB ID: 5ZIH)31, Chlamydomonas rein- hardtii channelrhodopsin-2 (CrChR2, PDB ID: 6EID)61, Channelrhodopsin-1/−2 chimæra (C1C2, PDB ID: 3UG9)62, Krokinobacter eikastus rhodopsin-2 (KR2, PDB ID: 6RF6)63, Gloeobacter rhodopsin (GR, PDB ID: 6NWD)64 and Coccomyxa subellipsoidea rhodopsin (CsR, PDB ID: 6GYH)65. Their sequences were aligned with ClustalO66. Standard protonation states were chosen for all amino acid resi- dues of the model, except for the counterions E136, D140, and E262, for which all eight possible permutations of deprotonation and protonation were examined. Eight inner-protein waters were added based on hydration analysis using the DOWSER 0.26 plugin67 in VMD 1.9.3 (https://www.ks.uiuc.edu)68. Characterization of the engineered cGMP-activated ion channel reporter. SthK is a potassium-selective cyclic nucleotide-gated channel from Spirochaeta thermophila with a high preference for cAMP over cGMP. Modiﬁcation of the C- linker and the cyclic nucleotide binding domain allow us to increase the ability of cGMP to open the channel. ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-19375-8 The extracellular solution contained (in mM):10 HEPES, 140 NaCl, 2.4 KCl, 2 CaCl2, 4 MgCl2, and 10 glucose, pH 7.4. Action spectra were measured on an Axiovert 100 Carl Zeiss microscope (Oberkochen, Germany). Light was delivered through an Objective W Plan-Apochromat 40X/1.0 DIC objective (Carl Zeiss), with a Polychrome V light source (TILL Photonics, Hillsboro, OR, USA); the illumination ﬁeld was 0.066 mm2. The half bandwidth was set to ±7 nm for all data points, and light exposure time was controlled with a shutter system (VS25 and VCM-D1, Vincent Associates). In order to obtain the same photon irradiance for all wavelengths (photon count = 2.34 × 1013 photons s−1), a motorized neutral density ﬁlter (NDF) wheel (Newport Corporation, Irvine, CA, USA) was placed between the Polychrome V and the microscope. For light titration the CoolLED pE-4000 illumination system (CoolLED, Andover, UK) was coupled to an Olympus IX73 Inverted Microscope for light stimulation through the 60X objective. In light titration experiments a 490-nm light-emitting diode (LED) was used for RGC2/ NeoR activation by 10 ms light ﬂashes. Light-intensity dependent activation curve was made using 0.3, 0.7, 1.8, 3.4, 6.2, 12.5, 17.1 and 19.0 mW mm−2 in presence or absence of 660 nm LED background light provided by the CoolLED pE-4000. Photocurrents (3 sweeps, 10 ms, 490 nm, 12.5 mW mm−2 activation light) were further measured before and after 15 s illumination with 660 nm light within the same cell using this setup. 660 nm illumination for 15 s was sufﬁcient for complete conversion of NeoR690 to NeoR367 as determined by the decrease of NeoR ﬂuor- escence. For seven independent experiments the averaged peak photocurrents of each cell, before and after red light treatment, as plotted in Supplemenary Fig. 3b were subjected to a one-sided, paired student’s t-test implemented in Origin 2017 (Originlab, Northampton, MA) resulting in t = −3.6458, d.f. = 6, p = 0.00538. 0.1 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES (pH 7.5), supplemented with 1 µM all-trans retinal, for 3–5 days. TEVC measurements at −40 mV were performed using a TURBO TEC-03X ampliﬁer (NPI Electronic GmbH, Tamm, Germany), pCLAMP v. 9.0 software (Molecular Devices, San Jose, CA, USA), a XBO 75 W Xenon lamp (Osram, Munich, Germany), and a UNIBLITZ LS3 shutter (Vincent Associates, Rochester, NY, USA). The initial slope of the photocurrents was linearly ﬁtted in Clampﬁt v. Methods P t i Protein expression and puriﬁcation. For insect cell expression (Sf21 cells (Gibco, Thermo Fisher Scientiﬁc, Waltham, MA, USA)), the rhodopsin domains of NeoR NATURE COMMUNICATIONS \| (2020) 11:5682 \| https://doi.org NATURE COMMUNICATIONS \| (2020) 11:5682 \| https://doi.org/10.1038/s41467-020-19375-8 \| www.nature.com/naturecommunications 6 References Tuning the electronic absorption of protein-embedded all- trans-retinal. Science 338, 1340–1343 (2012). 7. Rich, T. C., Tse, T. E., Rohan, J. G., Schaack, J. & Karpen, J. W. 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Nature 482, 369–U115 (2012). Acknowledgements We thank our technicians, Melanie Meiworm, Maila Reh, and Sandra Augustin, for their technical assistance. We also thank Oded Béjà, Peter Hildebrandt, and Ulrike Scheib for helpful discussion, Thomas Korte for microscopy support, and Wolfgang Bönigk for cloning support. Further we gratefully acknowledge the contribution of Saumik Sen and Rajiv K. Kar to the simulations. This work was supported by the German Research Foundation (DFG, SFB1078 (Grant No. 221545957) and Leibniz Project, PH) and the European Research Council (ERC) (Grant No. 693742 “MERA” and Grant No. 767092 “Stardust” (PH)). P.H. is a Hertie Professor for Neuroscience and supported by the Hertie Foundation. A.S., Y.A.B.S., and R.S. were supported by the German Research Foundation (DFG, SPP 1926 (Grant No. 315193289). S.A. thanks the Minerva Foun- dation for a postdoctoral fellowship. 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https://openalex.org/W2914921570	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0211867&type=printable	English	null	Suicide by persons with foreign background in Japan	PloS one	2,019	cc-by	6,366	Methods Citation: Ueda M, Yoshikawa K, Matsubayashi T (2019) Suicide by persons with foreign background in Japan. PLoS ONE 14(2): e0211867. https://doi.org/10.1371/journal.pone.0211867 Using the nationwide death records in the Vital Statistics of Japan, we first reported the age- adjusted suicide rates of 8 foreign nationals (Brazil, China, Korea, Peru, the Philippines, Thailand, the United Kingdom, and the United States) in Japan by sex from 1980 to the mid- 2010s. We also computed rate ratios to compare the suicide rate of each group with those of the Japanese population. Second, we focused on Koreans, who had the highest suicide rate in Japan. We compared the suicide rates of Koreans in Japan with Koreans in South Korea to examine whether their suicide rates were more closely related to those of their country of origin or those of their host country. Editor: Shang E. Ha, Sogang University (South Korea), REPUBLIC OF KOREA Received: September 19, 2018 Accepted: January 23, 2019 Published: February 6, 2019 Copyright: © 2019 Ueda 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. Results We found that the suicide rates of Koreans and Chinese in Japan were similar to or higher than those of Japanese, while other groups tended to show lower suicide rates. Most nota- bly, Koreans displayed consistently high suicide rates from 1980 to the mid-2010s, which were nearly twice as high as those of the Japanese population. Korean males and females in Japan had higher suicide rates than those in South Korea. Data Availability Statement: The data that we used in this study are individual death records and thus contain confidential information. Its use is restricted to researchers who obtain written approval from the Ministry of Health, Labour, and Welfare of Japan. Under the conditions of our contract with the Ministry, we are not allowed to share the data with third parties. Information on how to obtain these restricted data can be found at: https://www.mhlw.go.jp/english/database/link. html. Michiko UedaID1, Kanako Yoshikawa2, Tetsuya Matsubayashi2 1 Faculty of Political Science and Economics, Waseda University, Shinjuku, Tokyo, Japan, 2 Osaka School of International Public Policy, Osaka University, Toyonaka, Osaka, Japan 1 Faculty of Political Science and Economics, Waseda University, Shinjuku, Tokyo, Japan, 2 Osaka School of International Public Policy, Osaka University, Toyonaka, Osaka, Japan mueda@waseda.jp a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 This study examined trends in the suicide rates of persons with foreign background in Japan. This study examined trends in the suicide rates of persons with foreign background in Japan. OPEN ACCESS Citation: Ueda M, Yoshikawa K, Matsubayashi T (2019) Suicide by persons with foreign background in Japan. PLoS ONE 14(2): e0211867. https://doi.org/10.1371/journal.pone.0211867 Editor: Shang E. Ha, Sogang University (South Korea), REPUBLIC OF KOREA Received: September 19, 2018 Accepted: January 23, 2019 Published: February 6, 2019 Copyright: © 2019 Ueda et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RESEARCH ARTICLE Introduction 17H02541 (MU) and Innovative Research Program on Suicide Countermeasures Research Grant, AY2017, Grant Number 1-1 (MU), https://www. jsps.go.jp/english/index.html, http://irpsc-jssc.jp/ irpsc.php. The funders had no role in study, design, data collection and analysis, decision to publish, or preparation of the manuscript. Immigrants have become an important and growing segment of the population, accounting for almost 10 percent of the population in the OECD countries [1]. Immigrants often face chal- lenges in the process of acculturation and adjustment to a new environment, especially when their traditional culture and the host country’s mainstream culture conflict [2,3]. Immigrants may be more susceptible to mental disorders due to traumatic events prior to immigration or face adverse circumstances upon arrival to their country of destination [4,5]. They may also struggle with low socio-economic status, poor working conditions, impairments in physical health, and psychological distress [6–8]. Competing interests: The authors have declared that no competing interests exist. Past studies focused on suicide as a measure of immigrants’ well-being and compared immigrant suicide rates to those of their host country or country of origin; however, conclu- sions among such studies are inconsistent. One body of research has found a high degree of correspondence between immigrants’ and their country of origin’s suicide rates [3,9–14]. Another group of studies found that the suicide rates of foreign nationals converge to the sui- cide rate of the native population [15,16]. This study sought to uncover the patterns in suicides by persons with of foreign back- ground in Japan. To our knowledge, there has been only one systematic study, conducted in the 1980s, that investigated the suicide rates of non-Japanese citizens in Japan [17]. Such a dearth of research is unfortunate, because studying the suicide rates of non-Japanese citizens in Japan is important for at least two reasons. First, Japan is a country that has one of the high- est suicide rates in the world and studying the suicide mortality of foreign nationals can help us understand whether their suicide rates tend to “converge” to the suicide rate of the country where they currently reside. Second, the suicide rates of persons with foreign background may indicate their overall well-being and health in the country of residence. The number of foreign nationals in Japan has increased nearly three-fold since 1980 [18,19]. However, such a rapid increase has also brought a backlash against non-Japanese citizens among a subset of the Japanese population. Suicide by persons with foreign background in Japan Conclusions Immigrants in Japan were not necessarily influenced by the suicide rates of the host country. The high suicide rates among Korean residents in Japan might be explained by various dis- advantages and adversities that they face in Japan. Funding: This work was supported by JSPS Grants-in-Aid for Scientific Research Grant Number 1 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0211867 February 6, 2019 Data and method The individual death records in this study were obtained from the Vital Statistics of Japan, which lists all reported deaths in Japan. The Vital Statistics data were collected for administra- tive purposes by the Ministry of Health, Labour, and Welfare of Japan and were anonymized and de-identified by the Ministry prior to analysis. Individual death records between 1980 and 2014 were made available for research purposes with approval from the Ministry. The data in the Vital Statistics Report are based on death certificates issued by physicians and are subsequently reported to local governments where the residency of the deceased is reg- istered. The data include the nationality of the deceased, the date of death, and the underlying cause of death based on the International Classification of Diseases (ICD) 8/9 standard (up to 1994) and the ICD-10 standard (1995 to present). Foreigners who are in Japan for a short period, such as travelers, do not appear in the death record. This study analyzed deaths by sui- cide (ICD-8/9: E950-E959, ICD-10: X60-X84) that occurred in Japan. Using the Vital Statistics data, we first computed the number of suicides by sex and country of origin. As of 1980, the countries of origin reported in the Vital Statistics were Japan, Korea, China, the United States, and “others.” Five other countries of origin were added in 1992 (the Philippines, Thailand, the United Kingdom, Brazil, and Peru) to be listed separately from the “others” category. Accordingly, we computed the number of suicides for four countries of ori- gin from 1980 to 1991 (Japan, Korea, China, and the United States) and nine countries of origin from 1992 to 2014 (Japan, Korea, China, the United states, the Philippines, Thailand, the United Kingdom, Brazil, and Peru). Because the annual number of suicides among non-Japanese groups can be small, we aggregated the number of suicides over five-year periods (e.g., 1980– 1984 and 2010–2014) for each nationality. Note that the number of suicide deaths by those from Brazil, Thailand, the Philippines, Peru, and the United Kingdom were aggregated over three years (1992–1994) for the period 1990–1994 instead of five for the reason described above. Next, we calculated the age-standardized suicide rates per 100,000 by sex and nationality. We used the population counts by sex, nationality, and five-year age groups from the Popula- tion Census of Japan. Introduction Hate crime incidents, especially hate speech demonstrations against Koreans, have been increasing in Japan [20]. In addition, non-Japanese citizens, including long-term residents, face a various types of discrimination in areas of housing, employment, and marriage, to name a few [21]. Thus, the conditions that foreign nationals face in Japan are not necessarily positive. The well-being of Koreans in Japan is of particular interest, as they have a unique historical background. Many Koreans in Japan are not recent immigrants, but long-term residents who were born and raised in Japan. They typically have “special permanent resident status,” which is different from the conventional status permitted to foreigners seeking permanent residence in Japan; the former is granted to those who migrated to Japan when the Korean Peninsula was under Japan’s colonial rule. Although the Koreans under Japanese colonial rule had been considered Japanese citizens during the occupation period, they lost their Japanese citizenship and associated privileges after the War. The special permanent resident status was created in 1991 for them and their descendants to recognize the special historical circumstances sur- rounding them. As of 2016, 66.1% of Koreans residing in Japan are special permanent resi- dents [22]. Using large-scale data from the national death registry in Japan, this study reported the age- standardized suicide rates of foreign nationals in Japan by sex from 1980 to the mid-2010s. We also computed rate ratios to compare their suicide rates to those of the Japanese population. In addition, we focused on Koreans who have the highest suicide rate in Japan and compared the suicide rates of Koreans in Japan with Koreans in South Korea to examine whether their 2 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0211867 February 6, 2019 Suicide by persons with foreign background in Japan suicide rates were more closely related to those of their country of origin or those of their host country. suicide rates were more closely related to those of their country of origin or those of their host country. PLOS ONE \| https://doi.org/10.1371/journal.pone.0211867 February 6, 2019 Note: The total number of suicides aggregated over five-year periods are reported. The 1990–1994 data for Brazil, Thailand, the Philippines, Peru, and the United Kingdom were aggregated over three years (1992–1994). Data and method Regardless of nationality, those who had lived or were going to live in Japan for three months or longer as of the census date were included in the tabulation [23]. The Population Census was conducted every five years, and we used the latest available Census population data for years between Census years. For instance, we used the census population from 1990 to compute the suicide rates for 1990, 1991, 1992, 1993 and 1994. The suicide rates by sex and nationality were age-standardized because the age composition of the Japanese and non-Japanese population can be different. The standardization was based on the WHO World Standard Population [24]. We report the average of the age-standardized suicide rates over the five-year periods. Using the standardized suicide rates per 100,000, we also calculated the rate ratios and their confidence intervals for the Korean and Chinese popu- lation, with the suicide rate of Japanese as the reference. The rate ratios were not calculated for other nationalities as the number of suicide counts in each category was too small. Our final analysis compared the suicide rates in Japanese and Korean populations in Japan with those of Koreans in South Korea. We calculated the age-standardized suicide rates in South Korea in the same manner, using data on the annual number of suicides by five-year age groups between 1983 and 2014 and the population counts by age group between 1980 and 2010. The suicide and population data were obtained from Statistics Korea and Korea’s quin- quennial Population Census, respectively. 3 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0211867 February 6, 2019 Suicide by persons with foreign background in Japan Table 1. Population of foreign nationals in Japan: 1980–2015. Total Foreign Pop. Korea % China % USA % Brazil % Thailand % Philippines % Peru % UK % 1980 668675 400530 0.45 34779 0.04 14373 0.02 1985 720093 427911 0.45 50317 0.05 19792 0.02 1990 886397 457681 0.45 97152 0.10 28224 0.03 40613 0.04 7226 0.01 34729 0.03 5855 0.01 5622 0.01 1995 1140326 474480 0.45 155400 0.15 33032 0.03 119118 0.11 19948 0.02 64498 0.06 23448 0.02 7970 0.01 2000 1310545 466948 0.43 227046 0.21 32428 0.03 157724 0.15 22445 0.02 87345 0.08 27100 0.03 9171 0.01 2005 1555505 422441 0.39 322833 0.29 32325 0.03 178460 0.16 24860 0.02 114619 0.10 32022 0.03 8871 0.01 2010 1648037 391335 0.36 425547 0.39 33275 0.03 123535 0.11 28133 0.03 132653 0.12 29128 0.03 9225 0.01 2015 1752368 353030 0.32 458823 0.42 37643 0.03 102761 0.09 32479 0.03 155823 0.14 28040 0.03 10494 0.01 Source: Population Census of Japan 1980 2015 Table 1. Population of foreign nationals in Japan: 1980–2015. Source: Population Census of Japan, 1980–2015. 0 2015. eru, and the United Kingdom were not independently reported until the 1990 census. % indicates the percentage of each n of Japan. p J p , Note: Brazil, Thailand, the Philippines, Peru, and the United Kingdom were not independently reported until the 1990 census. % indicates the percentage of each population relative to the total population of Japan. https://doi.org/10.1371/journal.pone.0211867.t002 Source: Vital Statistics of Japan, 1980–2014. https://doi.org/10.1371/journal.pone.0211867.t002 Suicide by persons with foreign background in Japan Table 3. Standardized suicide rates by sex and nationality, 1980–2014. Table 3. Standardized suicide rates by sex and nationality, 1980–2014. Male Years Japan Korea China USA Brazil Thailand Philippines Peru UK 1980–1984 33.59 58.64 28.53 21.31 1985–1989 31.59 53.99 19.68 13.68 1990–1994 25.78 47.28 30.96 17.04 13.20 61.24 10.84 0.00 9.25 1995–1999 32.34 65.14 15.90 16.15 8.00 10.54 4.84 4.90 55.40 2000–2004 37.76 70.33 17.91 18.18 6.30 13.98 14.46 5.29 12.85 (20.8) (12.7) (16.5) (8.9) (23.7) (4.5) (8.8) (12.7) 2005–2009 37.65 73.64 16.39 11.19 7.52 9.58 5.20 3.78 19.44 (39.3) (9.0) (18.6) (9.0) (21.0) (5.7) (7.5) (10.6) 2010–2014 33.28 56.74 12.54 16.30 9.74 6.21 7.66 3.07 7.46 (31.4) (7.9) (20.5) (9.7) (21.7) (5.4) (7.6) (11.6) Female Years Japan Korea China USA Brazil Thailand Philippines Peru UK 1980–1984 15.89 20.22 23.61 7.77 1985–1989 15.48 22.87 23.92 8.65 1990–1994 11.98 20.87 11.62 10.59 9.77 35.35 9.76 0.00 60.34 1995–1999 12.54 23.55 15.20 17.74 3.38 17.63 1.32 1.02 8.43 2000–2004 12.80 23.50 11.47 10.55 1.16 16.54 9.74 0.00 2.78 (8.5) (15.6) (4.1) (2.2) (8.3) (1.8) (3.3) (3.8) 2005–2009 13.49 27.31 11.54 5.20 4.33 8.50 4.54 0.88 10.13 (18.7) (11) (5.2) (2.8) (5.5) (2.3) (2.5) (3.2) 2010–2014 12.89 29.88 9.40 10.10 1.88 6.01 5.24 1.88 0.00 (12.5) (8.5) (6.3) (2.9) (4.9) (2.3) (2.7) (3.7) S Vit l St ti ti f J 1980 2014 P l ti C f J 1980 2010 (f d t J id t ) W ld H lth O i ti (d t i h Source: Vital Statistics of Japan, 1980–2014, Population Census of Japan, 1980–2010 (for data on Japanese residents). World Health Organization (data in home countries) [26]. Note: Age-standardized suicide rates aggregated over five-year periods are reported. The 1990–1994 data for Brazil, Thailand, the Philippines, Peru, and the United Kingdom were aggregated over three years (1992–1994). The numbers in parentheses report the age-standardized suicide rates in the country of origin. The data were available only from 2000, and the numbers in 2000, 2010, and 2015 are shown. https://doi.org/10.1371/journal.pone.0211867.t003 among Koreans for both sexes throughout the study period. The number of suicides sharply increased between the periods 1995–1999 and 2000–2004 in the aftermath of the Asian Finan- cial Crisis, especially among Japanese and Korean males. By 2010–2014, the total suicide counts declined for both Japanese and Korean males. Results We first present the number of non-Japanese citizens in Japan by eight nationality groups (Table 1). During 1980–2015, the number of foreign nationals increased by more than 160%, with the largest increase observed among Chinese. As of 2015, Chinese and Koreans were the two larg- est non-Japanese groups. Koreans were the largest group at the beginning of the study period, but by 2010 they were outnumbered by Chinese. During 1980–2015, 263,628 Koreans naturalized [25] as Japanese citizens, which may partly explain their apparent population decrease. bl h l b f d d h d f d b Table 2 presents the total number of suicide deaths aggregated over five-year periods, by sex and nationality. The highest number of suicides among foreign nationals was observed Table 2. Total number of suicides in Japan over Five-year periods by sex and nationality. Male Years Japan Korea China USA Brazil Thailand Philippines Peru UK 1980–1984 71939 553 26 8 1985–1989 74428 543 23 7 1990–1994 66016 521 57 11 14 13 4 0 1 1995–1999 89933 757 47 15 34 7 4 5 6 2000–2004 110158 846 65 18 31 4 3 4 4 2005–2009 109691 828 66 14 32 4 5 3 8 2010–2014 94511 587 76 21 33 2 12 3 3 Female Years Japan Korea China USA Brazil Thailand Philippines Peru UK 1980–1984 38803 181 19 3 1985–1989 42351 233 32 4 1990–1994 36347 241 23 3 5 10 5 0 2 1995–1999 40435 301 50 10 9 15 7 1 1 2000–2004 42127 323 62 5 5 12 13 0 1 2005–2009 42810 345 70 3 21 12 20 1 2 2010–2014 40832 378 87 5 6 14 21 1 0 Table 2. Total number of suicides in Japan over Five-year periods by sex and nationality. PLOS ONE \| https://doi.org/10.1371/journal.pone.0211867 February 6, 2019 4 / 10 ) [ ] Note: Age-standardized suicide rates aggregated over five-year periods are reported. The 1990–1994 data for Brazil, Thailand, the Philippines, Peru, and the United Kingdom were aggregated over three years (1992–1994). The numbers in parentheses report the age-standardized suicide rates in the country of origin. The data were available only from 2000, and the numbers in 2000, 2010, and 2015 are shown. PLOS ONE \| https://doi.org/10.1371/journal.pone.0211867 February 6, 2019 Source: Vital Statistics of Japan, 1980–2014, Population Census of Japan, 1980–2010 (for data on Japanese residents). World Health Organization (data in home t i ) [26] Suicide by persons with foreign background in Japan Suicide by persons with foreign background in Japan 0 .5 1 1.5 2 2.5 Rate ratio 1980 1990 2000 2010 Year Korea male 0 .5 1 1.5 2 2.5 Rate ratio 1980 1990 2000 2010 Year Korea female 0 .5 1 1.5 2 2.5 Rate ratio 1980 1990 2000 2010 Year China male 0 .5 1 1.5 2 2.5 Rate ratio 1980 1990 2000 2010 Year China female Fig 1. The Rate ratios of standardized suicide rates. Source: Vital Statistics of Japan, 1980–2014, Population Census of Japan, 1980–2010. Note: The rate ratios were calculated with the suicide rate of Japanese citizens as a reference. https://doi.org/10.1371/journal.pone.0211867.g001 0 .5 1 1.5 2 2.5 Rate ratio 1980 1990 2000 2010 Year Korea male 0 .5 1 1.5 2 2.5 Rate ratio 1980 1990 2000 2010 Year Korea female Rate ratio 0 .5 1 1.5 2 2.5 Rate ratio 1980 1990 2000 2010 Year China female 0 .5 1 1.5 2 2.5 Rate ratio 1980 1990 2000 2010 Year China male Rate ratio Rate ratio Fig 1. The Rate ratios of standardized suicide rates. Source: Vital Statistics of Japan, 1980–2014, Population Census of Japan, 1980–2010. Note: The rate ratios were calculated with the suicide rate of Japanese citizens as a reference. https://doi.org/10.1371/journal.pone.0211867.g001 to 12.54 by the latest period in our study. Comparison of the suicide rates of non-Japanese citi- zens in Japan and rates of their country of origin reveals that for most nationalities, the suicide rates of residents in Japan tended to resemble those in the home country. However, there were some exceptions; notably Korean and Chinese males and Korean females in Japan had higher suicide rates compared to those in their home country. to 12.54 by the latest period in our study. Comparison of the suicide rates of non-Japanese citi- zens in Japan and rates of their country of origin reveals that for most nationalities, the suicide rates of residents in Japan tended to resemble those in the home country. However, there were some exceptions; notably Korean and Chinese males and Korean females in Japan had higher suicide rates compared to those in their home country. Fig 1 presents the rate ratios (RRs) of the suicide rates for the Korean and Chinese popula- tion by sex, with Japanese suicide rates as the reference group. The vertical bars represent 95% confidence intervals. Over the same period, the number of sui- cides among Japanese females also decreased. However, the number of suicides among Korean females in Japan remained high after 2010, even though their population in Japan had decreased. Table 3 reports the age-standardized suicide rates per 100,000 for residents in Japan by nationality to facilitate comparisons across nationalities. Except for the Japanese, the table also shows the age-standardized suicide rates in each group’s country of origin in parentheses. The data are available only for 2000, 2010, and 2015 [26]. According to Table 3, the suicide rates of Koreans living in Japan were by far the highest among all nationalities for both sexes. For example, during the 2005–2009 period, the suicide rates for Korean males and females in Japan were 73.64 and 27.31 per 100,000, respectively, which were approximately twice as high as the suicide rates of their Japanese counterparts (37.65 and 13.49). The suicide rate of Korean females rose since the 1995–1999 period, while the suicide rates of other groups had declined by the 2010–2014 period. The suicide rates of Chinese residents decreased from 28.53 in 1980 5 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0211867 February 6, 2019 PLOS ONE \| https://doi.org/10.1371/journal.pone.0211867 February 6, 2019 Suicide by persons with foreign background in Japan According to the top panel, which depicts the RRs for Koreans, the sui- cide rates of Korean males in Japan were more than 50% higher than those of Japanese males throughout the study period. In the 1995–1999 period, the RR was 2.014 (95% CI: 1.943– 2.086), which suggests that Korean males in Japan were twice as likely to die by suicide com- pared to Japanese males. Regarding Korean females, at the beginning of the study period, they were approximately 25% more likely to die by suicide than Japanese females, but the ratio gradually increased over time. By the 2010–2014 period, the RR reached 2.318 (95% CI: 2.217– 2.419), which indicates that the suicide rate among Korean females was c. 130% higher than that of Japanese females by the latest study period. We found different patterns among the Chinese population in Japan. According to the bot- tom panel of Fig 1, the suicide rates of Chinese males were similar to those of Japanese males until 1990–1994 but became significantly lower after 1995. The RRs for Chinese females also 6 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0211867 February 6, 2019 Suicide by persons with foreign background in Japan Koreans in Japan Koreans in Korea Japanese 0 20 40 60 80 Suicide rate 1980 1990 2000 2010 Year Male Koreans in Japan Koreans in Korea Japanese 0 10 20 30 Suicide rate 1980 1990 2000 2010 Year Female Fig 2. Trends in standardized suicide rates by sex and nationality and the country of residence. Source: Vital Statistics of Japan, 1980–2014, Population Census of Japan, 1980–2010, Statistics Korea, 1980–2014. Notes: The age- standardized suicide rates aggregated over five-year periods reported. https://doi.org/10.1371/journal.pone.0211867.g002 Koreans in Japan Koreans in Korea Japanese 0 20 40 60 80 Suicide rate 1980 1990 2000 2010 Year Male Female Koreans in Japan Koreans in Korea Japanese 0 10 20 30 Suicide rate 1980 1990 2000 2010 Year Female Fig 2. Trends in standardized suicide rates by sex and nationality and the country of residence. Source: Vital Statistics of Japan, 1980–2014, Population Census of Japan, 1980–2010, Statistics Korea, 1980–2014. Notes: The age- standardized suicide rates aggregated over five-year periods reported. https://doi.org/10.1371/journal.pone.0211867.g002 https://doi.org/10.1371/journal.pone.0211867.g002 statistically did not differ from a value of one, except for the first two periods, in which their suicide rates were approximately 50% higher than those of Japanese females. Suicide by persons with foreign background in Japan Finally, we compared the trends in the suicide rates for Koreans living in Japan, Koreans living in South Korea, and Japanese citizens, to determine whether the suicide rates of Koreans living in Japan were more closely related to the rates of Koreans in South Korea, which may arguably be considered their country of origin, or to the suicide rates of Japanese citizens. As shown in the top panel of Fig 2, the suicide rates of Korean males in Japan followed a similar trend to that of Japanese males. The suicide rates of Korean females in Japan and Japanese females in Japan also exhibited similar trends, but the difference between their rates increased toward the end of our study period. In contrast, the suicide rates of Korean females in Japan and those in South Korea became more similar in recent years. Finally, Korean males and females residing in Japan had higher suicide rates than Koreans in South Korea throughout the study period. However, the gap between Koreans in Japan and Koreans in South Korea narrowed in recent years. Suicide by persons with foreign background in Japan comprehensive data regarding suicides by non-Japanese citizens in Japan using large-scale data that include all reported deaths in Japan. We found that the suicide rates of non-Japanese citizens in Japan differ across nationalities. The suicide rates of Koreans and Chinese in Japan were similar to or higher than those of Japa- nese, while other groups tended to show lower suicide rates. This suggests that immigrants in Japan are not necessarily influenced by the suicide rates of the host country (i.e., Japan). For example, the suicide rate of the Japanese population sharply increased in the mid-1990s, but such an increase was not observed among foreign nationals living in Japan. The results reported in Table 3 suggest that immigrant suicide rates are thus more likely to be influenced by other factors, such as the suicide rates of their country of origin [3, 9–14]. However, this result should be interpreted with caution because the size of immigrant population in Japan is small and suicide is a rare event, as shown in Tables 1 and 2. As a result, the suicide rates tend to vary considerably over years, which makes it challenging to compare across nationalities or over time. We also found that the Korean population in Japan had consistently high suicide rates from 1980 to the mid-2010s, which were nearly twice as high as those of the Japanese population. Both Korean males and females exhibit high suicide rates, and the suicide rate of Korean females has been increasing over time. While the length of their residency in Japan may explain why the suicide rates of Koreans in Japan tended to follow a similar temporal pattern to those of the Japanese population, it does not explain why their suicide rates were consider- ably higher than the suicide rates of Japanese and Koreans in South Korea. There could be multiple factors underlying their relatively high suicide rates, but of particular relevance is that Korean residents tend to face various disadvantages and adversities in Japan. As noted above, many Koreans in Japan are special permanent residents, and the younger generation was born and educated in Japan with Japanese as their native language. However, their socioeconomic status is not equivalent to that of the Japanese native population. Discussion The present study’s contribution is that it examined long-term trends in suicide deaths among foreign nationals in Japan from eight countries of origin. This is the first study that provides 7 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0211867 February 6, 2019 PLOS ONE \| https://doi.org/10.1371/journal.pone.0211867 February 6, 2019 Author Contributions Formal analysis: Michiko Ueda, Tetsuya Matsubayashi. Formal analysis: Michiko Ueda, Tetsuya Matsubayashi. Investigation: Kanako Yoshikawa. Methodology: Tetsuya Matsubayashi. Supervision: Michiko Ueda, Tetsuya Matsubayashi. Writing – original draft: Michiko Ueda, Kanako Yoshikawa, Tetsuya Matsubayashi. Writing – review & editing: Michiko Ueda, Kanako Yoshikawa, Tetsuya Matsubayashi. Investigation: Kanako Yoshikawa. Investigation: Kanako Yoshikawa. Methodology: Tetsuya Matsubayashi. Supervision: Michiko Ueda, Tetsuya Matsubayashi. Writing – original draft: Michiko Ueda, Kanako Yoshikawa, Tetsuya Matsubayashi. Writing – review & editing: Michiko Ueda, Kanako Yoshikawa, Tetsuya Matsubayashi. Writing – review & editing: Michiko Ueda, Kanako Yoshikawa, Tetsuya Matsubayashi. Suicide by persons with foreign background in Japan In summary, circumstances facing Koreans living in Japan can be challenging. Despite the fact that many Korean residents in Japan have integrated with the local language and culture, their aggregate suicide rate is significantly higher than the suicide rate among Japanese citi- zens. Although we cannot immediately conclude that these high suicide rates are due to the conditions that Koreans face in Japan, it is possible that such conditions influence the deci- sions of some Koreans to kill themselves. Understanding the causes underlying their high sui- cide rates is an important future research agenda. Several limitations of this study should be noted. First, our analysis did not consider length of stay in Japan as it was not recorded in the Vital Statistics. As discussed above, Koreans in Japan can be short-term residents, such as college students, or long-term special permanent residents. We expect that suicide rates would vary depending on length of stay, but we could not assess this in the present analysis. Second, the census population that we used to calculate the standardized suicide rates may not be accurate. The census count only reflected population as of the census date and undercounting foreign nationals was also a possibility. Third, our analysis did not consider the role of socioeconomic status such as income and employment. For example, Koreans in Japan might exhibit higher suicide rates than Japanese because the former might be more likely than the latter to be unemployed. Despite these limitations, the present study provides important data regarding suicide rates among foreign nationals in Japan. Our study also has significant policy implications. The find- ings of this study strongly suggest that extra care should be taken to ensure the well-being of foreign nationals, even if they live in a host country for a sufficient period of time. As the high suicide rates of Koreans in Japan documented in this study indicate, language acquisition and acculturation are insufficient to ensure the well-being and health of foreign nationals. Their risk of suicide may remain high until the host country fully accepts them as members of society. 6. Gilliver SC, Sundquist J, Li X, Sundquist K. Recent research on the mental health of immigrants to Swe- den: A literature review. Vol. 24, Eur J Public Helth. 2014; 24 Suppl 1: 72–9. PLOS ONE \| https://doi.org/10.1371/journal.pone.0211867 February 6, 2019 Accord- ing to the Population Census of Japan in 2010, the unemployment rate of Korean males aged 15 or higher was 4.6 percentage points higher than that of Japanese males. Even among those aged 25–44, the difference was 4.0 percentage points [19]. The same census data also indicate that Japanese and Korean males engage in different types of work: 61% of Japanese males work as full-time office workers, whereas only 41% of Korean males work as such. In addition, long-term Korean residents in Japan face discrimination and hardship to date. For example, according to a survey administered at the request of the Ministry of Justice, 27.2% of respondents with a special permanent resident status answered that a housing appli- cation had been denied within the prior five years due to their nationality [21]. Note that nearly all (99%) special permanent residents are Koreans [22]. In the same survey, 16.1% of special permanent residents responded that their romantic relationship with a Japanese part- ner was opposed by the partner’s Japanese relatives because of the respondent’s nationality. Some Koreans also experience hardship at school and in the workplace: 19.3% of special per- manent residents answered that their relationships with others at school or in the workplace were negatively affected because their Japanese peers are prejudiced against foreigners, and 10.1% of respondents reported that they were bullied at school or in the workplace because of their nationality. Finally, the number of hate speech and street demonstrations against Koreans and Chinese in Japan has been increasing throughout Japan. A total of 378 such street rallies were recorded in 2014, some of which contained strong racist speech toward long-term Korean residents [20]. The spread of hate speech and rallies against foreigners was mentioned as a source of con- cern in a country report by the United Nations [27]. 8 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0211867 February 6, 2019 References References 1. OECD. Foreign-born population (indicator) [Internet]. 2018 [cited 2018 Feb 24]. Available from: https:// data.oecd.org/migration/foreign-born-population.htm 2. Sharma M, Bhugra D. Suicide among migrants. In: Wasserman D, & Wasserman C (Eds) Oxford text- book of suicidology and suicide prevention. OUP Oxford; 2009. 3. Kliewer E V., Ward RH. Convergence of immigrant suicide rates to those in the destination country. Am J Epidemiol. 1988; 127(3):640–53. PMID: 3341364 4. Chou KL. Psychological distress in migrants in Australia over 50 years old: A longitudinal investigation. J Affect Disord. 2007; 98(1–2):99–108. https://doi.org/10.1016/j.jad.2006.07.002 PMID: 16890296 5. Anikeeva O, Bi P, Hiller JE, Ryan P, Roder D, Han G-S. The health status of migrants in Australia: a review. Asia Pac J Public Health. 2010; 22(2):159–93. https://doi.org/10.1177/1010539509358193 PMID: 20457648 6. Gilliver SC, Sundquist J, Li X, Sundquist K. Recent research on the mental health of immigrants to Swe- den: A literature review. Vol. 24, Eur J Public Helth. 2014; 24 Suppl 1: 72–9. 1. OECD. Foreign-born population (indicator) [Internet]. 2018 [cited 2018 Feb 24]. Available from: https:// data.oecd.org/migration/foreign-born-population.htm 2. Sharma M, Bhugra D. Suicide among migrants. In: Wasserman D, & Wasserman C (Eds) Oxford text- book of suicidology and suicide prevention. OUP Oxford; 2009. 3. Kliewer E V., Ward RH. Convergence of immigrant suicide rates to those in the destination country. Am J Epidemiol. 1988; 127(3):640–53. PMID: 3341364 4. Chou KL. Psychological distress in migrants in Australia over 50 years old: A longitudinal investigation. J Affect Disord. 2007; 98(1–2):99–108. https://doi.org/10.1016/j.jad.2006.07.002 PMID: 16890296 5. Anikeeva O, Bi P, Hiller JE, Ryan P, Roder D, Han G-S. The health status of migrants in Australia: a review. Asia Pac J Public Health. 2010; 22(2):159–93. https://doi.org/10.1177/1010539509358193 PMID: 20457648 6. Gilliver SC, Sundquist J, Li X, Sundquist K. Recent research on the mental health of immigrants to Swe- den: A literature review. Vol. 24, Eur J Public Helth. 2014; 24 Suppl 1: 72–9. 9 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0211867 February 6, 2019 Suicide by persons with foreign background in Japan 7. Grant BF, Stinson FS, Hasin DS, Dawson DA, Chou SP, Anderson K. Immigration and lifetime preva- lence of DSM-IV psychiatric disorders among Mexican Americans and non-Hispanic whites in the United States: Results from the National Epidemiologic Survey on Alcohol and Related Conditions. Arch Gen Psychiatry. 2004; 61(12):1226–33. https://doi.org/10.1001/archpsyc.61.12.1226 PMID: 15583114 8. Tran T V, Khatutsky G, Aroian K, Balsam A, Conway K. References Living arrangements, depression, and health status among elderly Russian-speaking immigrants. J Gerontol Soc Work. 2000; 33(2):63–77. 9. Spallek J, Reeske A, Norredam M, Nielsen SS, Lehnhardt J, Razum O. Suicide among immigrants in Europe-a systematic literature review. Eur J Public Health. 2014; 25(1):1–9. 10. Voracek M, Loibl LM, Dervic K, Kapusta ND, Niederkrotenthaler T, Sonneck G. Consistency of immi- grant suicide rates in Austria with country-of-birth suicide rates: A role for genetic risk factors for sui- cide? Psychiatry Res. 2009; 170(2–3):286–9. https://doi.org/10.1016/j.psychres.2008.10.032 PMID: 19900719 11. Burvill PW. Migrant suicide rates in Australia and in country of birth. Psychol Med. 1998; 28(1):201–8. PMID: 9483697 12. Ide N, Kõlves K, Cassaniti M, De Leo D. 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https://openalex.org/W3006857910	https://europepmc.org/articles/pmc7332121?pdf=render	English	null	Impact of New Standardized Population for Estimating Cancer Incidence in Indian Context- an Analysis from National Cancer Registry Programme (NCRP)	Asian Pacific journal of cancer prevention	2,020	cc-by	6,171	Sathishkumar K, Vaitheeswaran K, Stephen S, Sathya N, Prashant Mathur* athishkumar K, Vaitheeswaran K, Stephen S, Sathya N, Prashant Math Abstract Objective: Standardization adjusts for variations in population age-distribution and provides a summary measure for the comparison of populations and comparisons of time-trends in population. Globally, several standard population were used by many countries for comparison of rates. Segi World Standard Population (WSP) is suitable for international comparison. However, national standard population would be more appropriate for Intra-national comparison as the standard should be similar to the population of interest. This study aimed to develop Indian Standard Population (ISP) for estimating Age Adjusted Rate (AAR) and, to understand the outcome on the cancer incidence rate using ISP in Population Based Cancer Registries (PBCRs). Methods: The Indian Standard Population was estimated based on the average age structure of three Censuses (1991,2001 and 2011). Age Adjusted Rate has been estimated using two standard populations of WSP and ISP for magnitude of change and comparison between 15 PBCRs in India. Results: The implementation of ISP led to overall 20% reduction in AAR for all sites of cancers in both sexes and minor relative changes in the ranking among PBCRs. Time trends in cancer incidence rate showed same temporal pattern curve using both ISP and WSP, AARs. Conclusion: The ISP would be more representative of the age-structure of Indian registries population and this would give more realistic comparison across the different PBCRs in India. Keywords: Indian standard population- age adjusted rate- age standardised Rrate- NCRP- national population eywords: Indian standard population- age adjusted rate- age standardised Rrate- NCRP- national populati Asian Pac J Cancer Prev, 21 (2), 371-377 A common age-structured population is used as standard population in direct standardization. This could be the existing population of the country (e.g., United States population, 1999, Australian Standard Population, 2001) or may be arbitrary. A common set of age specific rate is applied to the population’s age structure whose rates are to be standardized which is known as indirect standardization. The most common and simple technique is the direct age standardization method (Mausner and Bahn, 1974). DOI:10.31557/APJCP.2020.21.2.371 Indian Standard Population DOI:10.31557/APJCP.2020.21.2.371 Indian Standard Population RESEARCH ARTICLE Editorial Process: Submission:06/06/2019 Acceptance:01/28/2020 Asian Pacific Journal of Cancer Prevention, Vol 21 371 National Centre for Disease Informatics and Research, Indian Council of Medical Research (ICMR), Bangalore, India. For Correspondence: director@ncdirindia.org Asian Pacific Journal of Cancer Prevention, Vol 21 Materials and Methods Census of India is the single largest source of a variety of statistical information on various characteristics of the people of India. The source data for Indian standard population was derived from Census of India (Census of India, 2018). The ISP was conceptualized based on average age structure of Indian population with the age group of (0-4,5-9,10-14,…75+) of population of last three censuses (1991, 2001 and 2011).The last three census population by age group for both sexes were added and relative proportion were arrived at. Since cancer incidence rate multiplication factor is 100,000, the proportion of age groups are multiplied by 100,000 and rounded with nearest five hundreds. A crude incidence rate is the number of new cancers occurring in a specified population in a specified period, usually expressed per 100,000 population at risk. Age Specific Incidence Rate was the number of new cancers occurring in a given age group (eg. 10-14) in specified population and period. Age specific rate was multiplied by the standard population of that same age group. The age-specific results are added up to get the age-adjusted incidence rate for the area of study. This is called direct method of standardization (Naing, 2000). Table 2 provides the magnitude of change in ASR using two different population for stomach cancer (ICD-10: C16) by gender. There was no change in relative ranking for stomach cancers in PBCRs using both the standard population. Table 3. The leading site of breast cancer (ICD-10: C50) in females showed major changes in the ranking using ISP- standardized rate. The first five ranking based on WSP (Delhi, Chennai, Bangalore, Thiruvananthapuram and Mumbai) were changed to (Delhi, Chennai, Thiruvananthapuram, Patiala, and Bangalore). However, the difference in rates between PBCRs are very minimum. The second leading sites of cervical cancer in (ICD-10: C53) females showed relative change in rank at 11th position. Table 4. Prostate cancer (ICD-10: C61) does not show any change in the ranking using either standard population. Though myeloid leukaemia (ICD-10: C92-C94) in males showed changes in the ranking, the difference in rates are minor. The Spearman rank correlation (r) between the rates using WSP and ISP showed positive correlation in all sites and specific sites of cancer. Results Figure 1.ISP had higher population in younger age group compared with WSP except in (0-4) age group. Above 45 year age the lower population (20%) were seen in ISP compared with WSP (26%). Since, Segi standard population (Segi, 1960) age structure is not representative of Indian population structure, an attempt was made in this study to use Indian Standard Population (ISP) for estimating Age Standardised Rate (ASR)/Age Adjusted Rate (AAR) using age structure of Indian population and, to understand the outcome on the incidence rate of cancer using Indian standard population in Population Based Cancer Registries along with Segi WSP (Segi, 1960). p ( ) Table 1 provides Crude rate, Age Adjusted Rate using WSP and ISP for the first 15 PBCRs (based on AAR-WSP ranking) for all sites of cancer (ICD-10: C00-C97) by sex. The first 15 PBCRs were ranked based on AAR-WSP and AAR-ISP rate as well. There was only one position change in ranking otherwise we did not find major changes in relative ranking of the PBCRs using either WSP or ISP. However, there was a change in magnitude of standardized rate using ISP, for example the incidence rate Aizawl district of Mizoram State (North eastern state of India) using WSP 270.7 per 100,000 reduced to 210.2 per 100,000 using ISP in males. The Papumpare district reduced from ASR 249.0 per 100,000 to 200.5 per 100,000 using ISP in females. As an average there was 20% of reduction in AAR across all the registries. Introduction Clinics, Diagnostic Labs, Imaging centres, Hospices and Registrars of Births and Deaths (NCDIR, NCRP, 2018). We have used the “Three-year report of Population Based Cancer Registries (PBCR):2012-2014 data as the source to calculate Crude rate (CR), Age adjusted rate using WSP and ISP for all sites, stomach, breast, cervix, prostate and myeloid leukaemia as varied risk patterns across the age group (NCDIR, 2016). Based on the AAR of two standard populations, the ranking of the cancer registries were compared. We estimated the Spearman rank correlation (r) between the rates using WSP and ISP. Also, the percentage change between AAR of world standard population and ISP has been observed by cancer site wise. Clinics, Diagnostic Labs, Imaging centres, Hospices and Registrars of Births and Deaths (NCDIR, NCRP, 2018). been used are those of Canada; Nordic countries (NORDCAN population in 2014); Australia (Australian Standard Population, 2001); the International Network for the Demographic Evaluation of Populations and Their Health (INDEPTH) standard population for low and middle-income countries and the European standard population for all cause of death and cancer incidence (Waterhouse 1976; Government of Canada, 1991; Engholm et al., 2016; HealthStats NSW, 2017; Sankoh et al., 2014; Crocetti et al., 2016). been used are those of Canada; Nordic countries (NORDCAN population in 2014); Australia (Australian Standard Population, 2001); the International Network for the Demographic Evaluation of Populations and Their Health (INDEPTH) standard population for low and middle-income countries and the European standard population for all cause of death and cancer incidence (Waterhouse 1976; Government of Canada, 1991; Engholm et al., 2016; HealthStats NSW, 2017; Sankoh et al., 2014; Crocetti et al., 2016). The National Centre for Disease Informatics and Research (NCDIR), National Cancer Registry Programme (NCRP) in India was initiated by the Indian Council of Medical Research (ICMR) with a network of cancer registries across the country in December 1981 and it has expanded to 36 Population Based Cancer Registries (PBCRs). NCDIR-NCRP reports Age Adjusted Cancer incidence rates based on Segi World Standard Population (NCDIR, 2013, 2016). Introduction Comparing incidence and mortality rates in two or more different regions/areas is vital for the assessment of health status of the community (Naing, 2000). A comparison between crude rates would be less informative as they are not very informative about the health status of a population especially for cancer which is more dependent on age. Age is the highest risk for developing cancer and the risk of epithelial cancers, which comprise 90% of all cancers worldwide, increases approximately as a fifth power of age (Armitage and Doll, 1954). Age is the most common variable used for standardization. Globally, standardization is the procedure used to adjust for differences in population structure and provides a single summary measure for the comparison of different populations/areas. Generally, a National population is considered as “Standard Population” for comparing rates in different geographic regions/populations within the country. The world standard population was previously used by many studies for comparing cancer incidence rates in different regions (NCDIR, 2013; Torre et al., 2015). This was originally proposed in 1960 as the pooled population of 46 countries and, thereafter, revised in 1966.The new standard population in the year 2000 was constructed by World Health Organisation (WHO) as the average of projected world population age structure during the years 2000–2025 (Segi, 1960; Doll et al., 1966; Ahmad et al., 2000).The US has replaced the existing 1940 standard population with new standard of US 2000 population (Anderson and Rosenberg, 1998). Some other standard populations which have extensively There are two commonly used methods for calculating standardized rates that is direct and indirect standardization. 371 Asian Pacific Journal of Cancer Prevention, Vol 21 Sathishkumar K et al Clinics, Diagnostic Labs, Imaging centres, Hospices and Registrars of Births and Deaths (NCDIR, NCRP, 2018). We have used the “Three-year report of Population Based Cancer Registries (PBCR):2012-2014 data as the source to calculate Crude rate (CR), Age adjusted rate using WSP and ISP for all sites, stomach, breast, cervix, prostate and myeloid leukaemia as varied risk patterns across the age group (NCDIR, 2016). Based on the AAR of two standard populations, the ranking of the cancer registries were compared. We estimated the Spearman rank correlation (r) between the rates using WSP and ISP. Also, the percentage change between AAR of world standard population and ISP has been observed by cancer site wise. Materials and Methods The NCRP population Based Cancer Registries in India, collect all new cancers occurring in the resident population of defined geographic area through active case-finding procedures and principles advocated by the International Agency for Research on Cancer, the International Association of Cancer Registries (Jensen and Storm, 1991) based on multiple sources of data such as Government Hospitals, Private Hospitals, Nursing Homes, Table 5. In percentage distribution by age group of different registries population as per Census 2011, the PBCRs of Chennai, Kollam, Mumbai, Nagpur and Patiala had higher older age (65+) population than ISP 372 Asian Pacific Journal of Cancer Prevention, Vol 21 72 DOI:10.31557/APJCP.2020.21.2.371 Indian Standard Population Table 1. Crude Rate (CR) and Age Adjusted Rate (AAR) Per 100,000 Population Using World Standard Population (WSP) and Indian Standard Population (ISP) in Population Based Cancer Registries (PBCR) – Males and Females (All Sites) Table 1. Crude Rate (CR) and Age Adjusted Rate (AAR) Per 100,000 Population Using World Standard Population (WSP) and Indian Standard Population (ISP) in Population Based Cancer Registries (PBCR) – Males and Females (All Sites) Table 1. Materials and Methods Cancer Incidence: Crude Rate (CR) and Age Adjusted Rate (AAR) Per 100,000 Population Using World Standard Population (WSP) and Indian Standard Population (ISP) in Population Based Cancer Registries (PBCR) –Stomach (ICD-10:C16) - Males and Females Stomach-Males Stomach-Females Registry CR Rank AAR- WSP AAR- ISP Rank* ISP-W SP (AAR) (%) Registry CR Rank* AAR- WSP AAR- ISP Rank* ISP-WSP (AAR) (%) Papumpare 21.5 1 50.2 39.0 1 -22.3 Papumpare 11.4 1 29.2 22.9 1 -21.6 Aizawl 31.8 2 43.9 33.3 2 -24.1 Aizawl 17.4 2 23.7 18.0 2 -24.1 Mizoram State 27.3 3 41.1 31.1 3 -24.3 Mizoram State 13.7 3 20.1 15.5 3 -23.3 Naharlagun 14.3 4 26.7 20.9 4 -21.7 Naharlagun 8.1 4 16.3 12.7 4 -22.1 Pasighat 16.2 5 22.8 17.3 5 -24.1 Pasighat 8.4 5 12.2 9.8 5 -19.7 Nagaland 9.2 6 17.8 13.5 6 -24.2 Nagaland 5.9 6 11.1 8.7 6 -21.6 Sikkim State 11.3 7 14.8 11.2 7 -24.3 Kamrup Urban 6.1 7 8.6 6.8 7 -21.8 East K Hills 7.3 8 14.3 11.0 8 -23.1 East K Hills 5.1 8 7.6 6.1 8 -19.7 Kamrup Urban 9.6 9 14.1 10.6 9 -24.8 Meghalaya 4.0 9 6.8 5.5 9 -19.1 Meghalaya 6.1 10 12.5 9.7 10 -22.4 Sikkim State 4.5 10 6.8 5.1 10 -25.0 Chennai 10.8 11 10.8 8.3 11 -23.1 Chennai 5.4 11 5.1 4.0 11 -21.6 Bangalore 6.3 12 8.2 6.2 12 -24.4 Bangalore 3.8 12 4.7 3.6 12 -23.4 Dibrugarh 5.7 13 7.6 5.9 13 -22.4 Dibrugarh 3.6 13 4.3 3.6 13 -16.3 Kollam 6.9 14 5.5 4.2 14 -23.6 Imphal West 2.6 14 2.8 2.2 14 -21.4 Cachar 4.0 15 5.4 4.2 15 -22.2 Cachar 2.0 15 2.6 2.1 15 -19.8 Correlation (r) 1.00 Correlation (r) 1.00 East K.Hills - East Khasi Hills;Rank- Ranking status of PBCR based on WSP and ISP; ISP-WSP (AAR) (%) – Change in percentage between ISP and WSP AAR’s Stomach-Males WSP-AAR over the period (1982-2013). At the beginning period the CR was lower than ISP-AAR and it crossed over to higher side in the year 1997. The percentage of older age group (65+) population in Chennai PBCR increased to 6.3% in 2011 from 4.0% in 1991. which resulted CR becomes greater than ISP-AAR and Meghalaya had lower older age population than ISP which resulted CR becomes lesser than ISP-AAR. Materials and Methods Mizoram, Bhopal and Delhi percentage of older age population is closer to ISP which resulted CR and ISP-AAR becomes closer. Figure 2. Trends in Age Adjusted Rate using ISP and WSP for All sites of Cancer showed same temporal pattern curve and the ISP-AAR incidence rate is lesser than Materials and Methods Crude Rate (CR) and Age Adjusted Rate (AAR) Per 100,000 Population Using World Standard Population (WSP) and Indian Standard Population (ISP) in Population Based Cancer Registries (PBCR) – Males and Females (All Si ) Male Female Registry CR Rank AAR- WSP AAR- ISP Rank* ISP-WSP (AAR) (%) Registry CR Rank* AAR- WSP AAR- ISP Rank ISP-WSP (AAR) (%) Aizawl 204.6 1 270.7 210.2 1 -22.3 Papumpare 115.3 1 249.0 200.5 1 -19.5 Papumpare 103.5 2 230.4 181.1 2 -21.4 Aizawl 167.3 2 207.7 165.6 2 -20.3 East K.Hills 125.5 3 218.3 173.1 3 -20.7 Kamrup Urban 123.3 3 174.0 137.9 3 -20.7 Mizoram State 147.4 4 211.5 163.5 4 -22.7 Mizoram State 121.7 4 165.8 132.7 4 -20.0 Kamrup Urban 143.4 5 206.0 158.3 5 -23.2 Delhi 121.7 5 144.8 118.2 5 -18.4 Meghalaya 88.8 6 169.6 134.5 6 -20.7 Chennai 132.3 6 126.2 101.7 6 -19.4 Delhi 112.6 7 149.4 119.3 7 -20.1 Bangalore 106.5 7 125.9 100.4 7 -20.3 Thi'puram 160.7 8 132.0 104.2 8 -21.1 Thi'puram 153.9 8 120.4 99.6 8 -17.3 Nagaland 74.7 9 125.8 98.6 10 -21.6 Mumbai 117.0 9 118.5 94.6 9 -20.2 Cachar 96.2 10 125.4 98.9 9 -21.1 East K.Hills 75.0 10 117.0 92.4 10 -21.0 Kollam 148.0 11 120.5 94.7 11 -21.4 Patiala 112.8 11 111.2 91.1 11 -18.1 Chennai 115.5 12 116.1 92.3 12 -20.5 Bhopal 90.4 12 108.3 87.8 12 -18.9 Mumbai 98.1 13 113.1 88.8 13 -21.5 Imphal West 100.3 13 103.6 85.4 13 -17.6 Pasighat 83.1 14 107.4 86.7 14 -19.3 Kolkata 120.8 14 103.4 84.0 15 -18.8 Bangalore 82.8 15 105.4 82.1 15 -22.1 Kollam 130.3 15 101.7 84.5 14 -16.9 Correlation (r) 0.99 Correlation (r) 0.99 ( ) East K.Hills - East KhasiHills;Th'puram- Thiruvananthapuram; Change in rank are highlighted in bold; Rank- Ranking status of PBCR based on WSP and ISP; ISP-WSP (AAR) (%) – Change in percentage between ISP and WSP AAR’s East K.Hills - East KhasiHills;Th'puram- Thiruvananthapuram; Change in rank are highlighted in bold; Rank- Ranking status of PBCR based on WSP and ISP; ISP-WSP (AAR) (%) – Change in percentage between ISP and WSP AAR’s Table 2. Cancer Incidence: Crude Rate (CR) and Age Adjusted Rate (AAR) Per 100,000 Population Using World Standard Population (WSP) and Indian Standard Population (ISP) in Population Based Cancer Registries (PBCR) –Stomach (ICD-10:C16) - Males and Females Table 2. Discussion Asian Pacific Journal of Cancer Prevention, Vol 21 373 The main reason for using Indian standard population is Asian Pacific Journal of Cancer Prevention, Vol 21 373 The main reason for using Indian standard population is 373 Sathishkumar K et al Figure 1. Distribution of World Standard population (WSP) and Indian Standard Population (ISP) per 100,000 population (%) Figure 1. Distribution of World Standard population (WSP) and Indian Standard Population (ISP) per 100,000 population (%) Distribution of World Standard population (WSP) and Indian Standard Population (ISP) per 100,00 n (%) Figure 2. Trends in Crude Rate (CR), Age Adjusted Rate (AAR) using World Standard Population (WSP) and Indian Standard Population (ISP) for all sites of Cancer (Males) - Chennai (1982-2013) Figure 2. Trends in Crude Rate (CR), Age Adjusted Rate (AAR) using World Standard Population (WSP) and Indian Standard Population (ISP) for all sites of Cancer (Males) - Chennai (1982-2013) Table 3. Crude Rate (CR) and Age Adjusted Rate (AAR) Per 100,000 Population Using World Standard Population (WSP) and Indian Standard Population (ISP) in Population Based Cancer Registries (PBCR) – Breast(ICD-10:C50) and Cervix (ICD-10:C53) - Females Table 3. Crude Rate (CR) and Age Adjusted Rate (AAR) Per 100,000 Population Using World Standard Population (WSP) and Indian Standard Population (ISP) in Population Based Cancer Registries (PBCR) – Breast(ICD-10:C50) and Cervix (ICD-10:C53) - Females i 374 Table 3. Th'puram- Thiruvananthapuram; Ahmedabad U- Ahmedabad Urban; Barshi Exp- Barshi Expanded; Change in rank are highlight Ranking status of PBCR based on WSP and ISP; ISP-WSP (AAR)(%) – Change in percentage between ISP and WSP AAR’s Asian Pacific Journal of Cancer Prevention, Vol 21 4 Discussion Cancer Incidence: Crude Rate (CR) and Age Adjusted Rate (AAR) Per 100,000 Population Using World Standard Population (WSP) and Indian Standard Population (ISP) in Population Based Cancer Registries (PBCR) – Prostate (ICD-10:C61) and Myeloid Leukaemia (ICD-10:C92-C94) - Males Prostate Myeloid Leukaemia Registry CR Rank* AAR- WSP AAR- ISP Rank* I S P - W S P (AAR) (%) Registry CR Rank* AAR- WSP AAR- ISP Rank* ISP-WSP (AAR) (%) Delhi 7.6 1 12.4 9.1 1 -26.6 Delhi 2.8 1 3.2 2.9 1 -9.4 Kamrup 6.9 2 12.2 8.9 2 -27.0 Patiala 2.9 2 2.9 2.6 3 -10.3 Mumbai 7.7 3 9.8 7.2 3 -26.5 Imphal West 2.8 3 2.8 2.7 2 -3.6 Thi'puram 11.5 4 9.4 6.9 4 -26.6 Thi'puram 3.1 4 2.7 2.5 5 -7.4 Kolkata 9.8 5 8.2 6.0 5 -26.8 Kollam 3.0 5 2.7 2.5 4 -7.4 Bangalore 5.8 6 8.2 6.0 6 -26.8 Aizawl 2.2 6 2.7 2.2 7 -18.5 Chennai 6.2 7 6.7 4.9 7 -26.9 Bangalore 2.3 7 2.6 2.2 6 -15.4 Papumpare 1.4 8 6.6 4.9 8 -25.8 Ahmedabad U 2.4 8 2.4 2.2 8 -8.3 Patiala 5.8 9 6.3 4.7 9 -25.4 Chennai 2.4 9 2.3 2.0 10 -13.0 Pune 4.5 10 6.3 4.6 10 -27.0 Bhopal 2.2 10 2.2 2.1 9 -4.5 Kollam 7.6 11 6.1 4.5 11 -26.2 Kolkata 2.4 11 2.1 2.0 11 -4.8 Bhopal 4.0 12 5.6 4.1 12 -26.8 Sikkim State 1.9 12 2.0 1.8 13 -10.0 Ahmedabad U 3.3 13 4.4 3.2 13 -27.3 Wardha 2.1 13 1.9 1.9 12 0.0 Aizawl 2.6 14 3.5 2.6 14 -25.7 Mumbai 1.7 14 1.7 1.5 15 -11.8 Mizoram State (MZ) 2.0 15 3.0 2.2 15 -26.7 Barshi Rural 1.6 15 1.7 1.6 14 -5.9 Correlation (r) 0.99 Correlation (r) 0.97 Prostate (ICD 10:C61) and Myeloid Leukaemia (ICD 10:C92 C94) Males Th'puram- Thiruvananthapuram; Ahmedabad U- Ahmedabad Urban; Change in rank are highlighted in bold; Rank- Ranking status of PBCR based on WSP and ISP; ISP-WSP (AAR) (%) – Change in percentage between ISP and WSP AAR’s Th'puram- Thiruvananthapuram; Ahmedabad U- Ahmedabad Urban; Change in rank are highlighted in bold; Rank- Ranking status of PBCR based on WSP and ISP; ISP-WSP (AAR) (%) – Change in percentage between ISP and WSP AAR’s national standard population for estimating AAR. ISP will help in making sub-national comparison. Discussion Crude Rate (CR) and Age Adjusted Rate (AAR) Per 100,000 Population Using World Standard Population (WSP) and Indian Standard Population (ISP) in Population Based Cancer Registries (PBCR) – Breast(ICD-10:C50) and Cervix (ICD-10:C53) - Females Breast (ICD-10:C50) Cervix (ICD-10:C53) Registry CR Rank* AAR- WSP AAR- ISP Rank* ISP-WSP (AAR) (%) Registry CR Rank* AAR- WSP AAR- ISP Rank* ISP-WSP (AAR) (%) Delhi 34.8 1 41.0 33.2 1 -19.0 Papumpare 15.6 1 30.2 24.9 1 -17.5 Chennai 40.8 2 37.9 30.6 2 -19.3 Aizawl 26.0 2 28.0 24.4 2 -12.9 Bangalore 29.3 3 34.4 27.4 5 -20.3 Mizoram 19.4 3 23.1 20.1 3 -13.0 Th'puram 43.9 4 33.7 27.6 3 -18.1 Pasighat 18.7 4 22.5 19.6 4 -12.9 Mumbai 33.6 5 33.6 26.8 7 -20.2 Barshi rural 17.7 16.1 12.7 5 -21.1 Patiala 34.2 6 33.1 27.5 4 -16.9 Chennai 16.7 6 15.9 12.6 6 -20.8 Bhopal 28.2 7 33.0 27.1 6 -17.9 Delhi 13.2 7 15.5 12.5 7 -19.4 Nagpur 30.4 8 29.3 24.5 9 -16.4 Bangalore 13.1 8 15.3 12.2 8 -20.3 Papumpare 17.3 9 29.2 25.0 8 -14.4 Barshi Exp 15.6 9 14.7 11.9 9 -19.0 Aizawl 24.3 10 28.0 23.5 10 -16.1 Kamrup Urban 10.6 10 14.5 11.5 11 -20.7 Kollam 36.3 11 27.7 23.0 11 -17.0 Aurangabad 12.0 11 14.3 11.7 10 -18.2 Kamrup 21.5 12 27.1 22.1 12 -18.5 Bhopal 11.3 12 13.8 11.1 12 -19.6 Pune 23.4 13 26.4 21.0 13 -20.5 Nagaland 8.9 13 13.1 11.0 13 -16.0 Kolkata 30.7 14 25.5 21.0 14 -17.6 Nagpur 12.9 14 12.9 10.4 15 -19.4 Ahmedabad U 23.0 15 23.8 19.3 15 -18.9 Cachar 10.9 15 12.7 10.9 14 -14.2 Correlation (r) 0.97 Correlation (r) 0.99 Th'puram- Thiruvananthapuram; Ahmedabad U- Ahmedabad Urban; Barshi Exp- Barshi Expanded; Change in rank are highlighted in bold; *Rank- Ranking status of PBCR based on WSP and ISP; ISP-WSP (AAR)(%) – Change in percentage between ISP and WSP AAR’s Breast (ICD-10:C50) 374 DOI:10.31557/APJCP.2020.21.2.371 Indian Standard Population Table 4. Cancer Incidence: Crude Rate (CR) and Age Adjusted Rate (AAR) Per 100,000 Population Using World Standard Population (WSP) and Indian Standard Population (ISP) in Population Based Cancer Registries (PBCR) – Prostate (ICD-10:C61) and Myeloid Leukaemia (ICD-10:C92-C94) - Males Table 4. Discussion NCDIR-NCRP registries were using Segi world standard population since from its inception in 1981 (Segi, 1960; Doll et al., 1966). WHO adopted a standard based on the average world population age-structure for the period 2000-2025.The WHO standard has fewer children proportionally and a greater proportion of adults aged 70 years compared to the Segi world standard (Ahmad et al., 2000). The US-2000 and Canada-1992 standard population was projected based on the census (Anderson and Rosenberg, 1988; Government of Canada, 1991; Anderson and Rosenberg, 1998). European-1976 standard population was based on several Scandinavian population and the revision of European population was published in 2016 based on the 2011–2030 population projections of the unweighted average age structure of the populations of EU-27 member states plus the European Free Trade Association (Waterhouse et al., 1976; European Commission, 2013). Similarly, the ISP is based on average age structure of the India populations of last three censuses (1991, 2001 and 2011). The census data was considered to be a true estimate rather than using hypothetical projected population. Before comparing various different populations, a ‘pooled’ standard population (which is created by adding together the populations of the areas/period being compared) would reduce the variance of the standardized rates (Rosenberg et al., 1992; Choi et al., 1999). Norwegian registry has done major change in the 2014 report as preferred a Norwegian Standard Population, instead of the World Standard Population to estimate age-standardised rates. The higher weights in the oldest age groups of Norway 2014 reference population led to twice as high age-standardised rates (Cancer Registry of Norway, 2015). The use of Revised European Standard Population, shown slight impact on the pattern of time trends and the relative ranking of countries compared to European Standard Population (Crocetti et al., 2016).ii The artificial WSP population was defined in the 1960s, where 62% of the population was assumed to be below 35 years of age, and 38% above age 35. This age structure is different from India (as per Census, 2011), where a higher percentage of the population was below 35 (69%) and less population in above 35 (31%) years of age (Census of India, 2018). g ( , ) One should change the future standard population if the differences between the standard and actual population become problematic (Anderson and Rosenberg, 1998). Discussion A better local policy decision and rolling out programme can be done at our own population. GLOBOCAN has not replaced WSP with WHO due to various reasons (Bray et al., 2002). This study aimed to compare the WSP with ISP. Our result suggest that for comparison of AAR between Indian registries the ISP may be used and, for comparison of AAR between countries the WSP population may be used. The need for implementing ISP was due to the major difference in AAR between ISP and WSP, relative changes in the ranking and this is the national standard. overall rate, due to the strong positive association between age and mortality (Robson et al., 2007). health interventions between the regions / states can be re-prioritised in India. A better local policy decision and rolling out programme can be done at our own population. Standardization may be used to adjust for confounding variables in which age and gender are two of the most common variables. The standard population based on gender was not done because the differences in age-structure were not substantial. The standard population can affect the magnitude of the adjusted rate, the observed trends in adjusted rates over time and ranking of priorities (e.g., ranking of leading site of cancer). Since the standard population has a major impact on the results and the conclusions drawn, one should select the standard population very carefully before analyzing rates (Kitagawa, 1964; Choi et al., 1999). GLOBOCAN has not replaced WSP with WHO due to various reasons (Bray et al., 2002). This study aimed to compare the WSP with ISP. Our result suggest that for comparison of AAR between Indian registries the ISP may be used and, for comparison of AAR between countries the WSP population may be used. The need for implementing ISP was due to the major difference in AAR between ISP and WSP, relative changes in the ranking and this is the national standard. The time trends in cancer incidence rate with the effect of ISP and WSP on all sites of cancer for the period 1982-2013 showed that parallel curves as seen in other studies. A time trend in CR curve was lower than the ISP-AAR at the beginning period of 1982-1996 and later the CR over took ISP-AAR. This change in curve was due to gradual increase in the older age population in the registry area. Asian Pacific Journal of Cancer Prevention, Vol 21 76 Discussion that it is more representative of Indian registries population and the most recent available population data. The WSP give more weightage to the older population (most of the cancers occur) than ISP. The larger the difference between the age distributions of the standard population and the study population, the greater the difference in the crude and adjusted rates for the study population. Though WSP available to compare the rate with other population, we also need to have standard population which is of close to the real population. Therefore, many countries have used The results of this preliminary analysis using the Indian Standard Population on selected sites of cancer rates showed significant differences as compared with the Segi World population. There was a major difference in the age structure of WSP compared to ISP, especially in the older age group. In general, the use of a young standard (higher proportion of young population) leads to a low standardised mortality rate, and an old standard to a high Asian Pacific Journal of Cancer Prevention, Vol 21 375 Registry / Zone Age Group CR AAR-WSP AAR-ISP 0-14 15-34 35-64 65+ South ISP 34.2 34.3 26.6 4.9 - - - Chennai 21.5 36.5 35.7 6.3 123.9 120.6 96.6 Kollam 22.1 30.0 39.0 8.8 138.5 110.1 89.1 Central Bhopal 29.1 38.4 27.8 4.8 86.2 104.5 84.3 West Mumbai 22.1 39.2 33.2 5.5 106.7 115.1 91.0 Nagpur 23.4 37.1 33.6 5.8 90.2 91.6 75.6 North Delhi 27.1 39.0 29.9 4.0 116.4 146.4 118.2 Patiala 25.4 37.5 30.8 6.4 101.1 104.3 84.5 North East Meghalaya 40.5 35.7 20.8 3.0 71.5 130.4 103.7 Mizoram 32.5 37.6 25.9 4.0 134.6 188.8 148.2 Table 5. Percentage Distribution by Age Group of Population (Census 2011) with Cancer Incidence Rate (All sites of cancer - Both sexes) in Selected Population Based Cancer Registries and Indian Standard Population (ISP) CR, Crude Rate; WSP, World Standard Population; ISP, Indian Standard Population Table 5. Percentage Distribution by Age Group of Population (Census 2011) with Cancer Inciden cancer - Both sexes) in Selected Population Based Cancer Registries and Indian Standard Populat entage Distribution by Age Group of Population (Census 2011) with Cancer Incidence Rate (All sites of h sexes) in Selected Population Based Cancer Registries and Indian Standard Population (ISP) Sathishkumar K et al health interventions between the regions / states can be re-prioritised in India. References Anderson RN, Rosenberg HM (1998). Report of the second workshop on age adjustment National Center for Health Statistics. Vital Health Stat, 4, 1-37. g g Registry Programme, Indian Council of Medical Research. NCDIR, NCRP, (2018) Cancer Registration in India, Guidelines for setting up of Population Based and Hospital Based Cancer Registries. http://www.ncdirindia.org/Downloads/ NCDIR_Guidelines.pdf Accessed 11th April 2018 Anderson RN, Rosenberg HM (1998). Age Standardization of Death Rates: Implementation of the Year 2000 Standard. Nat Vital Stat Rep, 47, 1-16, 20. Robson B, Purdie G, Cram F, Simmonds S (2007). Age standardisation – an indigenous standard. Emerg Themes Epidemiol, 4, 3. Ahmad OE, Boschi-Pinto C, Lopez AD, et al (2000). Age standardization of Rates: A New WHO standard. In GPE Discussion Paper Series: no31, Geneva, World Health Organization. Rosenberg HM, Curtin LR, Maurer J, Offutt K (1992). Choosing a standard population: Some statistical considerations. Chapter 5, In: Feinleib, M., and Zarate, A.O.Reconsidering age-adjustment procedures: Workshop proceedings. Vital and Health Statistics, Series 4, number 29. Publication No. 93-1466. Armitage P, Doll R (1954). The age distribution of cancer and a multi-stage theory of carcinogenesis. Br J Cancer, 8, 1-12. Bray F, Guilloux A, Sankila R, Parkin DM (2002). Practical implications of imposing a new world standard population. Cancer Causes Control, 13, 175-82. Sankoh O, Sharrow D, Herbst K, et al (2014). The INDEPTH Standard population for low- and middle-income countries, 2013. Glob Health Action, 7, 23286. Bray F, Znaor A, Cueva P, et al (2015). Planning and developing population-based cancer registration in low- and middle-income settings. IARC Technical Publication No. 43. IARC, Lyon. Segi M (1960). Cancer Mortality for Selected Sites in 24 Countries (1950-57). Sendai, Tohoku University School of Medicine, Dept. of Public Health, Japan Cancer Society,1960. Cancer Registry of Norway (2015). Cancer in Norway 2014 - Cancer incidence, mortality, survival and prevalence in Norway. Oslo: Cancer Registry of Norway.i Torre LA, Bray F, Siegel RL, et al (2015). Global cancer statistics 2012. CA Cancer J Clin, 65, 87-108. Census of India. Office of the Registrar General and Census Commissioner. (2018): Socio Cultural Tables, C14, Population by Five Year Age Group, by Residence and Sex, 1991, 2001 and 2011. New Delhi, India. http://www. censusindia.gov.in Waterhouse J, Muir CS, Correa P, Powell J (1976). Cancer incidence in five continents. Lyon: IARC Scientific Publications, No.15. Choi BCK, Deguia NA, Walsh P (1999). Look before you leap: stratify before you standardize. with ISP-AAR. European Commission (2013). Revision of the European standard population: Report of the Eurostat’s task force. Eurostat, Luxembourg: Publications Office of the European Union. Conflict of Interest Conflict of Interest The authors declare no conflicts of interest. Conflict of Interest The authors declare no conflicts of interest. NCDIR (National Centre for Disease Informatics and Research) (2016). Three-Year Report of Population Based Cancer Registries 2012-2014. Bangalore, India, National Cancer Registry Programme, Indian Council of Medical Research. Discussion The revision of standard population would be based on the future release of Census of India, population, subject to major significant changes in the age structures. Statistical adjustment was performed in epidemiology to remove or reduce the confounding effects of extraneous confounding factor, for example age, when comparing Incidence or mortality rates in different populations. An actual measure of summary information of Age Adjusted rate using ISP may be better in health planning and policy for comparison of different population and time period. Since India sub-population are remarkably different from the Segi standard the simultaneous observation of crude rate, age adjusted rate using world standard and national standard would offer better information. The National Cancer Registry Programme has been in existence from 1982 with three PBCRs and today its coverage has expanded to 36 PBCRs with about 10% of the population in India being covered. Since the population coverage by cancer registries were expanding periodically, the total India census population as considered as standard rather than pooled PBCRs population. Many PBCRs (Aizawl, Bangalore, Bhopal, Cahcar, Delhi and Pasighat) CR close to ISP-AAR indicates that, especially the older age population structure of PBCRs similar to the ISP. Breast cancer is the leading site of cancer among women in India (NCDIR, 2016). The changing of rank was seen by changing the standard population in specific sites of cancer including breast. Population Based Cancer Registries can measure cancer incidence, its trend and mortality and have a unique role in planning and evaluating cancer control plans and reducing the cancer burden in the community (Bray et al., 2015). Based on the change in rank, planning cancer prevention, cancer control activities and allocation of resources for In conclusion, Indian Standard population would be more representative of the age structure for many Indian registries and this would give more realistic comparison between populations within India. This information will be useful for cancer surveillance, policy and program development. Achieving entire national coverage of cancer registration in India would result more closer crude rate 376 Asian Pacific Journal of Cancer Prevention, Vol 21 76 Acknowledgements Evelyn MK (1964). Standardized comparisons in population research. Demography, 1, 296-315. Authors acknowledge the contribution of 27 PBCRs (Ahmedabad Urban, Aurangabad, Bangalore, Barshi Expanded, Barshi Rural, Bhopal, Cachar District, Chennai, Delhi, Dibrugarh District, Kamrup Urban District, Kolkata, Kollam District, Manipur State, Meghalaya, Mizoram State, Mumbai, Nagaland, Nagpur, Naharlagun, Pasighat, Patiala District, Pune, Sikkim State, Thiruvananthapuram District, Tripura State, Wardha District) under NCRP to this study and it was approved by the Scientific Advisory Committee and Research Area Panel on Cancer members. We analysed the existing data available with NCDIR-NCRP and it is more of secondary data. Hence, we did not identify any ethical issues in this analysis and this study was carried out without funding. Government of Canada: Canadian Standard Population, 1991. Retrieved from http://www.statcan.gc.ca/ pub/82-003-x/2013008/article/11857/tbl/appa-eng.htm. Accessed 11th April 2018. HealthStats NSW (2017). Australian Standard Population, 2001. Retrieved from http://www.healthstats.nsw.gov.au/Indicator/ dem_pop_stan/dem_pop_stan.Accessed 11th April 2018. Jensen OM, Storm HH (1991). Purposes and uses of cancer registration. IARC Sci Publ, 95, 7–21. Mausner JS, Bahn AK (1974). Epidemiology: An Introductory Text (1st ed.). Philadelphia, London and Toronto, W. B. Saunders Co, 1974. Naing NN (2000). Easy Way to learn standardization: Direct and Indirect methods. Malays J Med Sci, 7, 10-5. NCDIR (National Centre for Disease Informatics and Research) (2013). Time trends in cancer incidence rates 1982-2010. Bangalore, India, National Cancer Registry Programme, Indian Council of Medical Research. This work is licensed under a Creative Commons Attribution- Non Commercial 4.0 International License. DOI:10.31557/APJCP.2020.21.2.371 Indian Standard Population DOI:10.31557/APJCP.2020.21.2.371 Indian Standard Population with ISP-AAR. References Am J Epidemiol, 149, 1087- 96. Crocetti E, Dyba T, Martos C, et al (2017). The need for a rapid and comprehensive adoption of the revised European standard population in cancer incidence comparisons. Eur J Cancer Prev, 26, 447-52. This work is licensed under a Creative Commons Attribution- Non Commercial 4.0 International License. This work is licensed under a Creative Commons Attribution- Non Commercial 4.0 International License. This work is licensed under a Creative Commons Attribution- Non Commercial 4.0 International License. Doll R, Payne P, Waterhouse J (1966). Cancer Incidence in Five Continents: A Technical Report. Berlin, Springer- Verlag. Danckert B, Engholm G, Ferlay J, et al (2016). NORDCAN: Cancer Incidence, Mortality, Prevalence and Survival in the Nordic Countries. Association of the Nordic Cancer Registries, Danish Cancer Society, Version 7.3(08.07.2016). http://www.ancr.nu. Asian Pacific Journal of Cancer Prevention, Vol 21 377
https://openalex.org/W2807860275	https://www.nii-kpssz.com/jour/article/download/390/309	Russian	null	CARDIOMARKERS EVALUATION AND MYOCARDIAL ASSESSMENT IN PATIENTS WITH RECANALIZATION OF CHRONIC TOTAL OCCLUSIONS USING HIGH DOSE ATORVASTATIN LOADING	Kompleksnye problemy serdečno-sosudistyh zabolevanij	2,018	cc-by	6,346	омплексные проблемы сердечно-сосудистых заболеваний К В обеих группах определялся тропонин I и МВ фракция креатинфосфокиназы (МВ-КФК) за 1 сутки до и через 24 часа после ЧКВ. Также оценивалось функциональное состояние миокарда с помощью эхокардиографии за 1 сутки до и через 24 часа после ЧКВ. В основной группе через 24 часа после операции у 3 (7,9%) больных отмечалось умеренное повы- шение уровня тропонинов. В контрольной группе у 11 (25,0%) человек было определено повыше- ние уровня тропонинов в 5 и более раз. Через сутки после эндоваскулярной реваскуляризации, в основной группе у 3 (7,9%) пациентов отмечалось повышение уровня МВ-КФК, в контрольной группе это параметр был повышен у 16 (36,4%) пациентов. Через 24 часа после эндоваскулярной реваскуляризации не было отмечено статистически достоверного изменения эхокардиографиче- ских параметров ни в одной из групп. Применение нагрузочной дозы аторвастатина в 80 мг обладает протективным эффектом и пре- дотвращает развитие периоперационной ишемии миокарда. При этом не оказывает значимого влияния на показатели центральной гемодинамики в ближайшем периоде. Поступила в редакцию: 03.12.17; поступила после доработки: 18.01.18; принята к печати: 26.01.18 омплексные проблемы сердечно-сосудистых заболеваний К 29 УДК 616.132.2-089 DOI 10.17802/2306-1278-2018-7-1-29-35 ОРИГИНАЛЬНЫЕ ИССЛЕДОВАНИЯ ORIGINAL STUDIES ОЦЕНКА УРОВНЯ КАРДИОМАРКЁРОВ И ФУНКЦИОНАЛЬНОГО СОСТОЯНИЯ МИОКАРДА У ПАЦИЕНТОВ С РЕКАНАЛИЗАЦИЕЙ ХРОНИЧЕСКИХ ОККЛЮЗИЙ КОРОНАРНЫХ АРТЕРИЙ ПРИ ПРИМЕНЕНИИ НАГРУЗОЧНЫХ ДОЗ АТОРВАСТАТИНА ¹ Бюджетное учреждение Ханты-Мансийского автономного округа - Югры «Окружной кардиологический диспансер «Центр диагностики и сердечно-сосудистой хирургии», Сургут, Россия ² Научно-исследовательский институт терапии и профилактической медицины – филиал Федерального государственного бюджетного научного учреждения «Федеральный исследовательский центр Институт цитологии и генетики Сибирского отделения Российской академии наук», Новосибирск, Россия А.С. Рагозина¹, И.В. Петренко¹, И.А. Урванцева¹, А.И. Горьков¹, К.Ю. Николаев² Поступила в редакцию: 03.12.17; поступила после доработки: 18.01.18; принята к печати: 26.01.18 Цель Исследовать влияние нагрузочных доз аторвастатина на снижение ассоциированного с чрескож- ными коронарными вмешательствами (ЧКВ) инфаркта миокарда и функциональное состояние миокарда при реканализации хронических окклюзий коронарных артерий (ХОКА). Материалы и методы В исследование вошло 82 пациента, подвергшихся реканализации ХОКА. В основную группу во- шло 38 пациентов с применением нагрузочной дозы аторвастатина 80 мг за сутки до операции, в контрольную 44 пациента без применения нагрузочной дозы. В обеих группах определялся тропонин I и МВ фракция креатинфосфокиназы (МВ-КФК) за 1 сутки до и через 24 часа после ЧКВ. Также оценивалось функциональное состояние миокарда с помощью эхокардиографии за 1 сутки до и через 24 часа после ЧКВ. Результаты В основной группе через 24 часа после операции у 3 (7,9%) больных отмечалось умеренное повы- шение уровня тропонинов. В контрольной группе у 11 (25,0%) человек было определено повыше- ние уровня тропонинов в 5 и более раз. Через сутки после эндоваскулярной реваскуляризации, в основной группе у 3 (7,9%) пациентов отмечалось повышение уровня МВ-КФК, в контрольной группе это параметр был повышен у 16 (36,4%) пациентов. Через 24 часа после эндоваскулярной реваскуляризации не было отмечено статистически достоверного изменения эхокардиографиче- ских параметров ни в одной из групп. Заключение Применение нагрузочной дозы аторвастатина в 80 мг обладает протективным эффектом и пре- дотвращает развитие периоперационной ишемии миокарда. При этом не оказывает значимого влияния на показатели центральной гемодинамики в ближайшем периоде. Ключевые слова Хронические окклюзии коронарных артерий • Реканализация • Аторвастатин Исследовать влияние нагрузочных доз аторвастатина на снижение ассоциированного с чрескож- ными коронарными вмешательствами (ЧКВ) инфаркта миокарда и функциональное состояние миокарда при реканализации хронических окклюзий коронарных артерий (ХОКА). В исследование вошло 82 пациента, подвергшихся реканализации ХОКА. В основную группу во- шло 38 пациентов с применением нагрузочной дозы аторвастатина 80 мг за сутки до операции, в контрольную 44 пациента без применения нагрузочной дозы. статинов [11-16]. Например, в исследовании, проведённом Nafasi L. с соавто- рами, изучался эффект нагрузочной дозы аторвастатина в 80 мг. Всего в исследование вошло 190 человек со стабильной ИБС, отобранных для проведения ЧКВ. Пациенты были разделены на две группы: в первой группе была предписана нагрузочная доза аторвастатина перед операцией, во второй группе пациенты по- лучили плацебо. В первой группе отмечалась достоверно более низкая частота периоперационного ИМ [15]. В современном мире одной из основных причин смертности являются сердечно-сосудистые заболевания (ССЗ), и, к сожале- нию, прогнозы весьма неутешительны. Считается, что к 2030 году около 23,3 миллионов человек умрёт от ССЗ, главным образом, от болезней сердца и инсульта [1]. Основное место в структуре ССЗ играет ишемическая болезнь сердца (ИБС) [2-4]. Учитывая зна- чимость ССЗ в структуре общей смертности, в настоящее время существует множество методов лечения данной патологии, кото- рые можно разделить на две основные группы – медикаментоз- ные и хирургические. При этом на фоне широкого распростране- ния хирургических методов лечения ИБС большое значение при- обретает необходимость максимально снизить риск оперативной коррекции и защитить миокард от возможных неблагоприятных событий, к одним из которых относится периоперационный ин- фаркт миокарда (ИМ), что существенно ухудшает ближайший и отдалённый жизненный прогноз [5, 6]. В исследовании ROMA изучался эффект нагрузочной дозы розувастатина (40 мг) на частоту периоперационного ИМ, а также на уровень главных неблагоприятных сердечно-сосуди- стых и цереброваскулярных событий в течение 30-дневного и 12-месячного периода наблюдения. В группе нагрузочной дозы розувастатина по сравнению с контрольной группой отмечался достоверно меньший процент ЧКВ-ассоциированного ИМ (26,4% против 8,7%), а также меньшее число неблагоприятных событий в течение ближайшего и отдалённого периода наблюдения [16]. Одной из самых интересных и спорных групп в современной эндоваскулярной кардиологии являются пациенты с хрониче- скими окклюзиями коронарных артерий (ХОКА) [7]. Основной проблемой при проведении чрескожного коронарного вмеша- тельства (ЧКВ) у пациентов с ХОКА является развитие периопера- ционного ИМ от 10 до 40 % случаев, что связано с увеличением смертности в ближайшем и отдалённом периоде [5, 6]. Однако при анализе современной научной литературы выявлено, что успешная реваскуляризация пациентов с ХОКА приводит к умень- шению функционального класса (ФК) стенокардии, улучшению функции левого желудочка (ЛЖ) и увеличению выживаемости в отдалённом периоде [8-10]. В исследовании AMERICA [17] доказано протективное влия- ние плейотропных эффектов статинов. К основным эффектам от- носятся: вазодилатирующий, коагуляционный, противовоспали- тельный, стабилизирующий (в отношении атеросклеротической бляшки). Кроме этого, статины оказывают антиишемический эф- фект, связанный с улучшением функции эндотелия и увеличени- ем выработки оксида азота. 30 Использование нагрузочных доз аторвастатина у пациентов с реканализацией ХОКА Results 3 patients (7.9%) in the study group demonstrated moderate elevation of troponin I levels. 11 (25%) patients in the control had over 5-fold increase from the baseline in troponin I levels. 24 hours after percutaneous coronary intervention, elevated CPK-MB was found in 3 (7.9%) patients from the study group and in 16 (36.4%) patients from the control group. There were no statistically significant differences found in echocardiography parameters between the groups. Conclusion Atorvastatin 80 mg has a protective effect and prevents periprocedural myocardial ischemia. However, it does not produce any effects on central hemodynamic parameters in the immediate postoperative period. Keywords Chronic total occlusions in coronary arteries • Recanalization • Atorvastatin 3 patients (7.9%) in the study group demonstrated moderate elevation of troponin I levels. 11 (25%) patients in the control had over 5-fold increase from the baseline in troponin I levels. 24 hours after percutaneous coronary intervention, elevated CPK-MB was found in 3 (7.9%) patients from the study group and in 16 (36.4%) patients from the control group. There were no statistically significant differences found in echocardiography parameters between the groups. 3 patients (7.9%) in the study group demonstrated moderate elevation of troponin I levels. 11 (25%) patients in the control had over 5-fold increase from the baseline in troponin I levels. 24 hours after percutaneous coronary intervention, elevated CPK-MB was found in 3 (7.9%) patients from the study group and in 16 (36.4%) patients from the control group. There were no statistically significant differences found in echocardiography parameters between the groups. Список сокращений ИБС ИМ КАГ КДО КСО ЛЖ МВ-КФК ОКД – – – – – – – – ишемическая болезнь сердца инфаркт миокарда коронарография конечно-диастолический объём конечно-систолический объём левый желудочек МВ фракция креатинфосфокиназы Окружной кардиологический диспансер ОКС ССЗ ТнI ФВ ФК ХОКА ЧКВ ЭхоКГ – – – – – – – острый коронарный сидром сердечно-сосудистые заболевания тропонин I фракция выброса функциональный класс хроническая окклюзия коронарных артерий чрескожное коронарное вмешательство эхокардиография CARDIOMARKERS EVALUATION AND MYOCARDIAL ASSESSMENT IN PATIENTS WITH RECANALIZATION OF CHRONIC TOTAL OCCLUSIONS USING HIGH DOSE ATORVASTATIN LOADING 30 Использование нагрузочных доз аторвастатина у пациентов с реканализацией ХОКА CARDIOMARKERS EVALUATION AND MYOCARDIAL ASSESSMENT IN PATIENTS WITH RECANALIZATION OF CHRONIC TOTAL OCCLUSIONS USING HIGH DOSE ATORVASTATIN LOADING A.S. Ragozina¹, I.V. Petrenko¹, I.A. Urvantseva¹, A.I. Gorkov¹, K.Y. Nikolaev² 1State-financed organization Khanty-Mansi Autonomous Region - Ugra «District Cardiac Dispensary «Centre of Diagnosis and Cardiovascular Surgery», Surgut, Russia ² Research Institute of Internal and Preventive Medicine, Novosibirsk, Russia A.S. Ragozina¹, I.V. Petrenko¹, I.A. Urvantseva¹, A.I. Gorkov¹, K.Y. Nikolaev² 1State-financed organization Khanty-Mansi Autonomous Region - Ugra «District Cardiac Dispensary «Centre of Diagnosis and Cardiovascular Surgery», Surgut, Russia ² Research Institute of Internal and Preventive Medicine, Novosibirsk, Russia A.S. Ragozina¹, I.V. Petrenko¹, I.A. Urvantseva¹, A.I. Gorkov¹, K.Y. Nikolaev² 1State-financed organization Khanty-Mansi Autonomous Region - Ugra «District Cardiac Dispensary «Centre of Diagnosis and Cardiovascular Surgery», Surgut, Russia ² Research Institute of Internal and Preventive Medicine, Novosibirsk, Russia Для корреспонденции: Рагозина Аурика Салаватовна, тел. +79227796574; e-mail: aurikasa@gmail.com; адрес: 628403, г. Сургут, просп. Ленина, 69/1 Corresponding author: Ragozina Aurika, tel. +79227796574; e-mail: aurikasa@gmail.com; address: Russian Federation, 628403, Surgut, 69/1, Lenin av. Aim To assess the effects of high dose atorvastatin loading on the reduction of the rate of myocardial infarction associated with percutaneous coronary interventions and to estimate functional state of myocardium in patients with recanalization of chronic total occlusions in coronary arteries. Methods 82 patients who underwent recanalization of chronic total occlusions in coronary arteries were enrolled in the study. The study group included 38 patients who received atorvastatin 80 mg before surgery. The control group consisted of 44 patients who did not receive atorvastatin. Troponin I and CPK-MB fraction were measured in both groups 1 day prior and 24 hours after percutaneous coronary intervention. Functional parameters of the myocardium were estimated with echocardiography 1 day before and 24 hours after percutaneous coronary intervention. To assess the effects of high dose atorvastatin loading on the reduction of the rate of myocardial infarction associated with percutaneous coronary interventions and to estimate functional state of myocardium in patients with recanalization of chronic total occlusions in coronary arteries. 82 patients who underwent recanalization of chronic total occlusions in coronary arteries were enrolled in the study. The study group included 38 patients who received atorvastatin 80 mg before surgery. The control group consisted of 44 patients who did not receive atorvastatin. Troponin I and CPK-MB fraction were measured in both groups 1 day prior and 24 hours after percutaneous coronary intervention. Functional parameters of the myocardium were estimated with echocardiography 1 day before and 24 hours after percutaneous coronary intervention. Материалы и методы Группы наблюдения Проведено проспективное, сравнительное исследование па- циентов с реканализацией ХОКА. Протокол исследования одобрен локальным этическим комитетом по месту его проведения. Основная группа: 38 пациентов, которым за сутки до опе- ративного вмешательства назначалась нагрузочная доза 80 мг аторвастатина. Перед оперативной коррекцией за сутки всем пациентам назначался клопидогрел в нагрузочной дозе 300 мг и аспирин в дозе 200 мг. После имплантации стента пациенты продолжили принимать двойную антиагрегантную терапию в течение 18 ме- сяцев – аспирин 100 мг в сутки и клопидогрел 75 мг в сутки. Группа сравнения: 44 пациента, которым до реканализации ХОКА аторвастатин в нагрузочной дозе (80 мг) не применялся. Таким образом, изучены лабораторные и инструментальные данные 82 пациентов, госпитализированных в Окружной кардио- логический диспансер «Центр диагностики и сердечно-сосудистой хирургии» г. Сургута в период с 2009 г. по 2015 г. со стабильной ИБС, направленных для проведения КАГ и оперативного лечения кардиологами или кардиохирургами Окружного кардиологиче- ского диспансера (ОКД) с поликлинического этапа. Пациентам в основной группе за сутки до эндоваскулярной реваскуляризации также назначался аторвастатин в нагрузочной дозе 80 мг. В последующем пациенты продолжили принимать аторвастатин в дозе 20 мг пожизненно. Критерии включения: документированная по коронарогра- фии (КАГ) ХОКА, стабильная стенокардия напряжения I-IV ФК и согласие пациентов на участие в исследовании (всеми участника- ми подписано информированное согласие на участие в исследо- вании), возраст от 30 до 65 лет. Основные клинические характеристики обследованных па- циентов представлены в Табл. 1. Оценка эффективности нагрузочной дозы аторвастатина в 80 мг оценивалась по выявлению ЧКВ-ассоциированного ИМ. Критериями периоперационного ИМ служили критерии ESC/ ACCF/AHA/WHF от 2013 г. В качестве маркёров некроза миокар- да использовались: ТнI (анализатор - IMMULITE, фирма Siemens, Критерии исключения: сахарный диабет, сердечная недо- статочность высокого ФК (3-4 ФК по NYHA) со снижением фрак- ции выброса (ФВ) менее 40 %, ОКС (нестабильная стенокардия и Таблица 1. Клиническая характеристика обследованных больных Table 1. Clinical and demographic data of the study population Характеристика / Группа Основная группа (n = 38) Группа сравнения (n = 44) Достоверность Возраст 54,6±9,5 51,2±6,4 р>0,05 Пол муж. 29 (76,3%) жен. 9 (23,7%) муж. 40 (90,9%) жен. статинов [11-16]. Улучшение вариабельности частоты сердечных сокращений связано с модуляцией функции автоном- ной нервной системы сердца [12, 18, 19]. С учётом анализа имеющейся на данный момент литературы, наиболее доказанным протективным действием обладает аторвастатин [12, 15, 20-22]. Обращает на себя внимание, что работ, которые бы исследовали эффективность предварительного применения аторвастатина у пациентов с реканализацией ХОКА, практически нет. В арсенале современной медицины имеется значительное количество средств, способствующих улучшению результатов операции, и немаловажную роль играет медикаментозная те- рапия. В настоящее время возрос интерес к протективной роли 31 А.С. Рагозина и др. Суммируя вышеизложенное, можно заключить, что протек- тивная роль статинов при выполнении ЧКВ у пациентов с ХОКА настоящее время не вызывает сомнений. Необходимо расширять показания для применения указанной группы препаратов, в том числе при реваскуляризации пациентов с ХОКА. ИМ), тяжёлая почечная (хроническая болезнь почек С3 и выше), печёночная (тяжёлой степени по Чайлд-Пью), дыхательная недо- статочность (дыхательная недостаточность 3 степени), исходно повышенные уровни тропонинов либо МВ-фракции креатинфос- фокиназы (МВ-КФК) . Референсные значения лабораторных пока- зателей: МВ-КФК менее 5 нг/мл, Тропонин I (ТнI) – менее 1 нг/мл (нормативы лаборатории ОКД «ЦД и ССХ»). ИМ), тяжёлая почечная (хроническая болезнь почек С3 и выше), печёночная (тяжёлой степени по Чайлд-Пью), дыхательная недо- статочность (дыхательная недостаточность 3 степени), исходно повышенные уровни тропонинов либо МВ-фракции креатинфос- фокиназы (МВ-КФК) . Референсные значения лабораторных пока- зателей: МВ-КФК менее 5 нг/мл, Тропонин I (ТнI) – менее 1 нг/мл (нормативы лаборатории ОКД «ЦД и ССХ»). ОРИГИНАЛЬНЫЕ ИССЛЕДОВАНИЯ ORIGINAL STUDIES Результаты Обе группы сопоставимы по возрасту, полу, сопутствующим заболеваниям, стажу проживания пациентов на Севере. В основ- ной группе отмечалось статистически значимое преобладание пациентов с ожирением, а также перенесших ИМ. В контрольной группе было статистически значимо больше пациентов с куре- нием в анамнезе, а также отмечалось больше пациентов со сте- нокардией напряжения 3-4 ФК. Исходный уровень ТнI и МВ-КФК статистически значимо не различался. Одной из целей нашего исследования было оценить влияние нагрузочной дозы аторвастатина на изменение функционального состояния миокарда. Эхокардиография (ЭхоКГ) выполнялась трансторакальным методом при поступлении и через 24 часа после операции. Для проведения ЭхоКГ использовался аппарат «Vivid 9» фир- мы General Electric, датчик 5 МГц. Использовалась стандартная од- номерная, а также двухмерная ЭхоКГ с определением линейных размеров и объема предсердий и желудочков в систолу и диасто- лу. Также применялся цветной доплеровский режим. Оценивае- мые параметры: ФВ ЛЖ, конечный систолический объём (КСО), конечный диастолический объём (КДО). В основной группе пациентов через 24 часа после реканали- зации окклюзии у 3 больных отмечалось умеренное повышение уровня тропонинов, не достигающее диагностических критериев ЧКВ-ассоциированного ИМ. В группе сравнения у 11 человек было определено диагностически значимое повышение уровня тропо- нинов – в 5 и более раз, что соответствует критериям ЧКВ-ассоци- ированного ИМ. Статистическая обработка проводилась с помощью пакета статистических программ SPSS, версия 11. Определялся характер распределения количественных признаков методом Колмогоро- ва – Смирнова. При сопоставлении количественных переменных применялись параметрические и непараметрические методы статистики (t-критерий Стьюдента, тест Манна-Уитни). При срав- нении дихотомических переменных использовался критерий χ². Количественные переменные представлены в таблицах в виде При оценке уровня МВ-КФК выявлено, что перед проведени- ем операции не было выявлено повышения уровня МВ-КФК ни у кого из обследуемых в обеих группах. Через сутки после эндова- скулярной реваскуляризации в основной группе у 3 пациентов от- мечалось повышение уровня МВ-КФК, не достигшее двух верхних границ нормы, в то время как в группе сравнения превышение Таблица 2. Лабораторная характеристика обследованных больных Table 2. Материалы и методы 4 (9,1%) р>0,05 Гипертония 37 (97,4%) 44 (93,0%) р>0,05 Поражение БЦА 18 (47,4 %) 24 (54,5%) р>0,05 Атеросклероз артерий нижних конечностей 2 (5,6%) 6 (13,6%) р>0,05 Нарушение ритма сердца 3 (7,9%) 6 (13,6%) р>0,05 Класс стенокардии 1 ФК – 8 (21,1%) 2 ФК – 10 (26,3%) 3 ФК – 19 (50,0%) 4 ФК – 1 (2,6%) 1 ФК – 0 (0%) 2 ФК – 10 (22,7%) 3 ФК – 34 (77,3%) 4 ФК – 0 (0 %) р<0,05 р>0,05 р>0,05 р>0,05 Курение Да – 10 (26,3%) Нет – 28 (73,7%) Да – 43 (97,7%) Нет – 1 (2,3%) р<0,05 Перенесённый инфаркт миокарда в анамнезе Да – 8 (21,1%) Нет – 30 (78,9%) Да – 0 (0%) Нет – 44 (100%) р<0,05 Масса тела 88,8±14,9 77,4±8,6 р<0,05 ИМТ 30,2±5,1 26,0±2,0 р<0,05 Стаж проживания пациента на Севере 21,2±13,6 19,7±8,8 р>0,05 Примечания: БЦА - брахиоцефальные артерии, ИМТ - индекс массы тела Note: BCA – brachiocephalic arteries, BMI – body mass index Таблица 1. Клиническая характеристика обследованных больных Table 1. Clinical and demographic data of the study population 32 High dose atorvastatin loading in patients with CTO recanalization M±SD (среднее значение ± среднее квадратичное отклонение), а также Ме (медиана), нижний (Q1) и верхний (Q3) квартили. Во всех процедурах статистического анализа критический уровень значимости нулевой статистической гипотезы (р) принимался равным 0,05. Для оценки сопоставимости двух групп по крите- рию «Перенесённый инфаркт миокарда» проведён парциальный корреляционный анализ. иммунометрический тест) и МВ-КФК (анализатор - COBAS Е411, фирма – ROCHE, принцип «сэндвича»). Референсные значения лабораторных показателей: МВ-КФК менее 5 нг/мл, ТнI – менее 1 нг/мл (нормативы лаборатории ОКД «ЦД и ССХ»). Показатели оце- нивались за 1 сутки до и через 24 часа после операции. КАГ проводилась трансфеморальным доступом на аппарате Allura Clarity FD20 фирмы Philips (Голландия). Для контрастиро- вания коронарных артерий во всех случаях использовали йодсо- держащий рентгенконтрастный препарат (ксенетикс 350), доза в среднем составила 300 мл. Результаты Особой пробле- мой при проведении ЧКВ у пациентов с ХОКА является развитие периоперационного ИМ в 10-40% случаев, что существенно ухуд- шает ближайший и отдалённый прогноз оперативной коррекции и связано с увеличением смертности в течение 30 дней после ЧКВ, а также в отдалённом периоде [5, 6]. В своём исследовании А. Prasad с соавт. [24] оценили влияние на жизненный прогноз изначально повышенных маркёров некроза миокарда. Был елан вывод, что изначальное повышение уровня тропонинов у паци- ентов с реканализацией ХОКА является мощным независимым предиктором смертности в отдалённом периоде и имеет даже большее прогностическое значение, чем ЧКВ-ассоциированный ИМ. ОРИГИНАЛЬНЫЕ ИССЛЕДОВАНИЯ ORIGINAL STUDIES Основные результаты исследования лабораторных данных представлены в Табл. 2. Исходный уровень ФВ, КДО и КСО статистически значимо не различался. Через 24 часа после эндоваскулярной реваскуляри- зации не было отмечено статистически значимого изменения ис- следуемых параметров ни в одной из групп. В Табл. 3 приведены основные результаты функциональных параметров миокарда. При анализе характера поражения основных магистральных артерий у прооперированных больных выявлено, что в большин- стве случаев в обеих группах преобладало поражение перед- ней межжелудочковой ветви: в основной группе у 21 пациента (55,3%), в группе сравнения у 22 пациентов (56,8%). Поражение правой коронарной артерии в первой группе выявлено у 11 чело- век (28,9%), во второй группе также у 11 человек (25,0%). Пора- жение огибающей ветви выявлено у 6 человек (15,8%) в основ- ной группе и у 8 человек (18,2%) в группе сравнения. Изменения значимо не различались. Следует отметить, что в проведённом нами исследовании па- циенты с изначальным повышением маркёров некроза миокар- да – тропонинов, были исключены из исследования, что является существенным отличием от вышеуказанной научной работы. В современных рекомендациях для оценки ишемии и некро- за миокарда используются кардиоспецифические ферменты – МВ -КФК и тропонины. До 2013 г. критерием ЧКВ-ассоциированного ИМ служило повышение уровня данных показателей в 3 раза. По- сле 2013 г., согласно принятому третьему универсальному опре- делению ИМ, диагноз периоперационного ИМ выставляется при превышении верхней границы референсных значений в 5 и бо- лее раз, и использование тропонинов для диагностики инфаркта является предпочтительным [25]. В нашем исследовании в каче- стве маркёров некроза миокарда использовались именно реко- мендуемые показатели: ТнI и МВ-КФК. Оценка уровня периопера- ционной ишемии осуществлялась согласно критериями третьего универсального определения ИМ. Использовались референсные значения маркёров некроза миокарда согласно данным лабора- тории учреждения. Тяжесть коронарного атеросклероза оценивалась по шкале SYNTAX. При анализе квартилей SYNTAX Score более 18.75 рассма- тривался как высокий (Q1 = 7, Q3 = 18.75). Результаты В основной группе к группе высокого риска относились 10 человек (26,3%). В группе контроля высокий риск по данной шкале имело аналогичное ко- личество пациентов – 10 (22,7%). Полученные результаты стати- стически значимо не различались. Летальных исходов в обеих группах в ближайшем послеопе- рационном периоде не отмечено. Результаты Laboratory data of the study and control groups Показатель / Группа Основная группа (n = 38) Группа сравнения (n = 44) Статистическая значимость До операции Тропонин I, нг/мл <1 нг/мл – 38 (100%) 0,39±0,18 <1 нг/мл - 44 (100%) 0,24±0,09 р>0,05 Mе = 0,47; Q1 = 0,20; Q3 = 0,59 Mе = 0,20; Q1 = 0,20; Q3 = 0,34 МВ-КФК, нг/мл <5 нг/мл – 38 (100%) 3,2±1,2 <5 нг/мл – 44 (100%) 2,7±1,3 р>0,05 Mе = 3,10; Q1 = 2,1; Q3 = 4,20 Mе = 3,00; Q1 = 2,00; Q3 = 3,80 Через 24 часа после операции Тропонин I, нг/мл >5 нг/мл – 0 (0%) 0,5±0,3 >5 нг/мл – 11 (25%) 5,4±13,9 р<0,05 Mе = 0,50; Q1 = 0,20; Q3 = 0,70 Mе = 0,27; Q1 = 0,20; Q3 = 4,80 МВ-КФК, нг/мл >5 нг/мл – 3 (8%) 3,0±1,4 >5 нг/мл – 16 (36,4%) 7,5±11,8 р<0,05 Mе = 3,00; Q1 = 1,80; Q3 = 4,0 Mе = 3,00; Q1 = 2,00; Q3 = 8,80 Примечания: МВ-КФК – МВ-фракция креатинфосфокиназы Note: CPK-MB – Creatine phosphokinase-MB Таблица 2. Лабораторная характеристика обследованных больных Table 2. Laboratory data of the study and control groups Примечания: МВ-КФК – МВ-фракция креатинфосфокиназы Note: CPK-MB – Creatine phosphokinase-MB 33 А.С. Ragozina et al. уровня МВ-КФК было отмечено у 16 пациентов, в 7 случаях из которых верхний уровень был превышен в 2 раза и более. В ос- новной группе не было выявлено значимой динамики уровня МВ-КФК в течение всего периода наблюдения. приводит к формированию развитой системы коллатералей, что закономерно вызывает множество вопросов по поводу целесоо- бразности реваскуляризации таких поражений. Особой пробле- мой при проведении ЧКВ у пациентов с ХОКА является развитие периоперационного ИМ в 10-40% случаев, что существенно ухуд- шает ближайший и отдалённый прогноз оперативной коррекции и связано с увеличением смертности в течение 30 дней после ЧКВ, а также в отдалённом периоде [5, 6]. В своём исследовании А. Prasad с соавт. [24] оценили влияние на жизненный прогноз изначально повышенных маркёров некроза миокарда. Был елан вывод, что изначальное повышение уровня тропонинов у паци- ентов с реканализацией ХОКА является мощным независимым предиктором смертности в отдалённом периоде и имеет даже большее прогностическое значение, чем ЧКВ-ассоциированный ИМ. приводит к формированию развитой системы коллатералей, что закономерно вызывает множество вопросов по поводу целесоо- бразности реваскуляризации таких поражений. Заключение По результатам оценки уровня кардиомаркёров, применение нагрузочной дозы аторвастатина в 80 мг перед реканализацией и стентированием ХОКА обладает протективным эффектом и пре- дотвращает развитие периоперационной ишемии миокарда. При этом высокая доза аторвастатина не оказывает значимого влия- ния на показатели центральной гемодинамики в ближайшем и отдалённом периоде. Применение статинов перед ЧКВ у пациентов даже с исходно повышенным уровнем тропонинов (не достигших критериев ИМ) значительно улучшает исход оперативной коррекции и приводит к уменьшению смертности и развития ИМ в послеоперационном периоде [27]. Конфликт интересов: Авторы заявляют об отсутствии кон- фликта интересов. В нашем исследовании исходное повышение уровня тропо- нинов служило критерием исключения, поскольку в зоне наших СПИСОК ЛИТЕРАТУРЫ/REFERENCES I.A., Nikolaev K.Ju. Vlijanie nekonvencionnyh faktorov riska na tjazhest' techenija ostrogo koronarnogo sindroma u pacientov, prozhivajushhih v uslovijah severa. Vestnik SurGU. Medicina. 2016; 3 (29): 59-61. (In Russ.)] I.A., Nikolaev K.Ju. Vlijanie nekonvencionnyh faktorov riska na tjazhest' techenija ostrogo koronarnogo sindroma u pacientov, prozhivajushhih v uslovijah severa. Vestnik SurGU. Medicina. 2016; 3 (29): 59-61. (In Russ.)] I.A., Nikolaev K.Ju. 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Lo N., Michael T.T., Moin D., Patel V.G., Alomar M., Papayannis A., Cipher D., Abdullah S.M., Banerjee S., Brilakis E.S. Periprocedural Myocardial Injury in Chronic Total Occlusion Percutaneous Interventions A Systematic Cardiac Biomarker Evaluation Study. Journal of the American College of Cardiology. 2014; 7(1): 47-54. doi: 10.1016/j.jcin.2013.07.011. 3. Отева Э.А., Николаева А.А., Егорова Н.А. Подходы к организации первичной профилактики сердечно-сосудистых заболеваний. Здравоохранение Российской Федерации. 2002; 1: 21-23. [Oteva Je.A., Nikolaeva A.A., Egorova N.A. Podhody k organizacii pervichnoj profilaktiki serdechno-sosudistyh zabolevanij. Zdravoohranenie Rossijskoj Federacii. 2002; 1: 21-23. (In Russ.)] 7. Калугина Л.С., Горьков А.И., Урванцева И.А. Эндоваскулярное лечение хронической окклюзии коронарных артерий при многососудистом поражении венечного русла. Вестник СурГУ. Медицина. 2016; 2 (28): 13-16. интересов лежали именно ХОКА. необходимость продолжения приёма статинов во время острого инфаркта миокарда. Так, Giancarlo Marenzi и соавторы в декабре 2015 г. опубликовали результаты исследования, в котором при- няли участие 230 пациентов с ОКС с подъёмом сегмента ST, по- лучивших инвазивное лечение посредством ЧКВ. 50 пациентов (22%) находились на длительной терапии статинами. У этой груп- пы отмечался более низкий уровень ТнI, меньший размер ИМ и меньшая степень обструкции микроциркуляторного русла [26]. Существует множество исследований, доказывающих про- тективный эффект статинов при проведении ЧКВ. В исследова- ние, выполненное Nafasi L. с соавторами, вошло 190 человек со стабильной ИБС, отобранных для проведения ЧКВ. Пациенты были разделены на две группы: в первой группе была предписа- на нагрузочная доза аторвастатина (80 мг) перед операцией, во второй группе пациенты получили плацебо. В первой группе от- мечалась достоверно более низкая частота периоперационного ИМ [15]. Полученные в указанном исследовании данные согласу- ются с результатами нашей работы: пациенты в основной группе, получившие нагрузочную дозу аторвастатина перед оперативной коррекцией, имели достоверно более низкий уровень маркёров некроза миокарда после ЧКВ, что свидетельствовало о меньшем уровне периоперационной ишемии и, следовательно, в будущем улучшало жизненный прогноз таких пациентов. Большой интерес вызвало исследование, проведённое Sun Fei с соавторами. Они разделили пациентов с нестабильной сте- нокардией, подвергшихся ЧКВ, на три группы. В первой группе пациенты за неделю до стентирования получали аторвастатин в дозе 80 мг, во второй группе пациенты продолжили принимать аторвастатин в стандартной дозе 20 мг, в третьей же группе па- циенты 6 дней получали 20 мг, а за сутки до операции получили нагрузочную дозу 80 мг. В первой группе была отмечена стати- стически более низкая частота периоперационной ишемии, оце- ниваемой по уровню ТнI и МВ-КФК, а также более низкий уровень воспаления, что оценивалось по уровню С-реактивного белка и Интерлейкина-6 [12]. В нашей работе получены достаточно обнадёживающие ре- зультаты, которые позволяют предположить, что применение нагрузочной дозы аторвастатина 80 мг обладает выраженным протективным эффектом на миокард при проведении рекана- лизации ХОКА и может служить основанием для расширения показаний к эндоваскулярной реканализации в такой сложной и неоднозначной группе пациентов. Однако в данные исследования были включены пациенты с ОКС, что предполагает наличие нестабильной бляшки либо острой тромбоокклюзии коронарной артерии. Данный факт су- щественно отличает эти исследования от нашего. Однако, учиты- вая результаты проанализированных работ и результаты нашего собственного исследования, можно прийти к выводу, что статины оказывают протективный эффект с одинаковой эффективностью как при ОКС, так и при стабильной ИБС. Обсуждение В настоящее время достаточно сложно выбрать оптимальную тактику лечения пациентов со стабильной ИБС и наличием ХОКА [23]. Как правило, хронические окклюзии формируются долгое время и к моменту диагностики существуют уже много лет, что В настоящее время существует достаточно много работ, по- свящённых протективному воздействию статинов. Доказана Таблица 3. Оценка уровня фракции выброса, конечного систолического объёма, конечного диастолического объёма у пациентов с реканализацией хронических окклюзий коронарных артерий в основной и контрольной группе Table 3. Estimation of ejection fraction, end-systolic volume, end-diastolic volume in patients with recanalization of chronic total occlusions in coronary arteries (the study and control groups) Показатель / Группа Основная группа (n = 38) Группа сравнения (n = 44) Статистическая значимость До операции ФВ, % 57,1±10,8 57,0±9,2 р>0,05 КСО, мл 55,2±29,1 58,3±28,6 р>0,05 КДО, мл 128,4±28,4 131,3±30,4 р>0,05 Через 24 часа после операции ФВ, % 57,3±9,7 57,0±8,2 р>0,05 КСО, мл 56,9±31,5 60,4±29,1 р>0,05 КДО, мл 128,2±35,8 131,9±32,4 р>0,05 Примечания: ФВ – фракция выброса; КСО – конечно-систолический объём; КДО – конечно-диастолический объём Note: EF – ejection fraction; ESV – end-systolic volume; EDV – end-diastolic volume блица 3. Оценка уровня фракции выброса, конечного систолического объёма, конечного диастолического объёма у пациентов с реканализацией хронических окклюзий коронарных артерий в основной и контрольной группе Table 3. Estimation of ejection fraction, end-systolic volume, end-diastolic volume in patients with recanalization of chronic total occlusions in coronary arteries (the study and control groups) Примечания: ФВ – фракция выброса; КСО – конечно-систолический объём; КДО – конечно-диастолический объём Note: EF – ejection fraction; ESV – end-systolic volume; EDV – end-diastolic volume 34 Использование нагрузочных доз аторвастатина у пациентов с реканализацией ХОКА СПИСОК ЛИТЕРАТУРЫ/REFERENCES Петренко И.В. Повышение эффективности реваскуляризации миокарда у пациентов с хроническими окклюзиями коронарных артерий. Дисс. канд.мед. наук. Сургут. 2010. [Petrenko I.V. Povyshenie jeffektivnosti revaskuljarizacii miokarda u pacientov s hronicheskimi okkljuzijami koronarnyh arterij [dissertation] Surgut; 2010. (In Russ] 10. Петренко И.В. Повышение эффективности реваскуляризации миокарда у пациентов с хроническими окклюзиями коронарных артерий. Дисс. канд.мед. наук. Сургут. 2010. [Petrenko I.V. 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Оценка уровня кардиомаркёров и функционального состояния миокарда у пациентов с реканализацией хронических окклюзий коронарных артерий при применении нагрузочных доз аторвастатина. Комплексные проблемы сердечно-сосудистых заболеваний. 2018; 7 (1): 29-35. DOI: 10.17802/2306-1278-2018-7-1-29-35 To cite: A.S. Ragozina, I.V. Petrenko, I.A. Urvantseva, K.Y. Nikolaev. Cardiomarkers evaluation and myocardial assessment in patients with recanalization of chronic total occlusions using high dose atorvastatin loading. Complex Issues of Cardiovascular Diseases. 2018; 7 (1): 29-35. DOI: 10.17802/2306-1278-2018-7-1-29-35 СПИСОК ЛИТЕРАТУРЫ/REFERENCES [Kalugina L.S., Gor'kov A.I., Urvanceva I.A. Jendovaskuljarnoe lechenie hronicheskoj okkljuzii koronarnyh arterij pri mnogososudistom porazhenii venechnogo rusla. Vestnik SurGU. Medicina. 2016; 2 (28): 13-16. (In Russ)] 4. Кожокарь К.Г., Урванцева И.А., Николаев К.Ю. 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https://openalex.org/W2893411682	https://www.jnma.com.np/jnma/index.php/jnma/article/download/3624/2759	English	null	Estimation of Stature from Percutaneous Hand Length Among the Students of A Medical College	Journal of Nepal Medical Association	2,018	cc-by	2,573	ORIGINAL ARTICLE ORIGINAL ARTICLE J Nepal Med Assoc 2018;56(211):687-90 CC BY ABSTRACT ABSTRACT Introduction: Anthropometry is commonly used technique for the measurement of bone and soft parts in Anatomy and other fields of science. It has practical implications mainly in the field of anatomy and forensic medicine. The present study is aimed to determine the average hand length with stature among the students of a medical college. Methods: A cross sectional study was done among 185 students including 98 females and 87 males of Devdaha Medical College and Research Institute. Hand length and stature were measured and collected data was entered and analyzed in SPSS 21. The differences in measurements of hand bone length and stature among males and females were calculated. The regression equation for the estimation of stature from hand length was calculated. Results: The mean hand length among the total population was 17.80±1.04 cm and stature was 160.50±6.73 cm. The mean hand length and stature were higher among males as compared to females. The range of hand length and stature among the total population is 16.00-21.20 and 146.00- 176.50 respectively. The regression equation for the estimation of stature from hand length revealed statistically significant value among the males and females. Conclusions: Our study reveals higher value of hand length and stature for males as compared to females. Also there is significant correlation between the hand length and the stature. The present values are obtained from the small population of an institute. Further studies have to be carried out to develop the regression formula for the Nepalese population with larger sample size. Correspondence: Anup Pandeya, Department of Anatomy, Devdaha Medical College and Research Institute, Devdaha, Rupandehi, Nepal. Email: anup.bpkihs@gmail.com, Phone: +977-9841461364. Estimation of Stature from Percutaneous Hand Length Among the Students of A Medical College Anup Pandeya,1 Alok Atreya2 1Department of Anatomy, Devdaha Medical College and Research Institute, Devdaha, Rupandehi, Nepal, 2Department of Forensic Medicine, Devdaha Medical College and Research Institute, Devdaha, Rupandehi, Nepal. Keywords: hand length; stature; students. INTRODUCTION Stature is regarded as one of the important factor to denote various parameters of a population including nutrition, health and genetics. Identification is the crucial part in forensic medicine and estimation of race, sex, age and stature is considered as the ‘big fours’ in forensic anthropology.1 The estimation of stature is based on two major methods: the anatomical method, which requires the complete skeleton, or the mathematical method, which employs regression formulae to estimate the stature.1,2 Body proportions and the dimensions of different body segments, including the vertebral column, long bones of the limbs and the bones of the hand and foot have been commonly used for stature estimation in different countries.3,4,5,6 There may be unavailability Correspondence: Anup Pandeya, Department of Anatomy, Devdaha Medical College and Research Institute, Devdaha, Rupandehi, Nepal. Email: anup.bpkihs@gmail.com, Phone: +977-9841461364. JNMA I VOL 56 I ISSUE 211 I MAY-JUN, 2018 Pandeya et al. Estimation of Stature from Percutaneous Hand Length Among the Students of A Medical College of middle finger, which denotes the longest length of the hand. of complete skeleton or complete long bones when the bodies are dismembered or mutilated in wars, mass disasters, and crimes. Therefore, a practical alternative is to develop new standards formula that helps in the identification of skeletal remains.7 The collected data was then entered and analyzed by using SPSS 21. The descriptive analysis was performed for frequency, mean and standard deviation (SD). Regression equations for the estimation of stature from hand length were calculated among the study participants. The inclusion criteria were: a) those participants who had no past history of hand bone fracture; b) those who had no congenital deformity of hand; and c) those who consented for the study. The exclusion criteria were: a) those having past history of hand bone fracture; b) those having congenital deformity of hand; and c) those participants who did not consent to participate. RESULTS The present study comprised of 185 students in a medical college. Among them 85 (47%) were males and 98 (53%) were females. The mean value of hand length and stature were higher in males as compared to females. Similarly, the range of hand length and stature is far higher in males with the mean (18.58±0.84) and (165.941±4.30) respectively (Table 1). The hand lengths were measured in centimetres (cm) from the transverse crease of the wrist to the distal end JNMA I VOL 56 I ISSUE 211 I MAY-JUN, 2018 688 Table 1. Mean and S.D. for hand bone length and stature among male and female students. Gender Total n (%) Hand length Stature Mean± SD Range Mean± SD Range Male 87 (47) 18.58±0.84 16.40-21.20 165.941±4.30 154.50-176.50 Female 98 (53) 17.10±0.62 16.00-18.60 155.68±4.40 146.00-169.00 Combined 185 (100) 17.80±1.04 16.00-21.20 160.50±6.73 146.00-176.50 The regression formula for the estimation of stature from hand length is Height(y) = Constant + Independent variable x Hand length(x). From the formula we can estimate the stature from hand length of males, females and combined population (Table 2, Figure 1, 2 and 3). Table 2. Regression equations for the estimation of stature from hand length. Gender Regression equation Standard error P Male 95.86+3.76 x hand length 0.372 <0.001 Female 79.41+4.46 x hand length 0.556 <0.001 Combined 62.59+5.50 x hand length 0.250 <0.001 Figure 1. Regression of height on hand length for males. 180.0 170.0 160.0 150.0 Height Hand_length 16.0000 17.0000 18.0000 19.0000 20.0000 21.0000 22.0000 Gender:Male O Observed Linear JNMA I VOL 56 I ISSUE 211 I MAY-JUN, 2018 688 Table 1. Mean and S.D. for hand bone length and stature among male and female students. Gender Total n (%) Hand length Stature Mean± SD Range Mean± SD Range Male 87 (47) 18.58±0.84 16.40-21.20 165.941±4.30 154.50-176.50 Female 98 (53) 17.10±0.62 16.00-18.60 155.68±4.40 146.00-169.00 Combined 185 (100) 17.80±1.04 16.00-21.20 160.50±6.73 146.00-176.50 The regression formula for the estimation of stature from hand length is Height(y) = Constant + Independent variable x Hand length(x). From the formula we can estimate the stature from hand length of males, females and combined population (Table 2, Figure 1, 2 and 3). Table 2. Regression equations for the estimation of stature from hand length. Gender Regression equation Standard error P Male 95.86+3.76 x hand length 0.372 <0.001 Female 79.41+4.46 x hand length 0.556 <0.001 Combined 62.59+5.50 x hand length 0.250 <0.001 Figure 1. METHODS A cross-sectional study was done among the students of Devdaha Medical College and Devdaha College of Science and Technology from December 2017 to June 2018. The ethical approval was taken from ethical committee of Devdaha Medical College and Research Institute prior to the study. There were 185 participants who participated in the study including 87 males and 98 females. The participants were informed about the study protocols and personal identifier was removed before the data collection. The study participants were asked to stand in the anatomical position with bare foot and the height was measured with the help of stadiometer. The hand length was measured by using Vernier callipers. The variables were recorded as age, sex, hand length and height. JNMA I VOL 56 I ISSUE 211 I MAY-JUN, 2018 RESULTS Regression of height on hand length for males. 180.0 170.0 160.0 150.0 Height Hand_length 16.0000 17.0000 18.0000 19.0000 20.0000 21.0000 22.0000 Gender:Male O Observed Linear Table 1. Mean and S.D. for hand bone length and stature among male and female students. 180.0 170.0 160.0 150.0 Height Hand length 16.0000 17.0000 18.0000 19.0000 20.0000 21.0000 22.0000 Gender:Male O Observed Linear Table 2. Regression equations for the estimation of stature from hand length. Gender Regression equation Standard error P Male 95.86+3.76 x hand length 0.372 <0.001 Female 79.41+4.46 x hand length 0.556 <0.001 Combined 62.59+5.50 x hand length 0.250 <0.001 Table 2. Regression equations for the estimation of stature from hand length. Figure 1. Regression of height on hand length for males. Figure 1. Regression of height on hand length for males. JNMA I VOL 56 I ISSUE 211 I MAY-JUN, 2018 688 JNMA I VOL 56 I ISSUE 211 I MAY-JUN, 2018 688 Pandeya et al. Estimation of Stature from Percutaneous Hand Length Among the Students of A Medical College Pandeya et al. Estimation of Stature from Percutaneous Hand Length Among the Students of A Medical College Jos medical school of Nigeria among male students shows significant correlation between the hand length and stature which is similar to our study.10 Similar study conducted in Egypt also reported the positive correlation between the hand length and stature among the students of different colleges.11 Furthermore, the study conducted in different colleges of New Delhi also reported significant correlation between hand length and stature which is similar with our study.12 Similarly, the studies conducted in National Institute of Technology, Korea and Sudanese adults revealed statistically significant differences in the measurements of males and females with the higher values in males which is in agreement with our study.13 This may be the fact that the males have genetically larger size as compared to females. CONCLUSIONS Our study reveals higher value of hand length and stature for males as compared to females. There is significant correlation between the hand length and the stature. The present values are obtained from the small population of an institute. Further studies have to be carried out to develop the regression formula for the Nepalese population with larger sample size. Figure 3. Regression of height on hand length for combined population. Figure 3. Regression of height on hand length for combined population. RESULTS The puberty in females occurs about two years earlier as compared to the males which cause earlier fusion of epiphysis and there will be less time for growth.11,14 However, several studies reported that proximal limb bones are the better marker of prediction of stature than distal limb bones.15.16 But this was not in agreement with the study done in Sudanese adults and the study on done in Turkish males, where forearm length was better predictor for estimation of stature than upper arm length.17,18 Although the study helped us to calculate the regression equation among the male and female students of a medical college. Further studies are required in large population in various states of the country to build national statistics for Nepalese population. Figure 2. Regression of height on hand length for females. 170.0 165.0 160.0 155.0 150.0 145.0 Height Hand_length 16.0000 17.0000 18.0000 19.0000 Gender:Female O Observed Linear Figure 3. Regression of height on hand length for combined population. 180.0 170.0 160.0 150.0 140.0 Height Hand_length O Observed Linear 16.0000 17.0000 18.0000 19.0000 20.0000 21.0000 22.0000 170.0 165.0 160.0 155.0 150.0 145.0 Height Hand_length 16.0000 17.0000 18.0000 19.0000 Gender:Female O Observed Linear Figure 2. Regression of height on hand length for females. Figure 3. Regression of height on hand length for combined population. 180.0 170.0 160.0 150.0 140.0 Height Hand_length O Observed Linear 16.0000 17.0000 18.0000 19.0000 20.0000 21.0000 22.0000 Figure 3. Regression of height on hand length for combined population. 180.0 170.0 160.0 150.0 140.0 Height Hand_length O Observed Linear 16.0000 17.0000 18.0000 19.0000 20.0000 21.0000 22.0000 ACKNOWLEDGEMENTS Personal identification is essential in medico-legal works. In cases of mass disaster where the body parts are dismembered, it is the role of forensic expert to identify the body and hand it over to the relatives for final rituals. Of this various methods of personal identification, estimation of stature from the measurement of body parts is widely used by forensic anthropologist.8 The estimation of stature from hand length and the derivation of regression equation was done in Punjabi males.9 The study performed in We would like to acknowledge all the participants who participated voluntarily in this study in Devdaha Medical College and Research Institute as well as Devdaha College of Science and Technology, Rupandehi, Nepal. Conflict of Interest: None. REFERENCES 1. Krishan K, Sharma A. Estimation of stature from dimensions of hands and feet in a North Indian population. J Forensic Leg Med. 2007;14(6):327-32. [PubMed \| DOI] 9. Bhatnagar DP, Thapar SP, Batish MK. Identification of personal height from the somatometry of the hand in Punjabi males. Forensic Sci Int. 1984;24(2):137-41. [PubMed \| DOI] 2. Lundy JK. The mathematical versus anatomical methods of stature estimate from long bones. Am J Forensic Med Pathol. 1985;6(1):73-6. [PubMed] 10. Saxena SK. A study of correlations and estimation of stature from hand length, hand breadth and sole length. Anthropol Anz. 1984;42(4):271-6. [PubMed \| DOI] 3. Hurley RS, Bartlett BJ, Witt DD, Thomas A, Taylor EZ. Comparative evaluation of body composition in medically stable elderly. J Am Diet Assoc. 1997;97(10):1105-9. [PubMed \| DOI] 11. Abdel-Malek AK, Ahmed AM, El-Sharkawi SA, El-Hamid NA. Prediction of stature from hand measurements. Forensic Sci Int. 1990;46(3):181-7. [PubMed \| DOI] 12. Sunil, Dikshit PC, Aggrawal A, Rani M. Estimation of stature from hand length. JIAFM. 2005:27(4);219-21. [Full Text] 4. Radoinova D, Tenekedjiev K, Yordanov Y. Stature estimation from long bone lengths in Bulgarians Homo. 2002;52(3):221-32. [PubMed] 13. Kim W, Kim YM, Yun MH. Estimation of stature from hand and foot dimensions in a Korean population. J Forensic Leg Med. 2018 Apr;55:87-92. [PubMed \| DOI] 5. Munoz JI, Linares-Iglesias M, Suarez-Penaranda JM, Mayo M, Miguens X, Roriguez-Calvo MS, et al. Stature estimation from radiographically determined long bone length in a Spanish population sample. J Forensic Sci. 2001;46(2):363–6. [PubMed] 14. Rastogi P1, Nagesh KR, Yoganarasimha K. Estimation of stature from hand dimensions of north and south Indians. Leg Med (Tokyo). 2008;10(4):185-9. [PubMed \| DOI] 15. Akhlaghi M1, Hajibeygi M, Zamani N, Moradi B. Estimation of stature from upper limb anthropometry in Iranian population. J Forensic Leg Med. 2012;19(5):280-4. [PubMed \| DOI] 6. Mall G, Hubig M, Buttner A, Kuznik J, Penning R, Graw M. Sex determination and estimation of stature from the long bones of the arm. Forensic Sci Int. 2001;117(1-2):23–30. [PubMed] 16. Vercellotti G, Agnew AM, Justus HM, Sciulli PW. Stature estimation in an early medieval (XI-XII c.) Polish population: testing the accuracy of regression equations in a bioarcheological sample. Am J Phys Anthropol. 2009;140(1):135-42. [PubMed \| DOI] 7. Menezes RG, Kanchan T, Kumar GP, Rao PP, Lobo SW, Uysal S, et al., Stature estimation from the length of the sternum in South Indian males: a preliminary study. Conflict of Interest: None. 689 JNMA I VOL 56 I ISSUE 211 I MAY-JUN, 2018 JNMA I VOL 56 I ISSUE 211 I MAY-JUN, 2018 Pandeya et al. Estimation of Stature from Percutaneous Hand Length Among the Students of A Medical College REFERENCES J Forensic Leg Med. 2009;16(8) 441–3. [PubMed \| DOI] 8. Pal A, De S, Sengupta P, Maity P, Dhara PC. Estimation of stature from hand dimensions in Bengalee population, West Bengal, India. Egypt J of Forensic Sci. 2016;6:90-8. [Full Text \| DOI] 17. Ahmed AA. Estimation of stature from the upper limb measurements of Sudanese adults. Forensic Sci Int. 2013;228(1-3):1-7. [PubMed \| DOI] 18. Ozaslan A, Koç S, Ozaslan I, Tuğcu H. Estimation of stature from upper extremity. Mil Med. 2006;171(4):288-91. [PubMed \| DOI] © The Author(s) 2018. © The Author(s) 2018. 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/ JNMA I VOL 56 I ISSUE 211 I MAY-JUN, 2018 690 JNMA I VOL 56 I ISSUE 211 I MAY-JUN, 2018
https://openalex.org/W4295292872	https://bmcpregnancychildbirth.biomedcentral.com/counter/pdf/10.1186/s12884-022-05029-8	English	null	A comprehensive evaluation of pre- and post-processing sperm parameters for predicting successful pregnancy rate following intrauterine insemination with the husband’s sperms	BMC pregnancy and childbirth	2,022	cc-by	8,955	Correspondence: xuxia503@126.com; clover_jato@163.com 1 Department of Obstetrics and Gynecology, Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150, China 3 Guangzhou Key Laboratory for Clinical Rapid Diagnosis and Early Warning of Infectious Diseases, Kingmed School of Laboratory Medicine, Guangzhou Medical University, Guangzhou 510182, China Full list of author information is available at the end of the article A comprehensive evaluation of pre‑ and post‑processing sperm parameters for predicting successful pregnancy rate following intrauterine insemination with the husband’s sperms Yumei Luo1,2,3†, Mingxing Liu1,2†, Shunhong Wu3†, Mimi Zhang1,2, Jingru Yuan1,2, Yufang Zhong3, Qing Li1,2, Xiaofang Sun1,2, Xia Xu3 and Detu Zhu1,2,3* Abstract Background: To determine the predictive values of sperm parameters pre- and post-processing by density gradient centrifugation for clinical pregnancy rates (CPRs) following artificial insemination by husband (AIH) in infertile Chinese couples. Methods: A total of 3,522 AIH cycles from 1,918 couples were retrospectively analyzed. The parameters were com- pared between the pregnant and non-pregnant groups and further between different etiological groups (Male-factor, Both-male-and-female-factor, and Other-factor). Multivariate logistic regression analysis was performed to create models for predicting the CPRs of each etiological group. Results: The overall CPR was 13.3%. There were significant improvements for most sperm parameters after DGC. Multivariate logistic regression analysis indicated that, in overall AIH cases, the top parameters significantly influencing the CPR of AIH were pre-STR (OR = 1.037; P = 0.048) and post-VSL (OR = 1.036; P = 0.011). In the Male-factor Group, the top influencing parameters were pre-VCL (OR = 2.096; P = 0.008), pre-LIN (OR = 1.930; P = 0.002) and post-VSL (OR = 1.316; P = 0.023). In the Both-factor Group, the top influencing parameters were pre-VCL (OR = 1.451; P = 0.008) and post-motility (OR = 1.218; P = 0.049). In the Other-factor Group, the top influencing parameters were pre-VAP (OR = 1.715; P = 0.024), pre-STR (OR = 1.20; P = 0.011) and post-VSL (OR = 1.04; P = 0.017). Moreover, receiver operat- ing characteristic analysis showed that the logistic regression models of the Male- and Both-factor Groups had greater powers for prognostic classification than those of other groups. †Yumei Luo, Mingxing Liu and Shunhong Wu contributed equally to this work. Correspondence: xuxia503@126.com; clover_jato@163.com Luo et al. BMC Pregnancy and Childbirth (2022) 22:703 https://doi.org/10.1186/s12884-022-05029-8 RESEARCH A comprehensive evaluation of pre‑ and post‑processing sperm parameters for predicting successful pregnancy rate following intrauterine insemination with the husband’s sperms Yumei Luo1,2,3†, Mingxing Liu1,2†, Shunhong Wu3†, Mimi Zhang1,2, Jingru Yuan1,2, Yufang Zhong3, Qing Li1,2, Xiaofang Sun1,2, Xia Xu3 and Detu Zhu1,2,3* Open Access Luo et al. BMC Pregnancy and Childbirth (2022) 22:703 https://doi.org/10.1186/s12884-022-05029-8 RESEARCH A comprehensive evaluation of pre‑ and post‑processing sperm parameters for predicting successful pregnancy rate following intrauterine insemination with the husband’s sperms Yumei Luo1,2,3†, Mingxing Liu1,2†, Shunhong Wu3†, Mimi Zhang1,2, Jingru Yuan1,2, Yufang Zhong3, Qing Li1,2, Xiaofang Sun1,2, Xia Xu3* and Detu Zhu1,2,3* Open Access Luo et al. BMC Pregnancy and Childbirth (2022) 22:703 https://doi.org/10.1186/s12884-022-05029-8 Background Owing to its minimal invasiveness, simple manipula- tion and low cost, intrauterine insemination (IUI) is cur- rently the first-line assisted reproduction treatment for infertile couples [1]. IUI can be classified into artificial insemination by husband (AIH) and artificial insemina- tion by donor (AID) according to the source of sperms. Prior to AIH, semen must be processed to separate via- ble spermatozoa with normal morphology and motility from unfavourable debris, non-sperm cells, and dead or immotile spermatozoa. The most widely used method for semen processing is density gradient centrifugation (DGC) [2]. This was a retrospective cohort study enrolling infertile Chinese couples that underwent AIH at the fertility clinic of the Third Affiliated Hospital of Guangzhou Medical University between September 2018 and May 2020 [7]. The study was approved by the Ethics Committee of the Third Affiliated Hospital of Guangzhou Medical Univer- sity (Protocol no. 2018–142). y ( ) Before the enrollment, the patients were diagnosed with the causes of infertility, with necessary assays to elicit etiologies. Female patients had hysterosalpingo- grams, and men had semen analyses. Causes of infertil- ity were grouped into: (1) male factors, (2) female factors (ovulatory dysfunction, cervical factors, immune factors, endometriosis, endometriosis after pelvic plastic sur- gery, etc.), (3) combined male and female factors, and (4) unexplained infertility. Demographic data such as the age of the couple, duration of infertility, semen param- eters before and after sperm processing, and the AIH outcomes were extracted from the patients’ records. All pregnancies were confirmed by positive beta-human chorionic gonadotropin (β-hCG) in the serum 14 days after AIH. Exclusion criteria: (1) Those whose record writing is not standard. (2) Those whose semen sample was collected with an incorrect container, partially lost, or was sent for examination after more than 30 min. (3) Those had undergone antibacterial treatment. (4) Those with incomplete data regarding the pregnancy outcome or missing data on pre- and post-processing sperm parameters. Finally, a total of 3,522 AIH cycles from 1,918 infertile Chinese couples were retrospectively ana- lyzed (Table 1). It is expected that DGC processing leads to signifi- cant improvements in most sperm parameters [3, 4]. A number of previous studies indicated that some semen parameters, including semen volume, sperm motility and sperm morphology, could predict the outcomes of IUI [5–7]. © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Luo et al. BMC Pregnancy and Childbirth (2022) 22:703 Page 2 of 11 Conclusions: This study demonstrated that some sperm parameters have a collinearity relationship in predicting the CPR following AIH. Moreover, the predictive capacity of a multivariate logistic regression model is better than those of individual parameters, especially for the Male- and Both-factor Groups. In these cases, pre-VCL is the common top influencing factor. Keywords: Assisted reproduction technology, Intrauterine insemination, Artificial insemination by husband, Semen processing, Density gradient centrifugation, Sperm parameters, Clinical pregnancy rate, Logistic regression Background Further, some studies demonstrated that the amount of sperm recovered after semen processing was significantly related to the clinical pregnancy rate (CPR) of AIH [8]. Also, there were predictive values of post-pro- cessing total motile sperm count (TMSC) and the normal form rate on AIH success [9–12]. In contrast, some stud- ies suggested that post-processing semen parameters are not greater than those pre-processing in predicting CPR following AIH [2, 13]. Some studies have shown that the TMSC did not have a predictive value for the CPR [14, 15]. Besides, there was a large volume of published stud- ies indicating that the normal rate of sperm morphol- ogy had nothing to do with the success rate of IUI [6, 14, 16]. Therefore, the prognostic values of pre- and pos- processing sperm parameters for AIH outcome remain controversial. To determine the prognostic values of sperm param- eters pre- and post-DGC processing for predicting CPRs following AIH in infertile Chinese couples, our study has retrospectively analyzed 1,918 Chinese infertile couples with a total of 3,522 AIH treatment cycles in the Third Affiliated Hospital of Guangzhou Medical University from September 2018 to May 2020, and provided a com- prehensive assessment on the predictive values of sperm parameters pre- and post-DGC processing for the CPR following AIH. Statistical analysish The SPSS v22.0 was used for statistical analysis [7]. The results are presented as mean ± standard deviation (SD). The analysis pipeline is shown in Fig. 1. Comparison of continuous variables between pregnant and non-preg- nant groups was performed using the Mann–Whitney test because the data distribution was not normal accord- ing to the Kolmogorov–Smirnov test. Categorical varia- bles were evaluated using the chi-square test. Spearman’s correlation coefficient analysis was performed to evaluate the correlation of absolute and relative changes of each variable and the CPR. Both categorical and continuous variables that might influence AIH pregnancy outcome were analyzed by backward binary multivariate logistic regression to identify predictive factors for different eti- ology groups. Receiver operating characteristic (ROC) curves were constructed to evaluate the prognostic clas- sification capacities and calculate the clinically acceptable cut-off values for the logistic regression models of each etiology group. Statistical significance was accepted as P < 0.05. Semen analysis For semen analysis, 10 μL of semen was transferred to a counting chamber (AIYX, Cat# AI-20–4) and analyzed by computer-assisted sperm analysis (CASA; Hamilton Thorne HTCasa II 1.10.3). The sperm concentration was optimized to be (2 ~ 50) × 106/mL. At least 200 sperms from 5 analyzed fields were counted. Sperm param- eters including concentration, motility, total progres- sive motile sperm count (TPMSC), curvilinear velocity (VCL), straight-line velocity (VSL), average path veloc- ity (VAP), linearity (LIN), straightness (STR), beat cross frequency (BCF), and amplitude of lateral head displace- ment (ALH), sperm head area were reported. Semen treatmenth The samples were then treated by DGC method. In brief, the gradient separation solution (Vitrolife) was prepared in test tube, the lower layer is 1 mL 90% separation solu- tion, the upper layer is 1 mL 45% separation solution. After the semen samples were evenly mixed, 1 mL of semen was taken and placed above the density gradient, centrifuged at 3000 r/min for 20 min. Then remove most of the supernatant from the upper layer. The sperm was suspended in 5 mL sperm culture medium and gently blown, and then centrifuged at 2000 r/min for 5 min and the supernatant was removed. Finally, 0.5 mL semen was used for sperm suspension, and sperm concentration, vitality and other semen parameters were tested. Semen samples with TPMSC > 2 × 10 are accepted to go for AIH treatment. Semen collection Semen processing was performed as previously described [7]. In brief, semen specimens were produced with mas- turbation in a collection room at the fertility clinic. The specimens were kept at 37℃ temperature and were examined within half an hour post collection. After com- plete liquefaction, all samples were evaluated in a blinded fashion by a qualified technician to prevent the interob- server variation based on WHO 2010 criteria. Luo et al. BMC Pregnancy and Childbirth (2022) 22:703 Luo et al. BMC Pregnancy and Childbirth (2022) 22:703 Page 3 of 11 Table 1 Patient demographics Pregnant Group (n = 468) Non-pregnant Group (n = 3,054) p-value Female age 31.02 ± 3.74 31.37 ± 4.05 0.133 Male age 32.93 ± 4.36 33.29 ± 4.75 0.402 Cause of infertility 0.585 Male-factor 82 (17.5%) 595 (19.5%) Both-male- and-female- factor 65 (13.9%) 427 (14.0%) Other-factor 321 (68.6%) 2,032 (66.5%) Type of infertility 0.988 Primary 287 (61.3%) 1,874 (61.4%) Secondary 181 (38.7%) 1,180 (38.6%) IUI cycle 0.049 1 265 (56.6%) 1,659 (54.3%) 2 143 (30.6%) 1,085 (35.5%) ≥ 3 60 (12.8%) 310 (10.2%) Artificial insemination by husband Artificial insemination by husband Table 1 Patient demographics i AIH was performed as previously described [7]. In brief, after the bladder was emptied, the patient took the blad- der lithotomy position, with the vulva washed by saline, and the vagina, cervix and fornix wiped by a large cot- ton swab. A 1-mL syringe and an artificial insemination tube were connected in the uterine cavity. Then 0.5 mL of the husband’s sperm suspension was aspirated and slowly placed in the uterine cavity through the tube. After the sperm suspension is slowly injected into the uterus for 3 to 5 s, the tube was slowly withdrawn, and the patient was kept in the position of lowering the head and hips for approximately 30 min. Results Sperm parameters were improved after semen processing The semen samples were processed by DGC before the AIH treatments. Semen parameters before and after processing were compared across the pregnant and non- pregnant groups (Table 2). In both groups, there were significant improvements in most sperm parameters after DGC processing, except for TPMSC (all P < 0.001). To evaluate the impacts of these changes on the AIH outcomes, the correlations between the change values of each parameter and the likelihood of pregnancy were analyzed by Pearson correlation coefficients (Table 3). The results showed that neither absolute changes nor relative changes of the parameters, including the decrease of TPMSC, had any correlation with the AIH pregnancy outcome. Luo et al. BMC Pregnancy and Childbirth (2022) 22:703 Page 4 of 11 Fig. Results 1 Experimental design Table 2 Comparison of semen parameters before and after processing Table 2 Comparison of semen parameters before and after processing Abbreviations: TPMSC Total progressive motile sperm count, VCL Curvilinear velocity, VSL Straight-line velocity, VAP Average pathway velocity, LIN Linearity, STR Straightness, BCF Beat cross frequency, ALH Amplitude of lateral head displacement Parameters Pregnant group (n = 468) Non-pregnant group (n = 3,054) Before After (After-Before) p-value Before After (After-Before) p-value Concentration (× 106/mL) 50.86 ± 31.47 71.14 ± 54.85 20.28 ± 37.72 < 0.001 52.21 ± 33.29 71.12 ± 54.49 18.91 ± 38.43 < 0.001 Motility (%) 61.69 ± 14.55 93.42 ± 7.56 31.73 ± 12.08 < 0.001 61.29 ± 14.07 93.43 ± 7.89 32.14 ± 11.69 < 0.001 TPMSC (× 106/mL) 75.04 ± 63.50 32.97 ± 26.79 -42.07 ± 37.64 < 0.001 75.60 ± 63.88 33.08 ± 26.60 -42.52 ± 37.99 < 0.001 VAP (µm/s) 7.82 ± 3.84 24.47 ± 5.78 16.65 ± 6.67 < 0.001 7.86 ± 3.69 23.94 ± 5.83 16.08 ± 6.70 < 0.001 VSL (µm/s) 4.42 ± 2.26 14.10 ± 3.75 9.69 ± 4.14 < 0.001 4.42 ± 2.14 13.66 ± 3.57 9.24 ± 4.04 < 0.001 VCL (µm/s) 14.32 ± 5.87 47.39 ± 10.38 33.07 ± 11.56 < 0.001 14.37 ± 5.72 46.35 ± 10.69 31.98 ± 11.82 < 0.001 ALH (µm/s) 4.16 ± 0.50 6.56 ± 0.79 2.40 ± 0.91 < 0.001 4.16 ± 0.49 6.49 ± 0.80 2.33 ± 0.92 < 0.001 BCF (Hz) 5.45 ± 1.20 7.24 ± 0.70 1.79 ± 1.36 < 0.001 5.43 ± 1.15 7.25 ± 0.75 1.81 ± 1.33 < 0.001 STR (%) 46.73 ± 9.05 72.54 ± 7.86 65.17 ± 19.48 < 0.001 46.43 ± 8.41 71.95 ± 8.75 65.55 ± 18.48 < 0.001 LIN (%) 34.32 ± 6.28 47.50 ± 5.15 13.18 ± 7.59 < 0.001 34.18 ± 5.81 47.19 ± 5.72 13.01 ± 7.84 < 0.001 Sperm head area (µm2) 5.08 ± 0.48 5.55 ± 0.58 0.47 ± 0.70 < 0.001 5.12 ± 0.49 5.55 ± 0.57 0.43 ± 0.72 < 0.001 Abbreviations: TPMSC Total progressive motile sperm count, VCL Curvilinear velocity, VSL Straight-line velocity, VAP Average pathway velocity, LIN Linearity, STR Straightness, BCF Beat cross frequency, ALH Amplitude of lateral head displacement Multivariate logistic regression analysis of the influencing factors for different etiological groups of AIH Variables that might have impacts on pregnancy outcome were included in the logistic regression analysis, with CPRs of overall AIH cases and different etiological groups as the dependent variables, and pre-processing sperm morphological parameters, pre- and post- processing semen parameters and sperm motion kinetic parameters as independent variables. Then multivariate binary logistic regression analysis (backward conditional) was performed. g y p The results showed that, in overall cases, after excluding confounding factors, the top influencing fac- tors were pre-STR (OR = 1.037; 95% CI: 1.000–1.076; P = 0.048) and post-VSL (OR = 1.036; 95% CI: 1.008– 1.064; P = 0.011) (Table 8). Further, in the Male-fac- tor Group, the top influencing factors were pre-VCL (OR = 2.096; 95% CI: 1.218–3.607; P = 0.008); pre- LIN (OR = 1.930; 95% CI: 1.276–2.919; P = 0.002) and post-VSL (OR = 1.316; 95% CI: 1.039–1.666; P = 0.023) (Table 9). In the Both-factor Group, the top influenc- ing factors were pre-VCL (OR = 1.451; 95% CI: 1.104– 1.906; P = 0.008) and post-motility (OR = 1.218; 95% CI: 1.000–1.484; P = 0.049) (Table 10). In the Other- factor Group, the top influencing factors were pre-VAP (OR = 1.715; 95% CI: 1.073–2.740; P = 0.024); pre- STR (OR = 1.20; 95% CI: 1.042–1.382; P = 0.011) and parameters except the sperm cytoplasm deformity of the Other-factor Groups were significantly lower than those in the Male- and Both-factor Groups (Table 7) (all P < 0.01). This was reasonable as the pre-process- ing sperm quality of the cases due to non-male factors should be better than those due to or partly due to male factors. Both pre‑ and post‑processing semen parameters were not correlated with the pregnancy outcome of overall AIH cases Assuming that the above semen parameters might be more relevant with male factors, we classified the AIH cases into Male-factor (n = 677), Both-male-and-female- factor (n = 492) and Other-factor (n = 2,353) Groups by etiologies. The comparison of the parameters among these three groups was performed using the Kruskal– Wallis test because data distribution was not normal according to the Kolmogorov–Smirnov test. The pre- and post- processing semen parameters and sperm motion kinetic parameters (Table 4), and pre- processing sperm morphological parameters (Table 5) were compared between the Pregnant (n = 468) and Non-pregnant (n = 3,054) Groups of overall AIH cases. The results showed that these parameters were not significantly different between the two groups. It was probably due to the confounding effects of influencing factors other than the male factors. Our results showed that all pre-processing semen parameters and sperm motion kinetic parameters of the Other-factor Groups were significantly higher than those in the Male- and Both-factor Groups (Table 6); meanwhile, all pre-processing sperm deformity Luo et al. BMC Pregnancy and Childbirth (2022) 22:703 Page 5 of 11 Table 3 Correlations between the changes of sperm parameters and overall AIH pregnancy rate Abbreviations: CPR clinical pregnancy rate, PCC Pearson correlation coefficient, TPMSC total progressive motile sperm count, VCL curvilinear velocity, VSL Straight-line velocity, VAP Average pathway velocity, LIN linearity, STR Straightness, BCF Beat cross frequency, ALH Amplitude of lateral head displacement Parameters absolute changes vs. overall CPR Relative changes vs. overall CPR PCC r p-value PCC r p-value Concentration (× 106/mL) 0.017 0.307 0.026 0.122 Motility (%) − 0.011 0.506 − 0.012 0.475 TPMSC (× 106/mL) 0.003 0.871 0.019 0.259 VAP (µm/s) 0.019 0.248 0.019 0.261 VSL (µm/s) 0.027 0.103 0.024 0.162 VCL (µm/s) 0.025 0.135 0.02 0.234 ALH (µm/s) 0.020 0.227 0.019 0.249 BCF (Hz) − 0.007 0.687 − 0.006 0.740 STR (%) 0.010 0.566 − 0.006 0.726 LIN (%) 0.008 0.634 0.008 0.620 Sperm head area (µm2) 0.022 0.190 0.023 0.175 Table 3 Correlations between the changes of sperm parameters and overall AIH pregnancy rate (Table 6) (all P < 0.05), reflecting an improvement of sperm quality by the DGC processing. Our results showed that most pre- and post-processing semen parameters were significantly different among the etiological groups; hence, it was necessary to count in the etiology when analyzing the influence of semen param- eters on AIH pregnancy. Abbreviations: TPMSC total progressive motile sperm count, VCL curvilinear velocity, VSL straight-line velocity, VAP average pathway velocity, LIN Linearity; STR Straightness, BCF Beat cross frequency, ALH Amplitude of lateral head displacement Multivariate logistic regression analysis of the influencing factors for different etiological groups of AIH Nevertheless, in post-processing parameters, only the sperm concentration, motility, TPMSC, VCL and BCF parameters of the Other-factor Group were significantly higher than those of the Male- and Both-factor Groups Table 4 Comparison of semen parameters before and after processing between pregnant and non-pregnant groups Abbreviations: TPMSC total progressive motile sperm count, VCL curvilinear velocity, VSL straight-line velocity, VAP average pathway velocity, LIN Linearity; STR Straightness, BCF Beat cross frequency, ALH Amplitude of lateral head displacement Parameters Before semen treatment After semen treatment Pregnant (n = 468) Non-pregnant (n = 3,054) p-value Pregnant (n = 468) Non-pregnant (n = 3,054) p-value Concentration (× 106/mL) 50.86 ± 31.47 52.21 ± 33.29 0.468 71.14 ± 54.85 71.12 ± 54.49 0.896 Motility (%) 61.69 ± 14.55 61.29 ± 14.07 0.546 93.42 ± 7.56 33.08 ± 26.60 0.582 TPMSC (× 106/mL) 75.04 ± 63.50 75.60 ± 63.88 0.986 93.42 ± 7.56 93.43 ± 7.89 0.983 VAP (µm/s) 7.82 ± 3.84 7.86 ± 3.69 0.703 24.47 ± 5.78 23.94 ± 5.83 0.213 VSL (µm/s) 4.42 ± 2.26 4.42 ± 2.14 0.751 14.10 ± 3.75 13.66 ± 3.57 0.105 VCL (µm/s) 14.32 ± 5.87 14.37 ± 5.72 0.779 47.39 ± 10.38 46.35 ± 10.69 0.107 ALH (µm/s) 4.16 ± 0.50 4.16 ± 0.49 0.698 6.56 ± 0.79 6.49 ± 0.80 0.192 BCF (Hz) 5.45 ± 1.20 5.43 ± 1.15 0.681 7.24 ± 0.70 7.25 ± 0.75 0.415 STR (%) 46.73 ± 9.05 46.43 ± 8.41 0.64 72.54 ± 7.86 71.95 ± 8.75 0.214 LIN (%) 34.32 ± 6.28 34.18 ± 5.81 0.837 47.50 ± 5.15 47.19 ± 5.72 0.299 Sperm head area (µm2) 5.08 ± 0.48 5.12 ± 0.49 0.086 5.55 ± 0.58 5.55 ± 0.57 0.743 son of semen parameters before and after processing between pregnant and non-pregnant groups Abbreviations: TPMSC total progressive motile sperm count, VCL curvilinear velocity, VSL straight-line velocity, VAP average pathway velocity, LIN Linearity; STR Straightness, BCF Beat cross frequency, ALH Amplitude of lateral head displacement Luo et al. fif Abbreviations: TPMSC Total progressive motile sperm count, VCL Curvilinear velocity, VSL Straight-line velocity, VAP Average pathway velocity, LIN Linearity, STR Straightness, BCF Beat cross frequency, ALH Amplitude of lateral head displacement Multivariate logistic regression analysis of the influencing factors for different etiological groups of AIH BMC Pregnancy and Childbirth (2022) 22:703 Page 6 of 11 Table 5 Comparison of sperm morphological parameters between pregnant and non-pregnant groups Abbreviations: SDI Sperm deformity index, TZI Teratozoospermia index, H Sperm headpiece deformity, M Sperm middle piece deformity, P Sperm principal piece deformity, C Sperm cytoplasm deformity Parameters Pregnant (n = 468) Non-pregnant (n = 3,054) p-value SDI 1.24 ± 0.11 1.24 ± 0.11 0.44 TZI 1.30 ± 0.10 1.30 ± 0.10 0.718 H (%) 94.69 ± 7.54 94.79 ± 8.64 0.328 M (%) 16.47 ± 5.23 16.57 ± 5.08 0.559 P (%) 5.15 ± 3.12 5.48 ± 3.31 0.076 C (%) 6.83 ± 4.74 6.84 ± 4.81 0.917 Normal forms (%) 4.82 ± 4.24 4.42 ± 3.79 0.141 Abnormal forms (%) 95.17 ± 4.24 95.58 ± 3.79 0.113 Table 5 Comparison of sperm morphological parameters between pregnant and non-pregnant groups 14.77% (Sensitivity: 32.7%; Specificity:77.7%). For compari- son, the cut-off values of the logistic regression equations of the Male-factor, Both-factor and Other-factor Groups were 12.46% (Sensitivity: 59.8%; Specificity: 65.7%), 8.74% (Sen- sitivity: 90.8%; Specificity: 33.0%) and 14.82% (Sensitivity: 35.8%; Specificity: 73.2%), respectively (Table 12). i The areas under ROC curves (AUC) of overall cases, the Male-factor, Both-factor and Other-factor Groups were 0.551, 0.668, 0.655 and 0.544, respectively (Table 12; Fig. 3). Therefore, the prognostic classification capacities of the logistic regression equations for the Male-factor and Both-factor Groups were greater than those for the other groups. And pre-VCL is the common top influenc- ing factor in these two groups. Abbreviations: SDI Sperm deformity index, TZI Teratozoospermia index, H Sperm headpiece deformity, M Sperm middle piece deformity, P Sperm principal piece deformity, C Sperm cytoplasm deformity Values on the same row with different superscripts are significantly different (P < 0.05) Discussion Although the AIH technology has the advantages of sim- ple operation, low cost, high safety, and low invasive- ness, a major problem with AIH treatment is the lower pregnancy rate (about 5.0 to 15.0%). In this study, 3,522 AIH cycles were achieved from 1,918 couples, result- ing in a CPR of 13.3%, which is comparable to those of other reports [17–19]. In recent years, there has been an increasing amount of literature on AIH; however, there has always been controversy about the factors that influ- ence the outcome of AIH. Therefore, it is particularly important to explore the factors that affect AIH preg- nancy and take corresponding interventions to improve the CPR. This study aimed to evaluate the prognostic capacities of semen parameters before and after sperm selection processing on AIH outcomes. post-VSL (OR = 1.04; 95% CI: 1.007–1.074; P = 0.017) (Table 11). Taken together, most of the significantly influencing factors were sperm motion kinetic parameters. Moreo- ver, there were collinearity relationships among these parameters affecting the AIH outcomes in different etiological groups (Fig. 2). Semen parameters pre‑ and post‑processing had greater prognostic powers in Male‑ and Both‑factor Groups of AIH The expected probability equations of overall AIH cases and of each etiological group created by the multivariate logistic regression models were further evaluated with ROC curves as predictors for AIH pregnancy. In overall cases, the cut-off value of the logistic regression equation was Semen parameters pre‑ and post‑processing had greater prognostic powers in Male‑ and Both‑factor Groups of AIHh BMC Pregnancy and Childbirth (2022) 22:703 Page 7 of 11 Table 7 Comparison of sperm morphological parameters between different etiology groups Values on the same row with different superscripts are significantly different (P < 0.05) Abbreviations: SDI Sperm deformity index, TZI Teratozoospermia index, H Sperm headpiece deformity, M Sperm middle piece deformity, P Sperm principal piece deformity, C Sperm cytoplasm deformity Parameters Male-factor (n = 677) Both-factor (n = 492) Other-factor (n = 2,353) p-value SDI 1.27 ± 0.11a 1.28 ± 0.11a 1.22 ± 0.11b < 0.001 TZI 1.31 ± 0.11ab 1.31 ± 0.10a 1.30 ± 0.10b 0.009 H (%) 97.02 ± 6.95a 97.32 ± 5.16a 93.60 ± 9.19b < 0.001 M (%) 17.23 ± 5.62a 17.52 ± 5.05a 16.17 ± 4.91b < 0.001 P (%) 5.64 ± 3.36a 5.91 ± 3.36a 5.28 ± 3.23b < 0.001 C (%) 7.19 ± 5.29 7.02 ± 5.01 6.70 ± 4.60 0.364 Normal forms (%) 2.48 ± 2.49a 2.38 ± 2.58a 5.49 ± 3.99b < 0.001 Abnormal forms (%) 97.52 ± 2.49a 97.62 ± 2.58a 94.51 ± 4.00b < 0.001 Table 7 Comparison of sperm morphological parameters between different etiology groups Table 10 Influencing factors for AIH pregnancy of the Both- factor Group identified by multivariate logistic regression analysis Abbreviations: VCL Curvilinear velocity, VAP Average pathway velocity, ALH Amplitude of lateral head displacement, P Sperm principal piece deformity, β Regression coefficient, CI Confidence interval Parameters β Odd Ratio (95% CI) p-value pre-VAP -0.634 0.53 (0.34–0.827) 0.005 pre-VCL 0.372 1.451 (1.104–1.906) 0.008 post-motility 0.197 1.218 (1.000–1.484) 0.049 post-VCL 0.186 1.204 (0.995–1.458) 0.056 post-ALH -2.209 0.11 (0.008–1.431) 0.092 P -0.102 0.903 (0.826–0.988) 0.026 Constant -1.054 0.349 0.828 Table 10 Influencing factors for AIH pregnancy of the Both- factor Group identified by multivariate logistic regression analysis Abbreviations: VCL Curvilinear velocity, VAP Average pathway velocity, ALH Amplitude of lateral head displacement, P Sperm principal piece deformity, β Regression coefficient, CI Confidence interval Table 11 Influencing factors for AIH pregnancy of the Other- factor Group identified by multivariate logistic regression analysis Table 11 Influencing factors for AIH pregnancy of the Other- factor Group identified by multivariate logistic regression analysis Abbreviations: VAP Average pathway velocity, VCL Curvilinear velocity, VSL, Straight-line velocity, LIN Linearity, STR Straightness, β Regression coefficient, CI Confidence interval Parameters β Odd Ratio (95% CI) p-value pre-VAP 0.539 1.715 (1.073–2.74) 0.024 pre-VCL -0.342 0.71 (0.537–0.94) 0.017 pre-STR 0.182 1.2 (1.042–1.382) 0.011 pre-LIN -0.283 0.754 (0.601–0.945) 0.014 post-VSL 0.039 1.04 (1.007–1.074) 0.017 Constant -0.528 0.59 0.602 Table 8 Influencing factors for overall AIH pregnancy identified by multivariate logistic regression analysis Abbreviations: VSL Straight-line velocity, STR Straightness, P Sperm principal piece deformity, β Regression coefficient, CI Confidence interval Parameters β Odd Ratio (95% CI) p-value pre-VSL -0.155 0.856 (0.740–0.991) 0.037 pre-STR 0.037 1.037 (1.000–1.076) 0.048 post-VSL 0.035 1.036 (1.008–1.064) 0.011 P -0.027 0.973 (0.943–1.004) 0.086 Abnormal forms -0.017 0.983 (0.966–1.001) 0.061 Constant -1.65 0.192 0.125 Table 8 Influencing factors for overall AIH pregnancy identified by multivariate logistic regression analysis To select high-quality sperms for AIH, the most widely used methods for semen treatment are DGC and swim-up. Semen parameters pre‑ and post‑processing had greater prognostic powers in Male‑ and Both‑factor Groups of AIHh Table 6 Comparison of semen parameters before and after processing between different etiological groups Values on the same row with different superscripts are significantly different (P < 0.05) Parameters Before sperm processing After sperm processing Male-factor (n = 677) Both-factor (n = 492) Other-factor (n = 2,353) p-value Male-factor (n = 677) Both-factor (n = 492) Other-factor (n = 2,353) p-value Concentration (× 106/mL) 44.02 ± 31.41a 44.81 ± 29.91a 55.85 ± 33.48b < 0.001 53.45 ± 47.32a 56.11 ± 45.65a 79.35 ± 56.28b < 0.001 Motility (%) 54.95 ± 14.31a 57.29 ± 14.14b 64.03 ± 13.26c < 0.001 90.26 ± 10.31a 91.45 ± 9.29a 94.75 ± 6.17b < 0.001 TPMSC (× 106/mL) 54.81 ± 53.70a 59.03 ± 55.44b 84.94 ± 66.01c < 0.001 24.06 ± 22.97a 25.69 ± 22.28a 37.20 ± 27.45b < 0.001 VAP (µm/s) 6.55 ± 3.53a 6.80 ± 3.45a 8.45 ± 3.68b < 0.001 23.66 ± 6.03 23.49 ± 5.61 24.23 ± 5.80 0.113 VSL (µm/s) 3.69 ± 2.08a 3.84 ± 2.00a 4.74 ± 2.14b < 0.001 13.46 ± 3.75 13.46 ± 3.35 13.84 ± 3.60 0.231 VCL (µm/s) 12.45 ± 5.38a 12.82 ± 5.37a 15.24 ± 5.71b < 0.001 45.96 ± 11.04ab 45.42 ± 10.37a 46.87 ± 10.58b 0.018 ALH (µm/s) 3.99 ± 0.46a 4.03 ± 0.46a 4.24 ± 0.49b < 0.001 6.46 ± 0.84 6.42 ± 0.78 6.53 ± 0.80 0.128 BCF (Hz) 4.97 ± 1.17a 5.11 ± 1.15a 5.64 ± 1.09b < 0.001 7.16 ± 0.78a 7.28 ± 0.72ab 7.26 ± 0.74b 0.025 STR (%) 43.24 ± 8.29a 44.21 ± 8.18a 47.87 ± 8.26b < 0.001 71.32 ± 9.02 72.05 ± 8.38 72.23 ± 8.57 0.104 LIN (%) 31.88 ± 5.67a 32.60 ± 5.59a 35.20 ± 5.73b < 0.001 46.74 ± 5.85 47.32 ± 5.50 47.36 ± 5.62 0.056 Sperm head area (µm2) 5.27 ± 0.57a 5.20 ± 0.50a 5.05 ± 0.44b < 0.001 5.61 ± 0.61a 5.57 ± 0.56ab 5.53 ± 0.57b 0.002 ble 6 Comparison of semen parameters before and after processing between different etiological groups Luo et al. Semen parameters pre‑ and post‑processing had greater prognostic powers in Male‑ and Both‑factor Groups of AIHh As instructed by the WHO, these treatment methods should be chosen according to the charac- teristics of the samples [20]. It was suggested to select swim-up in cases with normal sperms, while DGC for other cases, as the latter resulted in higher recovery rates [21]. Significantly higher CPRs were observed among couples with unexplained infertility using DGC than those using swim-up [22]. Besides, Karamahmu- toglu et al. found that in the cases of unexplained infer- tility, the DGC technique contributed to a significantly higher CPR compared to the swim-up technique as it could select sperms with better DNA and chromatin structures; however, in male subfertile patients, both techniques yield similar clinical outcomes [22]. Oguz et al. compared the two methods and found that the swim-up method significantly reduced sperm DNA fragmentation and mgiht have some prognostic value on IUI in patients with decreased sperm DNA integrity [23]. Recently, there is a new sperm selection method— microfluid sperm sorting chip [24], which adopts microfluidic devices [25–27] for sperm selection. It Table 9 Influencing factors for AIH pregnancy of the Male-factor Group identified by multivariate logistic regression analysis Abbreviations: VCL Curvilinear velocity, VSL Straight-line velocity, VAP Average pathway velocity, LIN Linearity, STR Straightness, BCF Beat cross frequency, H Sperm headpiece deformity, β Regression coefficient, CI Confidence interval Table 9 G d Group identified by multivariate logistic regression analysis Abbreviations: VCL Curvilinear velocity, VSL Straight-line velocity, VAP Average pathway velocity, LIN Linearity, STR Straightness, BCF Beat cross frequency, H Sperm headpiece deformity, β Regression coefficient, CI Confidence interval Parameters β Odd Ratio (95% CI) p-value pre-VAP -0.707 0.493 (0.247–0.984) 0.045 pre-VSL -1.765 0.171 (0.045–0.658) 0.01 pre-VCL 0.74 2.096 (1.218–3.607) 0.008 pre-BCF -1.487 0.226 (0.084–0.611) 0.003 pre-LIN 0.657 1.93 (1.276–2.919) 0.002 pre-Sperm head area -0.585 0.557 (0.294–1.056) 0.073 post-VAP -0.117 0.89 (0.797–0.993) 0.037 post-VSL 0.275 1.316 (1.039–1.666) 0.023 post-BCF 0.483 1.621 (0.991–2.652) 0.054 post-STR -0.062 0.939 (0.878–1.005) 0.071 H 0.354 1.424 (0.808–2.513) 0.222 Normal forms 0.476 1.61 (0.899–2.883) 0.109 Constant -46.353 0 0.116 Page 8 of 11 Luo et al. BMC Pregnancy and Childbirth (2022) 22:703 Fig. 2 Forest plot for the influencing factors of overall AIH cases and different etiological groups identified by multivariate logistic regression analysis for the influencing factors of overall AIH cases and different etiological groups identified by multivariate logistic regression Table 12 ROC analysis for multivariate logistic regression models of overall AIH cases and different etiology groups Abbreviations: ROC Receiver-operating characteristic, AUC Area under curve, CI Confidence interval Multivariate logistic regression models AUC 95% CI p-value Cut-off Sensitivity Specificity Overall 0.551 ± 0.014 0.523–0.58 < 0.001 14.77 32.7 77.7 Male-factor Group 0.668 ± 0.031 0.606–0.729 < 0.001 12.46 59.8 65.7 Both-male-and-female-factor Group 0.655 ± 0.034 0.588–0.722 < 0.001 8.74 90.8 33.0 Other-factor Group 0.544 ± 0.017 0.51–0.578 0.011 14.82 35.8 73.2 Table 12 ROC analysis for multivariate logistic regression models of overall AIH cases and different etio however, the decrease in TPMSC did not affect the pregnancy outcome of AIH (Table 3).h would be of interest to investigate the influences of dif- ferent semen processing methods, especially including the newly developed methods, on CPRs in future stud- ies, so as to choose a better method for assisted repro- duction treatment [3]. In this study, our results showed that DGC processing led to significant increases in most sperm parameters, except for TPMSC (Table 1); There was a large volume of published studies describ- ing the impacts of semen parameters on the success of IUI [28, 29]. It has been reported that sperm kinetic parameters of motility, including VAP, VSL, VCL, ALH and LIN, are all associated with fertility [4, 30–32]. Our Luo et al. BMC Pregnancy and Childbirth (2022) 22:703 Page 9 of 11 Fig. Table 9 G d 3 ROC curves for the multivariate logistic regression models of overall AIH cases and different etiological groups Fig. 3 ROC curves for the multivariate logistic regression models of overall AIH cases and different etiological groups Semen parameters with predictive powers have been extensively exploited for prognostic classification of infer- tile patients. For example, Hamilton et al. indicated that TMSC had a predictive value for CPRs following IUI in infertile cases associated with male or unexplained fac- tors, and considered TMSC > 5 × 106 /mL as the optimal stratifying criterion [38]. Youn et al. demonstrated that the composition of semen parameters such as RAPID 30.1%, motility 51.4%, and concentration 111 × 106 /mL before sperm preparation could be useful in predict- ing IUI outcome [31]. Oppositely, some researchers illu- minated that in the multivariable model, the predictive powers of sperm parameters were rather low [16, 36, 39], which might be due to a lack of prospective studies, a lack of standardization in semen testing methodology, and a huge heterogeneity of patient groups and IUI treatment strategies. More prospective cohort trials and prospec- tive randomized trials investigating the predictive value of semen parameters on IUI outcome are urgently needed [40]. In our study, we classified the cases into different eti- ological groups – Male-factor, Both-male-and-female-fac- tor and Other-factor. Our comparison analysis suggested that both pre- and post-processing parameters were quite different among the etiological groups (Tables 5 and 6); therefore, it is necessary to count in the etiology when analyzing the impacts of semen parameters on AIH preg- nancy. Further logistic regression analysis showed that models integrating multiple influencing factors exhibited much better predictive abilities on AIH outcomes than individual parameters (Tables 7, 8, 9, 10; Fig. 2). ROC curve analysis indicated that the prognostic capacities of the logistic regression models were greater in the Male- and Both-factor Groups than in the overall and Other- factor Groups (Tables 12; Fig. 3). This was reasonable as team’s previous research has shown that cycle treatment options, single/double IUI, female age, sperm VSL, SDI, and normal form rate could predict successful pregnancy following AIH in China. The multivariate logistic regres- sion equation exhibited a greater value for prognostic classification than single predictors [7]. Similarly, our logistic regression analysis showed that there were some sperm kinetic parameters of motility could affect AIH outcomes, though the effects were varied between differ- ent etiological groups (Table 4). Table 9 G d Besides, some research- ers ascertained that the total number of active sperm and concentration are related to pregnancy [31, 33]. However, our study demonstrated that semen volume, sperm con- centration and TPMSC before and after processing had no significant effect on AIH success. team’s previous research has shown that cycle treatment options, single/double IUI, female age, sperm VSL, SDI, and normal form rate could predict successful pregnancy following AIH in China. The multivariate logistic regres- sion equation exhibited a greater value for prognostic classification than single predictors [7]. Similarly, our logistic regression analysis showed that there were some sperm kinetic parameters of motility could affect AIH outcomes, though the effects were varied between differ- ent etiological groups (Table 4). Besides, some research- ers ascertained that the total number of active sperm and concentration are related to pregnancy [31, 33]. However, our study demonstrated that semen volume, sperm con- centration and TPMSC before and after processing had no significant effect on AIH success. if A growing body of evidences is showing the impacts of sperm morphological parameters on the successful rate of AIH. Several studies advised couples with ≤ 4% nor- mal sperm form to choose IVF or ICSI instead of IUI [5, 34]. Erdem et al. pointed out that the predictive value of morphological assessment in unexplained infertility is not reliable; however, in male subfertility, normal sperm form > 4.5% after processing could increase the CPR [35]. Louise et al. also stated that normal sperm form ≤ 4% is more important in couples with male infertility factors [16]. Contrary to the above research, a recent review sug- gested that sperm morphological parameters had low pre- dictive values for pregnancy success in both natural and assisted reproduction [36]. Kohn et al. also supported this conclusion and suggested that the current sperm mor- phology assessment is so strict that its predictive value for IUI has been lost [37]. In our study, the morphologi- cal parameters of different etiologies groups also had no significant influence on AIH pregnancy success (Table 5). Luo et al. BMC Pregnancy and Childbirth (2022) 22:703 Page 10 of 11 Page 10 of 11 infertility cases caused by non-male factors might be less impacted by the improvement of sperm quality.h Oct., 2018. Written informed consent was waived by the Ethics Committee of The Third Affiliated Hospital of Guangzhou Medical University. Acknowledgements ld l k h k 5. Monraisin O, Chansel-Debordeaux L, Chiron A, Floret S, Cens S, Bourrinet S, et al. Evaluation of intrauterine insemination practices: a 1-year prospec- tive study in seven French assisted reproduction technology centers. Fertil Steril. 2016;105:1589–93. https://doi.org/10.1016/j.fertnstert.2016.01.039. 5. Monraisin O, Chansel-Debordeaux L, Chiron A, Floret S, Cens S, Bourrinet S, et al. Evaluation of intrauterine insemination practices: a 1-year prospec- tive study in seven French assisted reproduction technology centers. Fertil Steril. 2016;105:1589–93. https://doi.org/10.1016/j.fertnstert.2016.01.039. We would like to thank all participants and their families. Author details 1 Department of Obstetrics and Gynecology, Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150, China. 2 Key Laboratory of Reproduction and Genetics of Guangdong Higher Education Institutes, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150, China. 3 Guangzhou Key Laboratory for Clinical Rapid Diagnosis and Early Warning of Infectious Diseases, Kingmed School of Laboratory Medicine, Guangzhou Medical University, Guangzhou 510182, China. In summary, our study showed that the DGC method prior to AIH significantly improved the sperm quality, but the change values were not correlated with the CPR. We found that some sperm parameters pre- or post-pro- cessing could predict the AIH outcome, and there was a collinearity relationship among these semen parameters. Moreover, we accredited that the prognostic capacities of multivariate logistic regression models were better than those of individual parameters, especially in cases caused or partly caused by male factors. In these cases, pre-pro- cessing VCL is the common top influencing factor. Received: 6 January 2022 Accepted: 30 August 2022 Received: 6 January 2022 Accepted: 30 August 2022 Funding h This research was funded by the National Natural Science Foundation of China (82002774), Guangdong Provincial Natural Science Foundation (2020A1515010065), Guangzhou City Science, Technology and Innovation Commission (201804010340, 202002030077), Guangzhou City Science and Technology Planning Project (202201020208, 202102010138), Guangdong Province Outstanding Youth Medical Talent Program (110217110) and Lin He’s Academician Workstation of New Medicine and Clinical Translation at The Third Affiliated Hospital of Guangzhou Medical University (2021HLKY05). 8. Montanaro Gauci M, Kruger TF, Coetzee K, Smith K, Van Der Merwe JP, Lombard CJ. Stepwise regression analysis to study male and female factors impacting on pregnancy rate in an intrauterine insemination programme. Andrologia. 2001;33:135–41. 9. Tan O, Ha T, Carr BR, Nakonezny P, Doody KM, Doody KJ. Predictive value of postwashed total progressively motile sperm count using CASA estimates in 6871 non-donor intrauterine insemination cycles. J Assist Reprod Genet. 2014;31:1147–53. https://doi.org/10.1007/ s10815-014-0306-0. Availability of data and materials The datasets used in this study are not publicly available due to the clinical data management policy of the health authority but are available from the corresponding authors upon reasonable request. 10. Dong Fl, Sun Yp, Su Yc, Guo Yh, Hu Ll, Wang F. Relationship between processed total motile sperm count of husband or donor semen and pregnancy outcome following intrauterine insemination. Syst Biol Reprod Med. 2011;57:251–5. https://doi.org/10.3109/19396368.2011.603792. Consent for publication Not applicable. Consent for publication Not applicable. Abbreviations CPR Cli i l CPR: Clinical pregnancy rate; PCC: Pearson correlation coefficient; TPMSC: Total progressive motile sperm count; VCL: Curvilinear velocity; VSL: Straight-line velocity; VAP: Average pathway velocity; LIN: Linearity; STR: Straightness; BCF: Beat cross frequency; ALH: Amplitude of lateral head displacement; SDI: Sperm deformity index; TZI: Teratozoospermia index; H: Sperm headpiece deform- ity; M: Sperm middle piece deformity; P: Sperm principal piece deformity; C: Sperm cytoplasm deformity; ROC: Receiver-operating characteristic; AUC: Area under curve; CI: Confidence interval. 3. Ruiter-Ligeti J, Agbo C, Dahan M. The impact of semen processing on sperm parameters and pregnancy rates after intrauterine insemination. Minerva Ginecol. 2017;69:218–24. https://doi.org/10.23736/S0026-4784. 16.04002-8. 4. Mollaahmadi L, Keramat A, Ghiasi A, Hashemzadeh M. The relationship between semen parameters in processed and unprocessed semen with intrauterine insemination success rates. J Turk Ger Gynecol Assoc. 2019;20:1–7. https://doi.org/10.4274/jtgga.galenos.2018.2018.0089. 4. Mollaahmadi L, Keramat A, Ghiasi A, Hashemzadeh M. The relationship between semen parameters in processed and unprocessed semen with intrauterine insemination success rates. J Turk Ger Gynecol Assoc. 2019;20:1–7. https://doi.org/10.4274/jtgga.galenos.2018.2018.0089. Declarations 11. Ombelet W, Dhont N, Thijssen A, Bosmans E, Kruger T. Semen quality and prediction of IUI success in male subfertility: a systematic review. Reprod Biomed Online. 2014;28:300–9. https://doi.org/10.1016/j.rbmo. 2013.10.023. References 1. Ye F, Cao W, Lin J, Du Y, Lan L, Dong Y, et al. The pregnancy outcomes of intrauterine insemination with husband’s sperm in natural cycles versus ovulation stimulated cycles: a retrospective study. Biosci Trends. 2018;12:463–9. https://doi.org/10.5582/bst.2018.01164. 1. Ye F, Cao W, Lin J, Du Y, Lan L, Dong Y, et al. The pregnancy outcomes of intrauterine insemination with husband’s sperm in natural cycles versus ovulation stimulated cycles: a retrospective study. Biosci Trends. 2018;12:463–9. https://doi.org/10.5582/bst.2018.01164. p g 2. Lemmens L, Kos S, Beijer C, Braat DDM, Nelen WLDM, Wetzels AMM. Tech- niques used for IUI: is it time for a change? Hum Reprod. 2017;32:1835– 45. https://doi.org/10.1093/humrep/dex223. 2. Lemmens L, Kos S, Beijer C, Braat DDM, Nelen WLDM, Wetzels AMM. Tech- niques used for IUI: is it time for a change? Hum Reprod. 2017;32:1835– 45. https://doi.org/10.1093/humrep/dex223. Authors’ contributions 6. Zhou H, Wu S, Tang X, Zhou G, Yuan J, Li Q, et al. Chlamydia trachomatis infection in the genital tract is associated with inflammation and hypo- spermia in the infertile male of China. As J Androl. 2021;23:1–6. https:// doi.org/10.4103/aja.aja_54_21. 6. Zhou H, Wu S, Tang X, Zhou G, Yuan J, Li Q, et al. Chlamydia trachomatis infection in the genital tract is associated with inflammation and hypo- spermia in the infertile male of China. As J Androl. 2021;23:1–6. https:// doi.org/10.4103/aja.aja_54_21. XX and DZ conceived and designed the study; YL, ML, MZ, JY, YZ, QL and XS collected the samples, performed the assays, and acquired the data; YL, ML, SW, XX and DZ analyzed the data; YL, ML, SW, XX and DZ wrote the manu- script. All authors read and approved the final manuscript. 7. Luo Y, Wu S, Yuan J, Zhou H, Zhong Y, Zhang M, et al. Evaluation of prognostic factors for clinical pregnancy rate following artificial insemina- tion by husband in the Chinese Population. Front Med. 2021;8: 638560. https://doi.org/10.3389/fmed.2021.638560. Table 9 G d There are still a few limitations in our current study. At present, the overall pregnancy rate of AIH is low. Due to various causes of infertility in both couples, the predic- tive value of a single predictor is low. Moreover, surgery needs to be performed immediately after sperm process- ing, which results in our study remaining in retrospective analysis. Further comprehensive studies are of need in the future. Ethics approval and consent to participate pp p p This study was performed in accordance with the Declaration of Helsinki and approved by the Ethics Committee of The Third Affiliated Hospital of Guang- zhou Medical University with the Ethics Review Report #2018–142 on 29th 12. Akl LD, Oliveira JB, Petersen CG, Mauri AL, Silva LF, Massaro FC, et al. Effi- cacy of the motile sperm organelle morphology examination (MSOME) 12. Akl LD, Oliveira JB, Petersen CG, Mauri AL, Silva LF, Massaro FC, et al. Effi- cacy of the motile sperm organelle morphology examination (MSOME) Luo et al. BMC Pregnancy and Childbirth (2022) 22:703 Page 11 of 11 in predicting pregnancy after intrauterine insemination. Reprod Biol Endocrinol. 2011;9:120. https://doi.org/10.1186/1477-7827-9-120. in predicting pregnancy after intrauterine insemination. Reprod Biol Endocrinol. 2011;9:120. https://doi.org/10.1186/1477-7827-9-120. 30. Aitken RJ. Sperm function tests and fertility. Int J Androl. 2006;29:69–75. https://doi.org/10.1111/j.1365-2605.2005.00630.x. 13. Luco SM, Agbo C, Behr B, Dahan MH. The evaluation of pre and post pro- cessing semen analysis parameters at the time of intrauterine insemina- tion in couples diagnosed with male factor infertility and pregnancy rates based on stimulation agent. A retrospective cohort study. Eur J Obstet Gynecol Reprod Biol. 2014;179:159–62. https://doi.org/10.1016/j.ejogrb. 2014.05.003. 31. Youn JS, Cha SH, Park CW, Yang KM, Kim JY, Koong MK, et al. Predictive value of sperm motility characteristics assessed by computer-assisted sperm analysis in intrauterine insemination with superovulation in couples with unexplained infertility. Clin Exp Reprod Med. 2011;38:47–52. https://doi.org/10.5653/cerm.2011.38.1.47. 32. Larsen L, Scheike T, Jensen TK, Bonde JP, Ernst E, Hjollund NH, et al. Computer-assisted semen analysis parameters as predictors for fertility of men from the general population. The danish first pregnancy planner study team. Hum Reprod. 2000;15:1562–7. 14. Deveneau NE, Sinno O, Krause M, Eastwood D, Sandlow JI, Robb P, et al. Impact of sperm morphology on the likelihood of pregnancy after intrau- terine insemination. Fertil Steril. 2014;102:1584-90.e2. https://doi.org/10. 1016/j.fertnstert.2014.09.016. 33. Ok EK, Doğan OE, Okyay RE, Gülekli B. The effect of post-wash total pro- gressive motile sperm count and semen volume on pregnancy outcomes in intrauterine insemination cycles: a retrospective study. J Turk Ger Gynecol Assoc. 2013;14:142–5. https://doi.org/10.5152/jtgga.2013.52280. 15. Hassan N, Agbo C, Dahan MH. Pregnancy rates unaffected by sperm count in intrauterine insemination: a retrospective cohort study. Minerva Ginecol. 2017;69:6–12. https://doi.org/10.23736/S0026-4784.16.03892-2. 16. Lemmens L, Kos S, Beijer C, Brinkman JW, van der Horst FAL, van den Hoven L, et al. Ethics approval and consent to participate Predictive value of sperm morphology and progressively motile sperm count for pregnancy outcomes in intrauterine insemina- tion. Fertil Steril. 2016;105:1462–8. https://doi.org/10.1016/j.fertnstert. 2016.02.012. 34. Nikbakht R, Saharkhiz N. The influence of sperm morphology, total motile sperm count of semen and the number of motile sperm inseminated in sperm samples on the success of intrauterine insemination. Int J Fertil Steril. 2011;5:168–73. 35. Erdem M, Erdem A, Mutlu MF, Ozisik S, Yildiz S, Guler I, et al. The impact of sperm morphology on the outcome of intrauterine insemination cycles with gonadotropins in unexplained and male subfertility. Eur J Obstet Gynecol Reprod Biol. 2016;197:120–4. https://doi.org/10.1016/j.ejogrb. 2015.12.014. 17. Vargas-Tominaga L, Alarcón F, Vargas A, Bernal G, Medina A, Polo Z. Associated factors to pregnancy in intrauterine insemination. JBRA Assist Reprod. 2020;24:66–9. https://doi.org/10.5935/1518-0557.20190060. 18. Cao S, Zhao C, Zhang J, Wu X, Zhou L, Guo X, et al. A minimum number of motile spermatozoa are required for successful fertilisation through arti- ficial intrauterine insemination with husband’s spermatozoa. Andrologia. 2014;46:529–34. https://doi.org/10.1111/and.12109. 36. Danis RB, Samplaski MK. Sperm morphology: history, challenges, and impact on natural and assisted fertility. Curr Urol Rep. 2019;20:43. https:// doi.org/10.1007/s11934-019-0911-7. 37. Kohn TP, Kohn JR, Ramasamy R. Effect of sperm morphology on preg- nancy success via intrauterine insemination: a systematic review and meta-analysis. J Urol. 2018;199:812–22. https://doi.org/10.1016/j.juro. 2017.11.045. 19. Gubert PG, Pudwell J, Van Vugt D, Reid RL, Velez MP. Number of motile spermatozoa inseminated and pregnancy outcomes in intrauterine insemination. Fertil Res Pract. 2019;5:10. https://doi.org/10.1186/ s40738-019-0062-z. 20. WHO. WHO laboratory manual for the Examination and processing of human semen. Geneva: World Health Organization; 2010. 21. Henkel RR, Schill WB. Sperm preparation for ART. Reprod Biol Endocrinol. 2003;1:108 https://doi org/10 1186/1477-7827-1-108 20. WHO. WHO laboratory manual for the Examination and processing of human semen. Geneva: World Health Organization; 2010. 38. Hamilton JAM, Cissen M, Brandes M, Smeenk JMJ, de Bruin JP, Kremer JAM, et al. Total motile sperm count: a better indicator for the severity of male factor infertility than the WHO sperm classification system. Hum Reprod. 2015;30:1110–21. https://doi.org/10.1093/humrep/dev058. 21. Henkel RR, Schill WB. Sperm preparation for ART. Reprod Biol Endocrinol. 2003;1:108. https://doi.org/10.1186/1477-7827-1-108. 22. Karamahmutoglu H, Erdem A, Erdem M, Mutlu MF, Bozkurt N, Oktem M, et al. The gradient technique improves success rates in intrauterine insemination cycles of unexplained subfertile couples when compared to swim up technique; a prospective randomized study. J Assist Reprod Genet. 2014;31:1139–45. https://doi.org/10.1007/s10815-014-0274-4. 39. Check JH, Bollendorf A. Publisher’s Note S N Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 24. Gode F, Bodur T, Gunturkun F, Gurbuz AS, Tamer B, Pala I, et al. Compari- son of microfluid sperm sorting chip and density gradient methods for use in intrauterine insemination cycles. Fertil Steril. 2019;112:842-8.e1. https://doi.org/10.1016/j.fertnstert.2019.06.037. 25. Zhu D, Zhao Z, Cui G, Chang S, Hu L, See YX, et al. Single-cell tran- scriptome analysis reveals estrogen signaling coordinately augments one-carbon, polyamine, and purine synthesis in breast cancer. Cell Rep. 2018;25:2285–98. https://doi.org/10.1016/j.celrep.2018.10.093. 26. Luo Y, Huang J, Tang Y, Luo X, Ge L, Sheng X, et al. Regional methylome profiling reveals dynamic epigenetic heterogeneity and convergent hypomethylation of stem cell quiescence-associated genes in breast can- cer following neoadjuvant chemotherapy. Cell Biosci. 2019;9:16. https:// doi.org/10.1186/s13578-019-0278-y. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? 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Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: 27. Zhu D, Rostami MR, Zuo W-L, Leopold PL, Crystal RG. Ethics approval and consent to participate The correlation of the degree of abnormal sperm morphology using strict criteria and pregnancy rates following intrauter- ine insemination (IUI). Clin Exp Obstet Gynecol. 2017;44:183–4. 40. Ombelet W, Dhont N, Thijssen A, Bosmans E, Kruger T. Semen quality and prediction of IUI success in male subfertility: a systematic review. Reprod Biomed Online. 2014;28:300–9. https://doi.org/10.1016/j.rbmo.2013.10.023. p g 23. Oguz Y, Guler I, Erdem A, Mutlu MF, Gumuslu S, Oktem M, et al. The effect of swim-up and gradient sperm preparation techniques on deoxyribo- nucleic acid (DNA) fragmentation in subfertile patients. J Assist Reprod Genet. 2018;35:1083–9. https://doi.org/10.1007/s10815-018-1163-z. Publisher’s Note S N Single-cell tran- scriptome analysis of mouse liver cell-specific tropism and transcriptional dysregulation following intravenous Administration of AAVrh.10 vectors. Hum Gene Ther. 2020;31:590–604. https://doi.org/10.1089/hum.2019.366. 28. Atasever M, Kalem MN, Hatırnaz Ş, Hatırnaz E, Kalem Z, Kalaylıoğlu Z. Factors affecting clinical pregnancy rates after IUI for the treatment of unexplained infertility and mild male subfertility. J Turk Ger Gynecol Assoc. 2016;17:134–8. https://doi.org/10.5152/jtgga.2016.16056. 29. Trussell JC, Coward RM, Santoro N, Stetter C, Kunselman A, Diamond MP, et al. Association between testosterone, semen parameters, and live birth in men with unexplained infertility in an intrauterine insemination population. Fertil Steril. 2019;111:1129–34. https://doi.org/10.1016/j.fertn stert.2019.01.034.
https://openalex.org/W2012805127	https://europepmc.org/articles/pmc3702586?pdf=render	English	null	Imaging Lymphatic System in Breast Cancer Patients with Magnetic Resonance Lymphangiography	PloS one	2,013	cc-by	7,092	Abstract Objective: To investigate the feasibility of gadolinium (Gd) contrast-enhanced magnetic resonance lymphangiography (MRL) in breast cancer patients within a typical clinical setting, and to establish a Gd-MRL protocol and identify potential MRL biomarkers for differentiating metastatic from non-metastatic lymph nodes. Materials and Methods: 32 patients with unilateral breast cancer were enrolled and divided into 4 groups of 8 patients. Groups I, II, and III received 1.0, 0.5, and 0.3 ml of intradermal contrast; group IV received two 0.5 ml doses of intradermal contrast. MRL images were acquired on a 3.0 T system and evaluated independently by two radiologists for the number and size of enhancing lymph nodes, lymph node contrast uptake kinetics, lymph vessel size, and contrast enhancement patterns within lymph nodes. Results: Group III patients had a statistically significant decrease in the total number of enhancing axillary lymph nodes and lymphatic vessels compared to all other groups. While group IV patients had a statistically significant faster time to reach the maximum peak enhancement over group I and II (by 3 minutes), there was no other statistically significant difference between imaging results between groups I, II, and IV. 27 out of 128 lymphatic vessels (21%) showed dilatation, and all patients with dilated lymphatic vessels were pathologically proven to have metastases. Using the pattern of enhancement defects as the sole criterion for identifying metastatic lymph nodes during Gd-MRL interpretation, and using histopathology as the gold standard, the sensitivity and specificity were estimated to be 86% and 95%, respectively. Conclusion: Gd-MRL can adequately depict the lymphatic system, can define sentinel lymph nodes, and has the potential to differentiate between metastatic and non-metastatic lymph nodes in breast cancer patients. Editor: Tone Frost Bathen, The Norwegian University of Science and Technology (NTNU), Norway Editor: Tone Frost Bathen, The Norwegian University of Science and Technology (NTNU), Norway Received December 17, 2012; Accepted June 17, 2013; Published July 5, 2013 Copyright: © 2013 Lu 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 is supported by National Natural Science Foundation of China (81271638); National Basic Research Program of China (973 Program: 2012CB932600); Frontier technology project of Shanghai Shen-kang Hospital Development Centre (SHDC12012112); and Shanghai Leading Academic Discipline Project (S30203). Abstract The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have read the journal's policy and have the following conflicts: author Yongming Dai is currently employed by MR Business, Philips Healthcare. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors. * E-mail: jhu@med.wayne.edu (J. Hua); Jill_huajia@163.com (J. Hu) well documented that post mastectomy radiation can benefit patients with axillary lymph node metastases [3]. Qing Lu1, Jia Hua1, Mohammad M. Kassir2, Zachary Delproposto3, Yongming Dai4, Jingyi Sun2, Mark Haacke2 Jiani Hu2 1 Department of Radiology, Shanghai Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China,, 2 Department of Radiology, Wayne State University; Michigan, United States of America,, 3 Department of Radiology, Henry Ford Hospital, Detroit, Michigan, United States of America, 4 MR Business, Greater China, Philips Healthcare, Shanghai, China Introduction For Gd- MRL, 3D fast spoiled gradient-recalled echo T1-weighted coronal images with a fat saturation (T1 high-resolution isotropic volume excitation, THRIVE) were acquired prior to the administration of Gd-DTPA with the following parameters: TR/TE: 3.5/1.7ms, flip angle: 25, FOV: 375×350mm2, matrix: 750×700, slices: 150, voxel size: 1.2×0.5×0.5 mm3, acquisition time: 3 min. After intradermal administration of the contrast material, the same imaging sequence (THRIVE) was repeated at 9, 12, 15, 18, 21 and 24 minutes. MIPs (Maximum Intensity Projections) were used to improve visualization of lymphatic vessels. Finally, dynamic fat suppressed axial high-resolution T1-weighted fast gradient echo images (THRIVE) were acquired (TE/TR 3.4/1.3 ms; flip angle of 10°; FOV 340×340 mm2, acquired voxel size 1×1×1.5 mm3); temporal resolution was 60s per dynamic acquisition, with a total of six dynamic acquisitions, one obtained prior to and five obtained immediately after intravenous administration of a bolus injection of 0.1 mmol/kg gadopentate dimeglumine followed by a 20 ml saline flush at an injection rate of 2 ml/s using an automatic injector. Total imaging time for each patient was about 45 minutes. Table 1. Total number of patients and number of axillae imaged within each group. Table 1. Total number of patients and number of axillae imaged within each group. node mapping with the administration of isosulfan blue (“blue dye”) is another commonly used technique to identify SLN, but is invasive and can only be performed during surgery. The possibility of tracer progression into other lymph node drainage pathways can obscure or mistakenly mimic the SLN [8]. Furthermore, while both methods can identify SLNs, neither method can differentiate metastatic from non-metastatic lymph nodes. node mapping with the administration of isosulfan blue (“blue dye”) is another commonly used technique to identify SLN, but is invasive and can only be performed during surgery. The possibility of tracer progression into other lymph node drainage pathways can obscure or mistakenly mimic the SLN [8]. Furthermore, while both methods can identify SLNs, neither method can differentiate metastatic from non-metastatic lymph nodes. Magnetic Resonance Lymphangiography (MRL) is a relatively new technique consisting of the acquisition of magnetic resonance images following the interstitial injection of a contrast agent. MRL with gadolinium (Gd)-based contrast agents (Gd-MRL) can generate high spatial resolution images of lymphatic vessels and lymph nodes [9–14]. Despite substantial progress in MRL techniques [15], the single published paper on breast Gd-MRL to date used only healthy human volunteers [16]. Introduction Breast cancer is the leading cause of cancer mortality in women worldwide [1]. Metastatic spread of malignancy can profoundly alter the prognosis and management of breast cancer [2]. Malignant spread to axillary lymph nodes is one of the most important predictors of survival in patients with breast cancer, increasing the 10-year recurrence rate from 20–30% to nearly 70% [2]. Since confirmation of metastatic lymph nodes alters both clinical and surgical management, and it has been Typically, breast cancer metastasizes to the sentinel lymph node (SLN), which is considered to be the first node to drain lymphatic fluid from the tumor [4,5]. The detection of a SLN metastasis is therefore critical for staging and prognosis. Lymphoscintigraphy, currently the most widely used methods to localize SLN, has some diagnostic and implementation disadvantages [6,7]. A radioactive tracer and associated precautions are required. Moreover, image quality and spatial resolution of scintigrams is generally poor. Intraoperative lymph PLOS ONE \| www.plosone.org 1 1 July 2013 \| Volume 8 \| Issue 7 \| e69701 MR Lymphography in Breast Cancer node mapping with the administration of isosulfan blue (“blue dye”) is another commonly used technique to identify SLN, but is invasive and can only be performed during surgery. The possibility of tracer progression into other lymph node drainage pathways can obscure or mistakenly mimic the SLN [8]. Furthermore, while both methods can identify SLNs, neither method can differentiate metastatic from non-metastatic lymph nodes. Table 1. Total number of patients and number of axillae imaged within each group. Group Injection volume Injection sites Total number of imaged axillae 1 1.0 ml 1 16 2 0.5 ml 1 16 3 0.3 ml 1 16 4 0.5 ml 2 16 placed in the prone position, head first in the scanner using a dedicated seven-channel phased-array breast coil. The imaging protocol consisted of an axial T1-weighted fast spin echo (T1-FSE) sequence, an axial diffusion-weighted imaging (DWI) echo-planar sequence (TE/TR 6548/65 ms; flip angle 90°, FOV 340×340 mm2, acquired voxel size 2 × 2 × 3 mm3; b values of 0 and 600 sec/mm2), and an axial and two sagittal T2-weighted fat suppressed sequence (Philips SPAIR; TE/TR 120/90 ms; inversion delay 125 ms; flip angle 90°; FOV 340× 340 mm2, acquired voxel size 1.01×1.31×3.0 mm3). Introduction The objective of this study is to investigate the feasibility of Gd-MRL for breast patients in a typical clinical setting, to establish an effective Gd-MRL protocol, and to identify potential MRL biomarkers for differentiating metastatic from non-metastatic lymph nodes. To optimize the breast MRL procedure, 32 patients were assigned to one of four groups (8 patients per group), with each group representing a different contrast administration or contrast dosing protocol. Both breasts were injected in each patient in all groups. For groups 1-3, gadolinium-based contrast was administered intradermally into the upper-outer periareolar area; only the amount of contrast delivered varied between the groups. Groups 1, 2, and 3 had 1.0 ml, 0.5 ml, and 0.3 ml of contrast administered, respectively. Patients in group 4 received two 0.5 ml intradermal periareolar injections with one injection in the upper-outer and one in the lower-inner quadrants. For all groups, following each injection, finger massage was applied for about 90 seconds at the injection site in order to facilitate the penetration of contrast into the lymphatic system. Table 1 summarizes the above mentioned information. Image analysis Gadopentate dimeglumine (Gd-DPTA) (Magnevist, Bayer Schering Pharma AG, Berlin, Germany) was mixed 10:1 with 1% mepivacaine hydrochloride, and used for all injections. Mepivacaine hydrochloride was added to alleviate pain during intradermal injection. All injections were performed using a 1 ml tuberculin syringe and a 26 G needle. Images were evaluated by two radiologists with over 15 years of body MRI experience. Morphological characteristics, the number of enhancing axillary lymph nodes, number of enhancing breast lymphatic vessels, and the average signal intensity for each lymph node were evaluated independently using a workstation (ViewForum, release 5.1; Philips Healthcare). Morphological analysis included evaluation of contrast enhancement defects and the presence of lymphatic vessel dilation. Contrast enhancement defects are foci of nonenhancement within an enhancing lymph node, using Patients The study was conducted after approval of the Shanghai Jiao tong University School of Medicine institutional review board, and was performed in accordance with the ethical guidelines of the Declaration of Helsinki. Written informed consent was obtained for each patient. From April 2012 to July 2012, a total of 32 patients (ages ranging from 27 years to 71 years; mean age: 51.5±9.2 years) were enrolled. Patient inclusion criteria were: (1) the presence of invasive breast cancer in a single breast, and (2) a treatment plan that included axillary lymphadenectomy. Pregnant patients, patients with previous axillary lymphadenectomy, and patients with renal insufficiency were excluded. MR Imaging Imaging was performed on a 3.0 T MR scanner (Achieva TX, Philips Medical Systems, Best, Netherlands). Patients were PLOS ONE \| www.plosone.org 2 July 2013 \| Volume 8 \| Issue 7 \| e69701 MR Lymphography in Breast Cancer criteria adopted from the literature [17–20]. For morphological characteristics of lymphatic vessels, we defined a lymphatic vessel as dilated if its diameter was more than 1.5 mm or at least 50% greater than the diameter of lymphatic vessels in the contralateral breast. Lymph nodes were further classified as Level 1, Level 2, or Level 3 based on anatomic location (Figure 1). The maximum transverse diameter (short axis diameter) of enhancing axillary lymph nodes and lymphatic vessels were measured and recorded. Contrast uptake kinetics for all enhancing lymph nodes were acquired from operator-defined regions of interest (ROI) drawn on both the pre- and post- contrast images. The signal intensity of air (ROI, 300 mm2) was used to estimate the background signal. All measurements were performed three times and each signal amplitude value was calculated as the mean of 3 separately-sampled ROIs. Time-signal intensity curves of lymph nodes were then constructed from signal intensity (SI) values obtained from the ROIs. The maximal enhancement time was defined at time point that the lymph node signal intensity began to reach plateau. The nodal signal-to-noise ratio (SNR, the maximum enhancement height) and nodal enhancement ratio (ER) at the maximal enhancement time were subsequently calculated. The nodal maximum enhancement height was determined by dividing the mean nodal signal intensity at the maximal enhancement time by the standard deviation (SD) of noise measured outside the patient. Nodal ER was determined by mean nodal SI at maximal enhancement time subtracting nodal SI at pre-contrast and divided by the nodal SI before contrast, and converted to percentages. MRL data from patients in group 1, 2, 4 were used to explore the potential of Gd-MRL for differentiating metastatic lymph nodes from non-metastatic ones. Table 2. Clinically observed side effects for all patients in group 1-4. Group Diameter of local redness and swelling (cm) Subsiding time of local redness and swelling (hours) Cases of local blister appearing Local infection 1 2.7 16.9 3 None 2 1.6 8.7 None None 3 1.2 5.7 None None 4 1.7 9.6 None None Table 2. Clinically observed side effects for all patients in group 1-4. Figure 1. Diagrammatic representation of axillary lymph node levels. MR Imaging Lymph nodes were classified as Level 1, Level 2, or Level 3 based on anatomic location. Level I: latissimus dorsi to lateral pectoralis minor; level II: posterior to pectoralis minor; level III: medial pectoralis minor to thoracic inlet. doi: 10.1371/journal.pone.0069701.g001 criteria adopted from the literature [17–20]. For morphological characteristics of lymphatic vessels, we defined a lymphatic vessel as dilated if its diameter was more than 1.5 mm or at least 50% greater than the diameter of lymphatic vessels in the contralateral breast. Lymph nodes were further classified as Level 1, Level 2, or Level 3 based on anatomic location (Figure 1). The maximum transverse diameter (short axis diameter) of enhancing axillary lymph nodes and lymphatic vessels were measured and recorded. Contrast uptake kinetics for all enhancing lymph nodes were acquired from operator-defined regions of interest (ROI) drawn on both the pre- and post- contrast images. The signal intensity of air (ROI, 300 mm2) was used to estimate the background signal. All measurements were performed three times and each signal amplitude value was calculated as the mean of 3 separately-sampled ROIs. Time-signal intensity curves of lymph nodes were then constructed from signal intensity (SI) values obtained from the ROIs. The maximal enhancement time was defined at time point that the lymph node signal intensity began to reach plateau. The nodal signal-to-noise ratio (SNR, the maximum enhancement height) and nodal enhancement ratio (ER) at the maximal enhancement time were subsequently calculated. The nodal maximum enhancement height was determined by dividing the mean nodal signal intensity at the maximal enhancement time by the standard deviation (SD) of noise measured outside the patient. Nodal ER was determined by mean nodal SI at maximal enhancement time subtracting nodal SI at pre-contrast and divided by the nodal SI before contrast, and converted to percentages. MRL data from patients in group 1, 2, 4 were used to explore the potential of Gd-MRL for differentiating metastatic lymph nodes from non-metastatic ones. Figure 1. Diagrammatic representation of axillary lymph node levels. Lymph nodes were classified as Level 1, Level 2, or Level 3 based on anatomic location. Level I: latissimus dorsi to lateral pectoralis minor; level II: posterior to pectoralis minor; level III: medial pectoralis minor to thoracic inlet. doi: 10.1371/journal.pone.0069701.g001 significance of the different results among the four groups. Results All breast cancer patients completed their examinations successfully without any unexpected adverse events. A small papular eruption typically appeared in the periareolar area at the site of contrast injection, and was most obvious about 30 minutes following the injection. All side effects are summarized in Table 2. Evaluation of lymph node and lymphatic vessel, including the number, diameter, and signal intensity showed excellent inter- observer agreement, with k values of 0.97, 0.88, and 0.96, respectively. Typical examples of enhancing Level 1 through Level 3 axillary lymph nodes on MRL are shown in Figures 2 and 3. The overall number and diameters of enhancing lymph nodes in each of the four groups are summarized in Table 3. Groups 1, 2, and 4 had a significantly greater number of enhancing lymph nodes than group 3 (F ratio = 26.41, p<0.001), but otherwise there was no significant difference between groups 1, 2 and 4. There were a significantly greater number of enhancing Level 1 lymph nodes in groups 1, 2, and 4 compared to group 3 (F ratio = 15.34, p<0.001), but no significant differences in the number of enhancing Level 2 or Level 3 lymph nodes between any of the four groups, and no Evaluation of lymph node and lymphatic vessel, including the number, diameter, and signal intensity showed excellent inter- observer agreement, with k values of 0.97, 0.88, and 0.96, respectively. Typical examples of enhancing Level 1 through Level 3 axillary lymph nodes on MRL are shown in Figures 2 and 3. The overall number and diameters of enhancing lymph nodes in each of the four groups are summarized in Table 3. After the MRL, a radiologist with 22 years of breast MRI experience and a surgeon with 15 years of breast surgery experience worked together to correlate the MR images with dissected lymph nodes based on location, size, morphological characteristics, and the number of metastatic lymph nodes in the patient by pathological evaluation. A pathologist with 8 years of experience who was blinded to the MRL results performed pathological evaluations using standard departmental procedure [21]. Groups 1, 2, and 4 had a significantly greater number of enhancing lymph nodes than group 3 (F ratio = 26.41, p<0.001), but otherwise there was no significant difference between groups 1, 2 and 4. Results There were a significantly greater number of enhancing Level 1 lymph nodes in groups 1, 2, and 4 compared to group 3 (F ratio = 15.34, p<0.001), but no significant differences in the number of enhancing Level 2 or Level 3 lymph nodes between any of the four groups, and no MR Imaging Inter-observer agreement was calculated by using the kappa test, with k values of 0.5-0.75 considered to indicate satisfactory agreement, and values higher than 0.75 were considered to indicate excellent agreement. A p value of < 0.05 was used as the cutoff for statistical significance. July 2013 \| Volume 8 \| Issue 7 \| e69701 5 July 2013 \| Volume 8 \| Issue 7 \| e69701 Discussion Previous human studies have demonstrated that Gd-MRL is a safe method for imaging lymphatic system, and can be used to delineate the anatomy of lymphatic vasculature in various parts of the body [9,13,22] including the breast [16]. The sub- areolar plexus (Sappey’s plexus) is known to have a rich lymphatic network. Periareolar intradermal injection of contrast agent was chosen for this study based on the consensus that it is an end point for dermal lymphatic drainage, and subsequently drains to the axilla [23,24]. Gadopentate dimeglumine is a commercially available, widely used extracellular, water-soluble paramagnetic contrast agent. The recommend dose for intravenous administration is 0.1 mmol per kilogram of body weight, which is about 5 times higher than the concentration used in this study. A favorable safety profile has been previously shown for intravenous administration [25]. Although the smaller dose and local injection into the breast lowers the risk of anaphylactoid reactions and renal toxicity, we excluded patients with renal failure as a general precaution. Animal studies indicate that gadopentetate dimeglumine causes moderate necrosis, hemorrhage, inflammation and edema that is not statistically different from meglumine diatrizoate when the agent is injected into the subcutaneous tissues of rats or mice [26,27]. Periareolar intradermal contrast injections can cause minor tissue injury, namely superficial erythema and blistering at the injection site. We found that tissue injury at the injection sites was temporary and reactions fell within the expectations of clinical experience [28] Moreover, no irreversible or unexpected adverse events have been reported in prior contrast-enhanced MRL studies, which is confirmed by our observations [10,12,13,29,30]. Figure 4. Axillary lymph nodes contrast-uptake kinetics of the four patient groups. Graph of enhancement profile over time as determined with signal-to-noise ratio (SNR) measurements in the axillary lymph node shows a different contrast -uptake kinetics of the four patient groups. ANOVA test results show that the SNR (at the maximum enhancement height) of the lymph nodes in group 1, 2 and 4 was significantly higher than that in group 3 (SNR: F ratio=17.613, p<0.001), though there was no significant difference between groups 1, 2 and 4. Error bars represent standard errors of the mean. doi: 10.1371/journal.pone.0069701.g004 Figure 4. Axillary lymph nodes contrast-uptake kinetics of the four patient groups. Graph of enhancement profile over time as determined with signal-to-noise ratio (SNR) measurements in the axillary lymph node shows a different contrast -uptake kinetics of the four patient groups. Statistical Analysis The number of lymph nodes, SNR, ER, and number of lymphatic vessels were compared between the four groups. The ANOVA test was used to determine the statistical PLOS ONE \| www.plosone.org July 2013 \| Volume 8 \| Issue 7 \| e69701 3 MR Lymphography in Breast Cancer Figure 2. MRL images of breast lymph nodes at different levels. (A) (coronal plane) and (B) (transversal plane) show typica MRL images illustrating breast lymph node enhancement at different nodal levels. Arrowhead: Level 1 axillary lymph nodes. Arrows Level 2 axillary lymph nodes. doi: 10.1371/journal.pone.0069701.g002 Figure 2. MRL images of breast lymph nodes at different levels. (A) (coronal plane) and (B) (transversal plane) show typical MRL images illustrating breast lymph node enhancement at different nodal levels. Arrowhead: Level 1 axillary lymph nodes. Arrows: Level 2 axillary lymph nodes. doi: 10.1371/journal.pone.0069701.g002 doi: 10.1371/journal.pone.0069701.g002 4 PLOS ONE \| www.plosone.org 4 MR Lymphography in Breast Cancer Figure 3. MRL images of breast lymph nodes at different levels. A typical MRL image shows enhancing breast lymph nodes at different nodal levels. Arrowhead: Level 1 lymph nodes. Arrow: Level 3 lymph nodes. doi: 10.1371/journal.pone.0069701.g003 Figure 3. MRL images of breast lymph nodes at different levels. A typical MRL image shows enhancing breast lymph nodes at different nodal levels. Arrowhead: Level 1 lymph nodes. Arrow: Level 3 lymph nodes. doi: 10.1371/journal.pone.0069701.g003 Table 3. Comparison of number and diameter of lymph nodes and lymph vessels in group 1-4. Table 3. Comparison of number and diameter of lymph nodes and lymph vessels in group 1-4. Table 3. Comparison of number and diameter of lymph nodes and lymph vessels in group 1-4. Enhancing axillary lymph nodes Lymphatic vessels in breast Group Axillas (N) Number (Median) Diameter (mm) Number Diameter (mm) Level 1 Level 2 Level 3 Total number Mean±SD Median Mean±SD 1 16 3.5 1 0.5 5 9.13 ± 2.29 3 1.42 ± 0.53 2 16 3.5 1 0.5 4.5 8.57 ± 1.97 3 1.39 ± 0.53 3 16 1.5 * 0 0 2 * 8.21 ± 1.67 1 * 1.34 ± 0.53 4 16 4 1 0.5 5.5 8.24 ± 2.19 3 1.62 ± 0.46 Note: * indicates a significant difference between group 3 and the other three groups 5 PLOS ONE \| www.plosone.org 5 MR Lymphography in Breast Cancer MR Lymphography in Breast Cancer Table 4. Differences in dynamic contrast-enhanced kinetics between groups 1-4. Statistical Analysis Group Time to maximal enhancement (minutes) Axially lymph node at maximal enhancement timeMean±SD) SNR ER (%) 1 12 211.23 ± 53.07 145.91 ± 69.15 2 15 202.67 ± 22.09 124.19 ± 31.16 3 24 124.63 ± 12.81 * 51.57 ± 27.99 * 4 12 217.63 ± 41.53 129.19 ± 49.78 Note: * indicates a significant difference between group 3 and the other three groups Using histopathology as the gold standard, the presence of contrast enhancement defects gave a sensitivity of 86% and specificity of 95%. In addition to SLN morphological analysis, 27 of 128 (21%) lymphatic vessels were found to be dilated. All patients with dilated lymphatic vessels were pathologically proven to have metastases. Figure 5 is a representative MRL for a patient with a normal-size metastatic lymph node, showing an enhancement defect pattern and a dilated lymphatic vessel. Figure 6 is another example, but with an enlarged metastatic lymph node. Excepting size, it has the same morphological characteristics as the lymph node in Figure 5. July 2013 \| Volume 8 \| Issue 7 \| e69701 Discussion ANOVA test results show that the SNR (at the maximum enhancement height) of the lymph nodes in group 1, 2 and 4 was significantly higher than that in group 3 (SNR: F ratio=17.613, p<0.001), though there was no significant difference between groups 1, 2 and 4. Error bars represent standard errors of the mean. doi: 10.1371/journal.pone.0069701.g004 significant difference in the number of enhancing Level 1 lymph nodes between groups 1, 2, and 4. Similarly, the number of enhancing lymphatic vessels visualized in groups 1, 2, and 4 was significantly greater than those of group 3 (F ratio = 9.643, p<0.001), but there was no significant difference in the number of enhancing lymph vessels between groups 1, 2, and 4. Comparison of results from the four groups suggest that a single (per breast), a 0.5 ml periareolar gadolinium injection is sufficient for imaging axillary lymph nodes. More specifically, for axillary lymph nodes, the maximum peak enhancement from a 0.5 ml gadolinium contrast injection is comparable to a 1.0 ml injection, but with fewer side effects. Conversely, insufficient enhancement was seen with the administration of 0.3 ml of contrast. The protocol for group 4, (two 0.5 ml periareolar injections) was not found to be superior to the single injection of 1.0 or 0.5 ml gadolinium contrast agent, though this method could be helpful during training to ensure an intradermal rather than a subdermal (subcutaneous) injection. The only statistically significant advantage for using the protocol 1 or protocol 4 over protocol 2 was about a 3 Figure 4 shows contrast uptake curves for all groups, and Table 4 summarizes the time needed for axillary lymph nodes to reach peak enhancement and the enhancement rate (ER). ANOVA results show that the SNR and ER of the lymph nodes in group 1, 2 and 4 were significantly higher than that in group 3 (SNR: F ratio=17.613, p<0.001; ER: F ratio=8.721, p<0.001), though there was no significant difference between groups 1, 2 and 4. Table 5 lists the results for MRL morphological characteristics of SLNs from patients in groups 1, 2 and 4. PLOS ONE \| www.plosone.org 6 July 2013 \| Volume 8 \| Issue 7 \| e69701 MR Lymphography in Breast Cancer Figure 5. MRL images of breast normally-sized metastatic lymph nodes. Example of normally-sized metastatic lymph nodes with an abnormal nodal enhancement pattern and lymphatic vasculature. Arrowheads: enhancement defects within non-enlarged lymph nodes. Discussion Long arrows: lymphatic vessels. Short arrows: veins. doi: 10.1371/journal.pone.0069701.g005 Figure 5. MRL images of breast normally-sized metastatic lymph nodes. Example of normally-sized metastatic lymph nodes with an abnormal nodal enhancement pattern and lymphatic vasculature. Arrowheads: enhancement defects within non-enlarged lymph nodes. Long arrows: lymphatic vessels. Short arrows: veins. doi: 10.1371/journal.pone.0069701.g005 lymph nodes, contrast enhancement defects could be an additional biomarker identifying metastatic lymph nodes. The observation of contrast enhancement defects within metastatic lymph nodes is in agreement with previous MRL studies on cervical lymph node metastases in a rabbit model [31], and malignant lymphoma in a mouse model [32]. Our results also suggest that lymphatic vessels that connect to metastatic lymph nodes can appear dilated compared to non-metastatic vessels, an observation consistent with previous studies using CT lymphography [33]. To our knowledge, the morphological features of lymph node enhancement defects and dilated lymphatic vessels have not been previously observed in breast cancer patients, and more importantly, these features show potential for differentiating metastatic from non-metastatic lymph nodes. The mechanism which underlies these observations is not fully known. Elucidating the mechanism requires further thorough scientific investigation and is beyond the scope of this study. Nevertheless, we can speculate based on our observations and related knowledge in the literature. In the normal lymphatic system, intradermal contrast material diffuses into lymphatic vessels and drains with lymph fluid through the lymphatic system to lymph nodes. In metastatic lymphatic systems, however, lymphatic vessels can be partially blocked by metastatic implants (perhaps dilating lymphatic vessels) or be completely absent due to metastatic invasion. This can cause the enhancement defects on MRL images as observed in this study. Finally, our results are also in agreement with the well-documented knowledge that metastatic lymph nodes can be enlarged [34–39]. Table 5. Summary of lymph node analysis for patients in group 1, 2 and 4. MRL findings Number of lymph nodes with contrast enhancement defects Number of lymph nodes without contrast enhancement defects Number of non- Metastatic lymph nodes Number of Metastatic lymph nodes Number of non- Metastatic lymph nodes Number of Metastatic lymph nodes SensitivitySpecificity Group 1 2 6 51 1 Group 2 1 10 58 2 86.2% 95.3% Group 4 2 9 55 1 Table 5. Summary of lymph node analysis for patients in group 1, 2 and 4. Table 5. Summary of lymph node analysis for patients in group 1, 2 and 4. July 2013 \| Volume 8 \| Issue 7 \| e69701 Discussion MRL findings Number of lymph nodes with contrast enhancement defects Number of lymph nodes without contrast enhancement defects Number of non- Metastatic lymph nodes Number of Metastatic lymph nodes Number of non- Metastatic lymph nodes Number of Metastatic lymph nodes SensitivitySpecificity Group 1 2 6 51 1 Group 2 1 10 58 2 86.2% 95.3% Group 4 2 9 55 1 minute faster time to reach maximum peak enhancement in axillary lymph nodes, which could be valuable in clinical settings where an abbreviated waiting period between intradermal injection and MRL data acquisition is required. The importance of identifying SLN metastases in breast cancer patients has long been recognized. The physiology of Gd-MRL makes the identification of SLNs readily apparent due to gadolinium-based contrast uptake within lymphatic vessels and lymph nodes following intradermal injection. As indicated in Table 5 and in Figures 5 and 6, Gd-MRL cannot only identify SLN, but also has the potential to differentiate metastatic from non-metastatic lymph nodes. Specifically, in addition to the well-known parameter of enlarged short axis dimension of This study has several limitations. First, there are technical challenges preventing precise node-by-node correlation, which is well-understood by the community. What we did was to correlate MR results with pathologic results based on 1) the 7 PLOS ONE \| www.plosone.org July 2013 \| Volume 8 \| Issue 7 \| e69701 MR Lymphography in Breast Cancer Figure 6. MRL of enlarged metastatic lymph nodes. Arrowhead: Irregular enlarged lymph nodes with enhancement (or contrast filling) defect pattern. Long thin arrows: enhancing and dilated lymphatic vessels. Short fat arrow: enhancing normal lymphatic vessels with a beaded appearance. Short arrows: vein. Hollow arrows: injection sites. doi: 10.1371/journal.pone.0069701.g006 Figure 6. MRL of enlarged metastatic lymph nodes. Arrowhead: Irregular enlarged lymph nodes with enhancement (or contrast filling) defect pattern. Long thin arrows: enhancing and dilated lymphatic vessels. Short fat arrow: enhancing normal lymphatic vessels with a beaded appearance. Short arrows: vein. Hollow arrows: injection sites. doi: 10.1371/journal.pone.0069701.g006 subareolar, subcutaneous over the primary tumor site, peritumoral, and intratumoral. As mentioned before, Gd-MRL for patients with breast cancer is an essentially unexplored area, and further thorough investigations would have to be performed prior to routine clinical use. size and location of the lymph node, 2) the number of metastatic lymph nodes as determined by pathological examination and 3) corresponding number of lymph nodes with the most suspicious MRL patterns. Discussion That is, only if the number of metastatic lymph nodes by pathological examination was not equal to the number of lymph nodes with Gd-MRL enhancement defects or dilated lymphatic vessels, did we consider the results to be a mismatch between MRL and pathology. Second, the 32 (8 patients in 4 groups) patients had four different MRL protocols. Although these protocol differences did not affect our ability to identify potential MRL biomarkers for differentiating metastatic from non-metastatic lymph nodes, they do affect the accuracy of our statistical results. We are actively seeking a better way to improve the correlation between MRI results and pathologic finding. We are also actively recruiting more patients in an effort to overcome the above limitations. Finally, we only evaluated the intradermal periareolar injection. Other possible injection sites include In conclusion, we have successfully acquired MRL images in breast cancer patients using a widely available Gd-based contrast agent in a typical clinical setting. Our results demonstrate the potential of Gd-MRL to identify sentinel lymph nodes and differentiate metastatic from non-metastatic lymph nodes. Author Contributions Conceived and designed the experiments: J. Hu J. Hua MH. Performed the experiments: QL J. Hua. Analyzed the data: QL MMK. Contributed reagents/materials/analysis tools: YD JS. Wrote the manuscript: QL ZD J. Hu. Conceived and designed the experiments: J. Hu J. Hua MH. Performed the experiments: QL J. Hua. Analyzed the data: QL MMK. Contributed reagents/materials/analysis tools: YD JS. Wrote the manuscript: QL ZD J. Hu. 1. Jemal A, Bray F, Center MM, Ferlay J, Ward E et al. (2011) Global cancer statistics. CA Cancer J Clin 61: 69-90. doi:10.3322/caac.20107. PubMed: 21296855. 2. 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https://openalex.org/W3202032184	https://dadun.unav.edu/bitstream/10171/66350/1/pdf.pdf	English	null	Plant-insect chemical communication in ecological communities: an information theory perspective	bioRxiv (Cold Spring Harbor Laboratory)	2,021	cc-by	6,940	Peng‐Juan Zu1,2* , Reinaldo García‐García3 , Meredith C. Schuman4 , Serguei Saavedra5 , an Carlos J. Melián2,6 1Department of Environmental Systems Science, ETH Zurich, Schmelzbergstrasse 9, Zurich CH‐8092, Switzerland 2Department Fish Ecology & Evolution, Swiss Federal Institute of Aquatic Science and Technology (Eawag), Seestrasse 79, Kastanienbaum l 1Department of Environmental Systems Science, ETH Zurich, Schmelzbergstrasse 9, Zurich CH‐8092, Switzerland 2Department Fish Ecology & Evolution, Swiss Federal Institute of Aquatic Science and Technology (Eawag), Seestrasse 79, Kastanienbaum 6 i l d 1Department of Environmental Systems Science, ETH Zurich, Schmelzbergstrasse 9, Zurich CH‐8092, Switzerland 2Department Fish Ecology & Evolution, Swiss Federal Institute of Aquatic Science and Technology (Eawag), Seestrasse 79, Kastanienbaum CH‐6047, Switzerland 1Department of Environmental Systems Science, ETH Zurich, Schmelzbergstrasse 9, Zurich CH‐8092, Switzerland 2Department Fish Ecology & Evolution, Swiss Federal Institute of Aquatic Science and Technology (Eawag), Seestrasse 79, Kastanienbaum CH‐6047, Switzerland 3Departamento de Física y Matemática Aplicada, Facultad de Ciencias, Universidad de Navarra, Irunlarrea 1, Pamplona 31008, Spain 4Departments of Chemistry and Geography, University of Zurich, Winterthurerstrasse 190, Zurich CH‐8057, Switzerland 5Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, 77 Massachusetts Av., Cambridge 02139, MA, USA 6Institute of Ecology and Evolution, University of Bern, Baltzerstrasse 6, Bern CH‐3012, Switzerland Author for correspondence. E‐mail: pengjuan.zu@gmail.com Received 30 September 2021; Accepted 15 February 2022; Article ﬁrst published online 23 February 2022 3Departamento de Física y Matemática Aplicada, Facultad de Ciencias, Universidad de Navarra, Irunlarrea 1, Pamplona 31008, Spain 4Departments of Chemistry and Geography, University of Zurich, Winterthurerstrasse 190, Zurich CH‐8057, Switzerland 5Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, 77 Massachusetts Av., Cambridge 02139, MA, USA 6Institute of Ecology and Evolution, University of Bern, Baltzerstrasse 6, Bern CH‐3012, Switzerland Author for correspondence. E‐mail: pengjuan.zu@gmail.com p p g @g Received 30 September 2021; Accepted 15 February 2022; Article ﬁrst published online 23 February 2022 Abstract Cross‐species communication, where signals are sent by one species and perceived by others, is one of the most intriguing types of communication that functionally links diﬀerent species to form complex ecological networks. Global change and human activity can aﬀect communication by increasing ﬂuctuations in species composition and phenology, altering signal proﬁles and intensity, and introducing noise. So far, most studies on cross‐species communication have focused on a few speciﬁc species isolated from ecological communities. Scaling up investigations of cross‐species communication to the community level is currently hampered by a lack of conceptual and practical methodologies. Peng‐Juan Zu1,2* , Reinaldo García‐García3 , Meredith C. Schuman4 , Serguei Saavedra5 , an Carlos J. Melián2,6 Here, we propose an interdisciplinary framework based on information theory to investigate mechanisms shaping cross‐species communication at the community level. We use plants and insects, the cornerstones of most ecosystems, as a showcase and focus on chemical communication as the key communication channel. We ﬁrst introduce some basic concepts of information theory, then we illustrate information patterns in plant–insect chemical communication, followed by a further exploration of how to integrate information theory into ecological and evolutionary processes to form testable mechanistic hypotheses. We conclude by highlighting the importance of community‐level information as a means to better understand the maintenance and workings of ecological systems, especially during rapid global change. Key words: chemical communication, ecological networks, functional traits, information theory, plant–insect interaction, volatile organic compounds, Zipf's law. shaped by hundreds of millions of years of co‐evolution (Ehrlich & Raven, 1964). © 2022 The Authors. Journal of Systematics and Evolution published by John Wiley & Sons Australia, Ltd on behalf of Institute of Botany, Chinese Academy of Sciences. JSE Journal of Systematics and Evolution JSE Journal of Systematics and Evolution Journal of Systematics and Evolution doi: 10.1111/jse.12841 doi: 10.1111/jse.12841 JSE Journal of Systematics and Evolution Peng‐Juan Zu1,2* , Reinaldo García‐García3 , Meredith C. Schuman4 , Serguei Saavedra5 , and C l J M liá 2 6 u1,2* , Reinaldo García‐García3 , Meredith C. Schuman4 , Serguei Saavedra5 , and 2 6 1 Introduction Communication is prevalent in nature. For example, honey- bees waggle to send information that guides other bees in the colony (Von Frisch, 1974); birds sing to mate or alert others in the ﬂock (Freeberg, 2008); and ﬂowers exhibit colors and scents that can attract pollinators or deter herbivores (Schoonhoven et al., 2005). Cross‐kingdom (Plantae and Animalia) plant–insect communication is of extreme interest not only because of its ubiquity and fundamental roles in both natural and agricultural systems (Seastedt & Crossley, 1984; Strong et al., 1984; Isbell et al., 2011; Potts et al., 2016; Ollerton, 2021) but also due to the extraordinarily diverse communication mediators Chemical communication is one of the most ancient and pivotal means for plants and insects. In fact, chemistry underlies color (pigments), shape (genetic encoding in nucleic acids and chemical inducers such as hormones), and scent (volatile organic compounds, or VOCs). Yet, chemical mediators of communication are most often considered from among the vast number of secondary metabolites produced by plants, with an estimate of 200 000 compounds including VOCs that have been extracted and identiﬁed (Kessler & Kalske, 2018; Robbins, 2000), which are not strictly required for plant growth and development but may have important defensive and attractive functions (Fraenkel, 1959; Ehrlich This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. May 2023 \| Volume 61 \| Issue 3 \| 445–453 © 2022 The Authors. Journal of Systematics and Evolution published by John Wiley & Sons Australia, Ltd on behalf of Institute of Botany, Chinese Academy of Sciences. Zu et al. 446 & Raven, 1964; Pichersky & Gershenzon, 2002). Insects, with their numerous chemoreceptors, are very sensitive to many plant chemicals (Schoonhoven et al., 2005; Kaupp, 2010; Hansson & Stensmyr, 2011) and depend on chemical signals for fundamental activities, such as foraging (Schiestl, 2010), mating (Alexander et al., 1997), and oviposition (Renwick & Chew, 1994). Presumably, plant‐ feeding insects and their natural enemies have been under strong selective pressure to perceive and interpret plant chemicals that are relevant for these essential activities. So far, most plant–insect chemical communication studies have focused on speciﬁc species, isolating them from the community context in which they are naturally embedded. 1 Introduction However, scaling up plant–insect communication from the species to the community level can be possible through the lens of information theory and communication systems (Shannon, 1948). the information is reproduced at the receiver's end, we can quantify the clarity or ambiguity of the communication (Shannon, 1948). That is, the better a message is reproduced, the greater amount of information (clarity) it contains, and the lower its entropy (ambiguity, uncertainty). This mathematical theory of communication is called information theory. Figure 1 summarizes key information quantities for a vector of signals (entropy and self‐information of each signal) and for an object‐signal matrix (conditional entropy and mutual informa- tion). Information theory was originally developed for man‐ made communication systems, such as telecommunication, and later applied to a wide range of communication‐related ﬁelds, for example, cybernetics (Gabor, 1954), cryptography (Ahlswede & Csiszár, 1993), linguistics (Zipf, 1949; Ferrer‐i‐ Cancho & Solé, 2003), and neurobiology (Sharpee et al., 2014). Information theory brings at least two important new features that make it a promising tool for various systems and scales. First, information theory translates diverse communication signals into information. Thus, it unites diﬀerent signals by extracting their information content without being over- whelmed by their diﬀerent identities. Second, information Communication can be considered as a process of information transfer from a sender who encodes the information (e.g., visual, vocal, or olfactory), to a receiver who decodes the information (Fig. 1). Depending on how well Fig. 1. Information transferring process in communication and formula quantifying information in communication systems. Fig. 1. Information transferring process in communication and formula quantifying informatio J. Syst. Evol. 61(3): 445–453, 2023 J. Syst. Evol. 61(3): 445–453, 2023 Plant–insect chemical communication using information theory 447 theory measures the reproducibility of information between two states based on probabilistic concepts (Boso & Tartakovsky, 2018). This nonparametric perspective allows the scaling of information across diﬀerent dimensions. (Zu et al., 2020). Additionally, we also use a review study by Farré‐Armengol et al. (2020), who compiled a ﬂoral VOC dataset from 305 species. In the review study, the authors also categorized chemical groups of VOCs, families of plant species, and pollination systems of these plant species. Not surprisingly, information theory has long been proposed as a conceptual and quantitative framework to study information transfer across ecological systems (Margalef, 1968; Ulanowicz, 2001; O'Connor et al., 2019). However, concrete applications to plant–insect communica- tion are just starting to emerge (Zu et al., 2020). In this paper, we illustrate the application of information theory as a means to increase our understanding of plant–insect communication. In particular, we propose to consider plant VOCs as communication signals and explore the emerging information patterns of plant–insect chemical communica- tion. We then discuss potential future directions to uncover the underlying mechanisms of plant–insect communication by the integration of information theory with ecological and evolutionary processes. Note that here we use “signals” in an information theory framework to simply refer to all plant VOCs that form the repository (regardless of whether they have demonstrated attractant or deterrent functions, or not). By analyzing the distributions of VOCs in these studies and in diﬀerent categories (chemical groups, plant families, pollination groups), we found that most cases follow a heavy‐tail distribution where a few VOCs are predominant whereas many other VOCs occur rarely (Fig. 2). In ﬁve cases (out of the 16 examined cases and categories), a power–law distribution ﬁts either the whole data (in one community study PV_k, and the group of N‐, S‐containing compounds, Fig. 2, Table 1) or part of the data (in Orchidaceae, insect‐pollinated ﬂowers, and the overall review data, Fig. 2, Table 1). The ﬁtting was performed using the R package “poweRlaw” (Clauset et al., 2009; Gillespie, 2017). Many of the remaining cases appear to have power–law‐like distributions, but a ﬁt cannot be statistically supported (Table S1). Indeed, although power–law distributions are thought to be common, it is not easy to achieve a strict ﬁt to many empirical data (Clauset et al., 2009) partially due to limited sample size and sample bias on abundant vs. rare signals. 2 Patterns of Plant–Insect Chemical Communication In the ﬁve cases in which a full or partial ﬁt can be made, the slopes of the distribution range from k = 1.94 to k = 2.68, which is steeper than observed in human languages. These patterns suggest that plant VOC languages may be more redundant than human language. Following information theory, the entropy of VOCs in all these cases is very high, ranging from 0.78 to 0.99 (H(V) in Table 1A). 2.1 Zipf's law Zipf's law (Zipf, 1932, 1949) in linguistics describes that the frequency of a word decays rapidly proportional to its rank, or frequency of usage, following a power–law distribution (p(r) = ar−k, where r denotes rank). Taking the English language for example, “the” is the most frequent word in corpora, occurring at a rate of 6% in British National Corpus, followed by “of,” which only occurs half as often (3%) (Leech et al., 2014). And there is a large number of words that occur in very low frequency, leading to a heavy tail distribution. The power–law decay distribution is also related to the Pareto principle (or 80‐20 rule) known in social sciences: that is, roughly 80% of outcomes result from 20% of causes (Pareto, 1964). Importantly, the power–law exponent describing the speed of decay (i.e., the shape of this tailed distribution) and the redundancy of information in communication systems is around k = 1 in many human languages (Zipf, 1949; Piantadosi, 2014). Zipf's law has also been explored in various biological systems (Semple et al., 2021). Note that throughout the whole paper, we used stand- ardized entropy so that it ranges between 0 and 1, where the higher the value (closer to 1), the higher the uncertainty. Entropy calculation formulas can be found in Fig. 1. 2.2 Coding, decoding, and interactions 2.2 Coding, decoding, and interactions In the previous section, we only focus on the putative signals (VOCs) themselves. In this section, we place these into the context of communication: the coding and decoding of signals, and species interactions. Following the work by Zu et al. (2020), we can use the associations of VOCs (V) with plants (P) in a community to describe the plant coding process (PV‐matrix), the associations of VOCs with insects (or animals, A) to describe insect decoding process (AV‐matrix), and associations of animals with plants (AP‐matrix) for insect‐plant interactions. Empirically, to screen how each insect reacts to each of the VOCs in the community requires extensive experimental manipulations under both controlled and natural conditions. Therefore, it is challenging to generate the whole insect decoding matrix from ﬁeld and experimental work. If we consider a plant chemical “language,” we can think of plants emitting diﬀerent chemical “words” to their environment. Studies on plant VOCs have revealed that some VOCs are more frequent than others (Knudsen et al., 2006; Farré‐Armengol et al., 2020), but the detailed information structures have not been explicitly studied. To explore the redundancy of plant chemical language and whether the frequency of its vocabulary (VOCs) follows Zipf's law, we gathered the data from the only four community‐level plant VOC studies so far, three of which focused on ﬂoral VOCs in plant‐pollinator networks (Filella et al., 2013; Kantsa et al., 2017; Burkle & Runyon, 2019) and one on leaf VOCs in a plant‐herbivore (caterpillar) network From the four community‐level plant VOC studies that documented a PV‐matrix, three of them (Burkle & Runyon, 2019; Kantsa et al., 2019; Zu et al., 2020) also document insect‐plant interaction networks (AP‐matrix) in the same community. We found that plant‐coding patterns vary from community to community (Fig. 3, Table 1). In the case of the leaf VOC community (PV_z), VOCs are more commonly shared among plants (H(P\|V) = 0.94), whereas in J. Syst. Evol. 61(3): 445–453, 2023 www.jse.ac.cn 17596831, 2023, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jse.12841 by Universidad de Navarra, Wiley Online Library on 17596831, 2023, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jse.12841 by Universidad de Navarra, Wiley Online Library on [23/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are 448 t f ﬂ VOC (PV f PV k) th d i di ti i li ti (H(P \| V) Fig. 2. 2.2 Coding, decoding, and interactions Volatile organic compound (VOC) frequency vs rank (log–log scale) from four community‐level study papers (top row) and a review paper (second to fourth rows) that summarized VOC frequency in diﬀerent chemical groups (second row), four representatives of plant families (third row), and plants with diﬀerent pollination groups and the overall review data (fourth row). Black lines in the cases of “Community: PV_k,” “N‐, S‐, compounds,” “Orchidaceae,” “Overall” indicate that data can be described by power–law distribution (k values represent the exponent of the power–law distribution p(r) = ar−k), although log‐normal distribution can describe the data similarly well (tests see Table S1). N‐, S‐, compounds: nitrogen or sulfur‐ containing volatile compounds. Number of sampled plant species (N_plant) and number of VOCs (N_VOC) in each case can be found in Table 1. 448 Zu et al. Fig. 2. Volatile organic compound (VOC) frequency vs rank (log–log scale) from four community‐level study papers (top row) and a review paper (second to fourth rows) that summarized VOC frequency in diﬀerent chemical groups (second row), four representatives of plant families (third row), and plants with diﬀerent pollination groups and the overall review data (fourth row). Black lines in the cases of “Community: PV_k,” “N‐, S‐, compounds,” “Orchidaceae,” “Overall” indicate that data can be described by power–law distribution (k values represent the exponent of the power–law distribution p(r) = ar−k), although log‐normal distribution can describe the data similarly well (tests see Table S1). N‐, S‐, compounds: nitrogen or sulfur‐ containing volatile compounds. Number of sampled plant species (N_plant) and number of VOCs (N_VOC) in each case can be found in Table 1. around 0.5 indicating some specialization (H(P \| V) = 0.53, H(V \| P) = 0.41, Table 1A). We observe specialized patterns for all these insect‐plant interactions as revealed by entropy values much less than 1 (H (A\|P) around 0.5, H(P\|A) around 0.2, Table 1B, Fig. 4). The asymmetric values of H(A\|P) and H(P\|A) indicate that insects are two cases of ﬂower VOCs (PV_f, PV_k), there are more unique VOCs (H(P \| V) = 0.41, 0.51 respectively), and the third case of ﬂower VOCs is in between (H(P\|V) = 0.75). around 0.5 indicating some specialization (H(P \| V) = 0.53, H(V \| P) = 0.41, Table 1A). We observe specialized patterns for all these insect‐plant interactions as revealed by entropy values much less than 1 (H (A\|P) around 0.5, H(P\|A) around 0.2, Table 1B, Fig. 4). The asymmetric values of H(A\|P) and H(P\|A) indicate that insects are 2.2 Coding, decoding, and interactions We also analyzed plant coding patterns based on the review study and found these to vary from category to category (Table 1A), with the overall (from the whole dataset) value of around 0.5 indicating some specialization (H(P \| V) = 0.53, H(V \| P) = 0.41, Table 1A). We observe specialized patterns for all these insect‐plant interactions as revealed by entropy values much less than 1 (H (A\|P) around 0.5, H(P\|A) around 0.2, Table 1B, Fig. 4). The asymmetric values of H(A\|P) and H(P\|A) indicate that insects are J. Syst. Evol. 61(3): 445–453, 2023 J. Syst. Evol. 61(3): 445–453, 2023 www.jse.ac.cn 449 Plant–insect chemical communication using information theory Plant–insect chemical communication using information theory 449 Table 1 Entropy measures based on (A) plant‐VOC associations (PV‐matrix based) and (B) insect‐plant networks (AP‐matrix based) in the four community‐level studies papers and a review paper that compiled plant‐VOC publications A A B H(V) entropy of VOCs. H(·\|·) conditional entropy. Calculation formulas see Fig. 1. Details of statistical tests for power–law, see Table S1. VOC, volatile organic compound. A B H(V) entropy of VOCs. H(·\|·) conditional entropy. Calculation formulas see Fig. 1. Details of statistical tests for power–law, see Table S1. VOC, volatile organic compound. B B H(V) entropy of VOCs. H(·\|·) conditional entropy. Calculation formulas see Fig. 1. *Details of statistical tests for power–law, see Table S1. VOC, volatile organic compound. more specialized on plants than plants specialized on insects (H (P \| A) are lower). In addition, the interaction patterns seem to be indistinguishable between mutualistic (pollinator‐plant) and antagonistic (herbivore‐plant) networks based on these few studies. Indeed, a global analysis of more than 250 mutualistic and antagonistic networks has revealed that the network structures (e.g., generality, nestedness) per se cannot separate mutualistic from antagonistic networks (Michalska‐Smith & Allesina, 2019) unless one includes environmental factors (Song & Saavedra, 2020). more clearly with their pollinators than with their herbivores, while herbivores must try harder than pollinators to decode information from their host plants. While we recognize that further conclusions are likewise currently limited by a shortage of data, we can still ask why plant–insect chemical communication is structured in the way it is, and not otherwise. In this section, we highlight two approaches to investigate the potential mechanisms driving these chemical communication patterns between plants and insects. 2.2 Coding, decoding, and interactions Recall the diﬃculties of building a complete decoding matrix (how each insect decodes each of the VOCs) empirically. Here, we theoretically construct insect decoding from two approaches: (i) using a top‐down logic assuming that the insect can decode all the VOCs from a plant as long as they can feed on that plant; that is, AV = AP × PV and (ii) use a bottom‐up logic by hypothesizing the relationships between VOC abundance and functionality (i.e., eﬀects on insects). In both directions, we integrate information theory into ecological and evolutionary theories to generate testable hypotheses. 3 From patterns to mechanisms We have shown the information structures of plant–insect chemical communications as revealed by the apparent power–law behavior in some cases (but where these conclusions are limited by limited data availability), and by the ubiquitous decrease in entropy of VOC associations to plants as opposed to total entropy of VOCs. The latter is not surprising, as not all plants produce all VOCs. Interestingly, the conditional entropy of VOCs (H(P\|V)) involved in plant‐ herbivore interactions seems to be generally higher than the entropy of those involved in plant‐pollinator interactions, but this is not reﬂected in the conditional entropy of plant‐animal associations (H(A\|P))—which is similar among these inter- action types. This suggests that plants try to communicate 3.1 Top‐down: Interactions shape communication This direction follows the inspiration of studies exploring how Zipf's law patterns emerge in linguistics. Various hypotheses have been proposed to explain this intriguing pattern in human languages. Among these, the “least eﬀort” hypothesis (Zipf, 1949; Ferrer‐i‐Cancho & Solé, 2003) is a compelling hypothesis that nicely recovers the Zipf's law J. Syst. Evol. 61(3): 445–453, 2023 www.jse.ac.cn Fig. 3. Patterns of plant‐volatile organic compound (VOC) associations (PV‐matrix) in the four community studies (details see top four rows in Table 1A). Each row and column represents plant species and VOCs, respectively. Black and white squares show that a given VOC was present or absent in a given plant, respectively. 450 Zu et al. Zu et al. 450 Fig. 3. Patterns of plant‐volatile organic compound (VOC) associations (PV‐matrix) in the four community studies (details see top four rows in Table 1A). Each row and column represents plant species and VOCs, respectively. Black and white squares show that a given VOC was present or absent in a given plant, respectively. Fig. 3. Patterns of plant‐volatile organic compound (VOC) associations (PV‐matrix) in the four community studies (details see top four rows in Table 1A). Each row and column represents plant species and VOCs, respectively. Black and white squares show that a given VOC was present or absent in a given plant, respectively. This is a successful case of integrating other known evolutionary and ecological theories into information theory to build a framework that aims to disentangle mechanisms driving plant–insect interactions and communication at the community level. In general, there are three steps to construct communication frameworks for a given system. First, we must deﬁne who are the speakers and who are the listeners. 3 From patterns to mechanisms Second, we must deﬁne proper ﬁtness functions for both parties based on their ecological and evolutionary relation- ships. Third, we must deﬁne the rules of optimization for each party in the information game. Both the second and the third steps require very careful examination by integrating evolu- tionary and ecological processes. We also want to emphasize that the “ﬁtness” function at the community level, which deﬁnes success in terms of passing into the next “round” in a game, is not the same as the Darwinian ﬁtness proxies that biologists normally use to assess individual success in survival and reproduction but rather refers to broader beneﬁts, which can be assessed at the level of communities rather than individuals. This corresponds as well to the broader use of the term “signal” introduced at the beginning of this paper. This approach is compatible with the evaluation of individual‐level ﬁtness but aims at criteria that can be used at the community level. We hope more studies will be inspired by this information perspective to provide insights into other plant–insect chemical communication types (e.g., plant‐ pollinator, plant‐herbivore‐parasitoid). distribution as a natural consequence from the conﬂicting interests between speakers (who are thought to aim for “brevity and phonological reduction” to minimize their eﬀort to speak) and listeners (who should desire “explicitness and clarity” to minimize their eﬀort to understand). The successful application of information theory in studying the structure and emergence of human language has inspired studies on animal vocal communication (e.g., reviews in McCowan et al., 1999, 2008; Kershenbaum et al., 2021), and indicates its potential to be extended in studying the chemical “language” in plant–insect communities (Zu et al., 2020). distribution as a natural consequence from the conﬂicting interests between speakers (who are thought to aim for “brevity and phonological reduction” to minimize their eﬀort to speak) and listeners (who should desire “explicitness and clarity” to minimize their eﬀort to understand). The successful application of information theory in studying the structure and emergence of human language has inspired studies on animal vocal communication (e.g., reviews in McCowan et al., 1999, 2008; Kershenbaum et al., 2021), and indicates its potential to be extended in studying the chemical “language” in plant–insect communities (Zu et al., 2020). Indeed, Zu et al. (2020) aimed to borrow the framework to test whether plant‐herbivore chemical communication patterns can be explained by conﬂicting interests of plants (speakers) and herbivores (listeners). 4 Conclusion We have found from the limited studies that plant VOC frequencies follow a heavy‐tail distribution with a few predominant compounds and many rare compounds; all the three documented plant–insect interaction matrices are highly specialized, whereas plant–VOC coding patterns seem to vary from case to case, with diﬀerent plant communities and perhaps, interaction types, resulting in signals that are encoded more or less redundantly. We also provide a top‐ down and a bottom‐up direction for constructing theoretical frameworks to integrate information theory in studies of plant–insect chemical communication. By proposing both top‐down and bottom‐up frameworks, we hope we can draw attention to, and inspire more studies on the information perspective of trophic networks in ecosystems. Fig. 4. Patterns of insect‐plant interactions (AP‐matrix) in the three community studies (details see Table 1B). Each row and column represents insect species and plant species, respectively. Black and white squares show that a given insect was, or was not observed feeding on a given plant, respectively. plant–insect communication. Given that there are rare and common VOCs (frequency), it is natural to ask whether rare VOCs or common VOCs can be decoded more easily by insects (functionality) and how much information can be gained by decoding the VOCs. In the face of more serious and more frequent threats to ecological systems, plant–insect interactions are vulnerable, yet particularly important aspects of ecosystem functioning: insect pollinators are vital for agricultural productivity as well as for the survival of wild plant communities (Ollerton, 2021; Wei et al., 2021); herbivores support all higher trophic levels that depend on plants (Price et al., 1980; Harvey et al., 2003; Moreira et al., 2016), and their predators and parasitoids perform pest control services (Schmidt et al., 2003). We still struggle to control these interactions with relatively crude tools that carry heavy collateral damage, such as physically uprooting and transporting communities of pollinators to agricultural ﬁelds and broadly prescribed spraying of pesticides. Integrating information theory has the potential to help us gain a better understanding of the underlying mechanisms, and thus promises novel insights to better understand and manage these interactions. For example, novel chemicals of exotic plants have been suggested to be the main mechanism for invading the local communities (Cappuccino & Arnason, 2006; Macel et al., 2014). Plant–insect chemical communication using information theory 451 Fig. 4. Patterns of insect‐plant interactions (AP‐matrix) in the three community studies (details see Table 1B). Each row and column represents insect species and plant species, respectively. Black and white squares show that a given insect was, or was not observed feeding on a given plant, respectively. decoding common VOCs, lead to more generalized plant–insect interactions; whereas negative‐frequency dependence, preferentially decoding rare VOCs, may result in more specialized interactions? How could this be understood in terms of the co‐evolution of plant VOCs and insect olfaction? Within this framework, one can hypothesize diﬀerent speciﬁc relationships between VOC frequency and informa- tion functionality (VA‐matrix), to test how these give rise to plant–insect interaction network structures (using PA = PV × VA). Speciﬁcally, information functionality can be modeled with two elements: one, how easily a VOC can be decoded (VA_func); two, how much information can be gained by decoding the VOC (V_info). Therefore, the information functionality matrix (VA) can be treated as an information‐ weighted decoding matrix, generated by (VA = V_info × VA_func). We can use a drift process mimicking frequency‐ independent decoding, which might act as a control for testing the strength of frequency‐dependent decoding occurring in the empirical data. 3 From patterns to mechanisms It is well‐known that the chemical arms race between plants and herbivores has been playing out for hundreds of millions of years, where herbivores must constantly adapt to plant defense chemicals while plants must keep producing novel chemicals (Ehrlich & Raven, 1964). Zu and colleagues (Zu et al., 2020) translated this chemical arms race into an information arms race: plants code the (chemical) information in a way (changes in PV‐matrix) to make the decoding (AV given by AP × PV) diﬃcult (high entropy) for herbivores, whereas herbivores interact with plants in a way (changes in AP‐matrix) to make the decoding easier (low entropy). With repeated cycles of information optimization by plants and herbivores, they found that an equilibrium stage is reached in which a plant‐VOC redundancy matrix and herbivore‐ plant specialization matrix have emerged. These information patterns at the equilibrium stage matched their ﬁeld data collected in a tropical dry forest. 3.2 Bottom‐up: Connect VOC frequency with “information functionality” 3.2 Bottom‐up: Connect VOC frequency with “information functionality” A diﬀerent, bottom‐up approach would connect VOC frequency with information functionality to study www.jse.ac.cn J. Syst. Evol. 61(3): 445–453, 2023 www.jse.ac.cn Plant–insect chemical communication using information theory References structures are more resilient than others. We can further- more ask how climate change, pollution (including pesticide), and habitat fragmentation will aﬀect communication signal proﬁles, dynamics, and signal‐to‐noise ratios and thus rewire plant–insect networks. Overall, understanding the eﬀects of communication on species interactions and vice versa has the potential to improve our ability to assess impacts of anthropogenic disturbances on ecosystems, and assist conservation and restoration practices. For instance, instead of focusing on taxonomic diversity, chemical diversity and chemical information structure may be shown to be more crucial and informative for ecosystem biodiversity (Schuman et al., 2016). Ahlswede R, Csiszár I. 1993. Common randomness in information theory and cryptography. I. Secret sharing. IEEE Transactions on Information Theory 39: 1121–1132. Alexander RD, Marshall DC, Cooley JR. 1997. Evolutionary perspectives on insect mating. In: Choe JC, Crespi BJ eds. The evolution of mating systems in insects and arachnids. Cambridge: Cambridge University Press. 4–31. Boso F, Tartakovsky DM. 2018. Information‐theoretic approach to bidirectional scaling. Water Resources Research 54: 4916–4928. Burkle LA, Runyon JB. 2019. Floral volatiles structure plant–pollinator interactions in a diverse community across the growing season. Functional Ecology 33: 2116–2129. Information approaches can provide the scalability to integrate further dimensions to achieve a better and potentially more uniﬁed understanding of information ﬂows and their eﬀects in ecosystems (Deng et al., 2021). For instance, we can expand these frameworks by including diﬀerent sets of VOCs (e.g., from ﬂowers, leaves, and roots); diﬀerent sets of partners and trophic levels (e.g., pollinators, microbes, parasitoids, and predators); and even diﬀerent information forms (e.g., visual, vocal). Information theory abstracts all kinds of signals (or functional traits) as information and thus has the potential to gather the multi‐ layer functional maps into a uniﬁed framework. In summary, integrating information theory to existing theories in ecology and evolution has the potential to unveil central biological mechanisms driving the formation and maintenance of the functionality of species interactions and thus of entire ecosystems (Margalef, 1968; Kessler & Kalske, 2018; O'Connor et al., 2019), as has recently been argued more generally for plant chemistry (Walker et al., 2022). Cappuccino N, Arnason JT. 2006. Novel chemistry of invasive exotic plants. Biology Letters 2: 189–193. Clauset A, Shalizi CR, Newman ME. 2009. Power‐law distributions in empirical data. SIAM Review 51: 661–703. D'Alfonso S. 2011. On quantifying semantic information. Information 2: 61–101. Deng J, Angulo MT, Saavedra S. 2021. 4 Conclusion From the perspective of information theory, we can ask what are the eﬀects of invasive species on the information landscape of the current community and whether some information Shannon's original mathematical theory of communica- tion (Shannon, 1948) was mostly based on the “Inverse Relationship Principle” (D'Alfonso, 2011), which states that the less probable a signal is, the more information it bears. In our plant–insect communication systems, insects face an environment with VOCs at diﬀerent frequencies. On the one hand, specializing in decoding rare VOCs brings advantages if substantial information is thereby gained to access a niche that can be exploited by specialists. However, specialization comes with risks because rare VOCs (emitted by only one plant species or a few phenotypes) can go extinct more easily, locally or globally. On the other hand, decoding common VOCs is likely to be safer but less informative, meaning that insects might need to decode more signals to gather enough informa- tion to identify their plant hosts. Which strategy do insects use and how will diﬀerent strategies aﬀect the interaction network structure? For example, will positive‐frequency‐ dependent decoding structure: that is, preferentially J. Syst. Evol. 61(3): 445–453, 2023 www.jse.ac.cn Zu et al. 452 Conﬂicts of Interest Hansson BS, Stensmyr MC. 2011. Evolution of insect olfaction. Neuron 72: 698–711. The authors declare no competing ﬁnancial interest. Harvey JA, Van Dam NM, Gols R. 2003. Interactions over four trophic levels: Foodplant quality aﬀects development of a hyper- parasitoid as mediated through a herbivore and its primary parasitoid. Journal of Animal Ecology 72: 520–531. 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https://openalex.org/W1974314529	https://europepmc.org/articles/pmc4015768?pdf=render	English	null	Comparison of clinical and dental panoramic findings: a practice-based crossover study	BMC oral health	2,013	cc-by	6,740	© 2013 Moll 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: Aim was to compare clinical findings with x-ray findings using dental panoramic radiography (DPR). In addition, type and frequency of secondary findings in x-rays were investigated. Methods: Patients were selected on the basis of available DPRs (not older than 12 months). No therapeutic measures were permitted between the DPR and the clinical findings. The clinical findings were carried out by several investigators who had no knowledge of the purpose of the study. A calibrated investigator established the x-ray findings, independently and without prior knowledge of the clinical findings. The evaluation parameters for each tooth were: missing, healthy, carious, restorative or prosthetically sufficient or insufficient treatment. Type and frequency of additional findings in the DPR were documented, e.g. quality of a root canal filling and apical changes. Results: Findings of 275 patients were available. Comparison showed a correspondence between clinical and radiographic finding in 93.6% of all teeth (n = 7,789). The differences were not significant (p > 0.05). Regarding carious as well as insufficiently restored or prosthetically treated teeth, respectively there were significant differences between the two methods (p < 0.05). The DPRs showed additional findings: root fillings in 259 teeth and 145 teeth with periapical changes. Conclusions: With reference to the assessment of teeth, there was no difference between the two methods. However, in the evaluation of carious as well as teeth with insufficiently restorative or prosthetic treatment, there was a clear discrepancy between the two methods. Therefore, it would have been possible to have dispensed with x-rays. Nevertheless, additional x-ray findings were found. : Dental findings, Radiographic findings, Dental panoramic radiography (DPR), Clinical examination Comparison of clinical and dental panoramic findings: a practice-based crossover study Marc A Moll1, Miriam Seuthe1, Constantin von See2,3, Antonia Zapf4, Else Hornecker1, Rainer F Mausberg1 and Dirk Ziebolz1* Moll et al. BMC Oral Health 2013, 13:48 http://www.biomedcentral.com/1472-6831/13/48 Moll et al. BMC Oral Health 2013, 13:48 http://www.biomedcentral.com/1472-6831/13/48 Open Access * Correspondence: dirk.ziebolz@med.uni-goettingen.de 1Department of Preventive Dentistry, Periodontology and Cariology, University Medical Centre, Robert-Koch Str. 40, Goettingen D-37075, Germany Full list of author information is available at the end of the article Background In order to complete the dental findings x-rays are recommended [2]. Dental panoramic radiography (DPR) provides an overview, and represents a sensible and fre- quently used radiological basis; it enables an assessment of the hard tissue structures of the facial area [3]. In this way, clinical findings can be verified and supplemented by important information. At the same time, however, the value should be greater than the potential risk of genotoxic effects caused by x-rays [2,4]; in this connec- tion, the quality of the x-rays is of great importance [5]. Precise dental ascertainment of findings is the key to achieving adequate diagnostics, as well as the therapy based on this and the best possible treatment of the pa- tient. In addition to a comprehensive anamnesis, the documentation of extra- and intra-oral clinical findings is necessary. The clinical dental examination includes, in addition to assessment of the mucous membranes, the condition of the teeth (healthy or carious), the restora- tive and prosthetic treatment of the teeth (sufficient or insufficient), as well as sensitivity testing and determin- ation of the periodontal situation [1]. In Germany, the relevance of DPR in providing an overview is undisputed [6]. Already in the 1970s, the routine preparation of a DPR in dental practices was promoted for the pre-treatment examination: It enables the early diagnosis of tooth and jaw anomalies [7], and the treatment costs may be reduced in the long-term [8]. Page 2 of 8 Moll et al. BMC Oral Health 2013, 13:48 http://www.biomedcentral.com/1472-6831/13/48 Moll et al. BMC Oral Health 2013, 13:48 http://www.biomedcentral.com/1472-6831/13/48 for carrying out the study was obtained from the Bundesministerium für Verteidigung (German Ministry of Defense), Fü San I/1, (File NO.: 42-13-05 dated 18.07.06). for carrying out the study was obtained from the Bundesministerium für Verteidigung (German Ministry of Defense), Fü San I/1, (File NO.: 42-13-05 dated 18.07.06). Rushton and Horner (1996), in contrast, have questioned the importance of x-ray findings for dental diagnostics in the routine recording of clinical dental findings [9]. They argue that the differences between tactile-visual findings and x-ray findings are too small and may cause an unnecessary exposure of the patient to x-rays [9]. Moreover, the diagnostic precision has been questioned by some other authors [10,11]. The European Guideline on Radiation in Dental Radiography Issue No. 136 says that in adult patients DPR may be indicated in a limited number of dental problems [12]. Participants/x-rays Making use of the x-ray control books of the participat- ing centers, all DPRs from soldiers dating back to the years 2007/2008 were determined. The x-rays had to have been prepared within a maximum of 12 months be- fore the dental clinical (check-up) examination. No add- itional DPR was prepared for the study. Up until now, the question of the extent to which sup- plementary x-rays provide additional and valuable infor- mation during the clinical dental examination has not been unambiguously clarified. Thus, it is conceivable that, based on a proficient, reliable acquisition of clinical findings during the first (check-up) examination in everyday practice, the sense of an additional DPR is questionable. Accordingly, it is possible that either no x- rays are taken or, if it is, only an inadequate assessment will take place instead of obtaining detailed x-ray find- ings. However, the consequence may be the lack of or inadequate diagnostics and possibly inadequate therapy. In particular problems, i.e. suspicion on carious lesions and periapicale problems bitewing radiographs and/or periapicale radiographs, respectively, are the method of choice [13]. Nevertheless, DPRs nowadays are com- monly used. The reason may be that additional findings can be detected; however, this does not justify routinely preparing DPRs. For this reason, it seems appropriate to systematically examine the question of the additional value of DPR in the context of the first dental (check- up) examination. The following inclusion criteria were defined: current and evaluable DPR and complete clinical dental finding, no therapeutic measures having taken place between preparation of the DPR and documenting of the clinical finding. Only dental records of male soldiers who had signed up for a fixed period (minimally 4 years) or pro- fessional soldiers were included. The selection of the DPRs was carried out in accordance with the inclusion and exclusion criteria, respectively, shown in Table 1. Furthermore, subjects with removable dental restoration were excluded. Clinical examination ( earrings or piercings • Standardized setting of the x-ray equipment: chin support, bite bar, voltage (70 kV) and current (9 mA) • Asymmetrical positioning of the patient • False positioning of the patient’s head • Improper presentation of the jaws and the teeth • Comparable/good image quality: (no film overexposure and no soiling of the film) • recognizable movement of the patient while the picture was being taken • Presentation of jaws and teeth distortion-free as much as possible • overlaying effects Table 1 Quality criteria for the x-rays (DPR) in the study Inclusion criteria Exclusion criteria • DPR at the time of the study not older than 12 months • Metallic foreign bodies in the head region, e.g. earrings or piercings • Standardized setting of the x-ray equipment: chin support, bite bar, voltage (70 kV) and current (9 mA) • Asymmetrical positioning of the patient • False positioning of the patient’s head • Improper presentation of the jaws and the teeth • Comparable/good image quality: (no film overexposure and no soiling of the film) • recognizable movement of the patient while the picture was being taken • Presentation of jaws and teeth distortion-free as much as possible • overlaying effects Clinical examination ( Clinical findings (visual-tactile) were recorded on one occasion under standardized conditions (mirror, dental probe, illumination) during a routine first (check-up) examination in those dental centers participating in the study. The findings were randomly taken from two den- tists per dental center, all dentists were skilled in dental examination; they were not calibrated and had no know- ledge in the study. At the time of the clinical examin- ation, the radiographic findings of the soldiers were not known to the six dentists. The aim of this study was to compare the clinical den- tal findings of the first (check-up) examination in dental practices with currently available DPRs in terms of an assessment of healthy or carious, as well as restorative and prosthetically sufficient or insufficiently treated teeth. Also, additional x-ray findings were investigated, in particular, sufficient and insufficient root canal fill- ings, teeth with apicectomy or periapical changes, im- pacted teeth, as well as shadowing of the maxillary sinus. The following hypotheses were formulated: clinical findings and x-ray findings based on DPR show only minor assessment differences. However, DPR enables the detection of a number of additional information which is needed for comprehensive diagnostics. The following parameters were recorded: missing teeth, healthy and carious teeth, sufficient and insuffi- cient fillings (amalgam, composite, inlay), as well as Table 1 Quality criteria for the x-rays (DPR) in the study Inclusion criteria Exclusion criteria • DPR at the time of the study not older than 12 months • Metallic foreign bodies in the head region, e.g. earrings or piercings • Standardized setting of the x-ray equipment: chin support, bite bar, voltage (70 kV) and current (9 mA) • Asymmetrical positioning of the patient • False positioning of the patient’s head • Improper presentation of the jaws and the teeth • Comparable/good image quality: (no film overexposure and no soiling of the film) • recognizable movement of the patient while the picture was being taken • Presentation of jaws and teeth distortion-free as much as possible • overlaying effects Table 1 Quality criteria for the x-rays (DPR) in the study Table 1 Quality criteria for the x-rays (DPR) in the study Inclusion criteria Exclusion criteria • DPR at the time of the study not older than 12 months • Metallic foreign bodies in the head region, e.g. Moll et al. BMC Oral Health 2013, 13:48 http://www.biomedcentral.com/1472-6831/13/48 Moll et al. BMC Oral Health 2013, 13:48 http://www.biomedcentral.com/1472-6831/13/48 Moll et al. BMC Oral Health 2013, 13:48 http://www.biomedcentral.com/1472-6831/13/48 Moll et al. BMC Oral Health 2013, 13:48 http://www.biomedcentral.com/1472-6831/13/48 Page 3 of 8 Periapical region: periodontal ligament space widened, periapical change, apical radiolucency, root-tip-resection. prosthetic treatment (crowns/partial crowns), combin- ation of findings: filling or restoration with caries in the sense of a primary or secondary caries, as well as im- plant treatment. Because of irregular and imprecise de- tails or information, respectively, the sensitivity test, initial caries lesions and the periodontal situation were not taken into account. prosthetic treatment (crowns/partial crowns), combin- ation of findings: filling or restoration with caries in the sense of a primary or secondary caries, as well as im- plant treatment. Because of irregular and imprecise de- tails or information, respectively, the sensitivity test, initial caries lesions and the periodontal situation were not taken into account. Further findings: impacted and ectopic tooth, root resi- due, shadowing of the maxillary sinus. Participants A total of 302 patients’ records were available for the period under investigation. According to the inclusion criteria for the x-rays 275 patients’ records could be ex- amined and analyzed. The age of the subjects ranged from 25 to 35 years. Results The assessment of the DPRs was carried out under standardized conditions in a shaded room with an x-ray film viewer (light box) capable of functioning under such conditions; the clinical findings were not known to this investigator. The inclusion and exclusion criteria for the panoramic radiographs are given in Table 1. Each DPR was assessed twice at a 3-week interval. In the case of deviation between the two assessments, the final finding was established by a third assessment. Radiographic examination The findings of the DPRs were taken by a sole inves- tigator (MS) who was calibrated in advance (Kappa value ≥0.8). Statistical analysis Statistical analysis was performed with the commercially available program SPSS 14.0 (SPSS, Inc., Chicago, IL, USA). The statistical comparison of the clinical and radiological findings was made using the paired-rank test. The level of significance was set at p < 0.05. (Parameter for findings: in accordance with Material and Methods, n.s.: not significant = p > 0.05). Methods The study was a cross-sectional investigation based on available patients’ records (clinical findings and x-ray findings (DPR)) from three dental practices of the German Forces (locations: Wilhelmshaven (navy), Mun- ster (army) and Köln-Wahn (air-force).a Authorization • overlaying effects Clinical examination For the assessment of the x-rays, the same parameters as for the clinical findings were used. Fillings and resto- rations were judged to be sufficient if there was a smooth transition between restoration and tooth. Over- hanging fillings and crown margins, as well as radio- lucent gaps between restoration and tooth indicating deficient marginal fit in the sense of a secondary caries were judged to be insufficient. Implants were simply reg- istered without assessment. The results of the clinical findings are given in Table 2. Acting on the assumption of 32 teeth in the perman- ent dentition per participant, in total there were max- imally 8,800 teeth to assess, of these 1,007 teeth were clinically lacking, 553 of these were wisdom teeth. 4,650 teeth were classed as being healthy (52.8%), and 318 teeth showed carious lesions (3.6%). 2,424 teeth (27.5%) showed sufficient fillings (n = 1978) or sufficient pros- thetic restoration (n = 446). In contrast, 128 teeth (1.5%) were treated insufficiently (restorative: n = 125, prosthet- ically: n = 3). 136 (1.5%) teeth showed fillings or restora- tions as well as caries lesions. In the case of 121 teeth (1.38%), the clinical examination revealed “suspected caries”: 16 teeth had been replaced by an implant. In addition, various additional findings were recorded: Endodontic findings: proper root canal filling, deficient root canal filling based on the following criteria: insuffi- ciently filled or overfilled, coronal leakage, improper homogeneity of the filling material. Table 2 Summary of the dental findings and x-ray findings, as well as the number of agreements Parameter for findings Clinical findings (n = 8800)/[%]) X-ray findings (n = 8800/[%]) Number of accordance [%] Significance level (p value) Missing teeth 1007 (11.4) 1011 (11.5) 996 (99.6) n.s. Healthy teeth 4650 (52.8) 4858 (55.2) 4587 (95.7) n.s. Carious teeth 318 (3.61) 212 (2.41) 176 (66.6) p < 0.05 Tooth sufficiently filled 1978 (22.5) 2009 (22.8) 1873 (98.5) n.s. Tooth insufficiently filled 125 (1.42) 116 (1.32) 71 (92.8) p < 0.05 Tooth sufficiently prosthetically treated 446 (5.07) 429 (4.88) 425 (96.2) n.s. Tooth insufficiently prosthetically treated 3 (0.003) 16 (0.018) 3 (18.8) p < 0.05 Implant 16 (0.018) 16 (0.018) 16 (100.0) n.s. Combination (restoration and carious) 136 (1.55) 133 (1.51) 91 (97.8) n.s. Clinically: suspected caries 121 (1.38) – – – (*Parameter for findings: in accordance with Material and Methods, n.s.: not significant = p > 0.05). Comparison of clinical and radiographic findings It was possible to establish agreement in 93.6% of cases of all parameters between clinical and radiological find- ings (Table 4). There was no significant difference be- tween the two methods of assessment (p > 0.05). In 6.4% of the teeth discrepancies between the two method were found, and these were distributed mainly regarding the following findings: caries and insufficient restorative or prosthetic treatment (Tables 2 and 4). Within this group, there were significant differences be- tween clinical findings and x-ray findings (p < 0.05; Table 2). Clinically, there were 130 carious lesions present that could not be established by the x-ray find- ings. In contrast, 23 lesions in the sense of a carious le- sion were only identified by means of DPR findings. In the assessment of the existing restorations, 54 teeth showed a clinically sufficient filling (n = 43) or sufficient prosthetic treatment (n = 11) that were judged with ref- erence to x-rays to be insufficient. This contrasted with 51 clinically insufficient fillings that were assessed by x- rays to be sufficient. Details of additional findings are summarized in Table 3. Endodontic findings and assessment of the periapical region: 259 of all teeth evaluated (2.9%) showed root canal fillings; in accordance with the laid-down criteria, of these teeth 184 (71.0%) were assessed to have suffi- cient endodontic treatment. In the case of the root canal fillings considered to be insufficient. (n = 75), the major- ity (n = 42, 56%) showed inadequately filled canals. Inde- pendent of endodontic treatment and/or the quality of the existing root canal filling, in total, 145 teeth showed periapical changes (1.6%). About half of them (n = 72; 0.8%) were root-canal-filled teeth. For the other parameters (healthy tooth, filling insuffi- cient, prosthetic sufficient), there was no significant dif- ference between the two methods of assessment (p > 0.05; Table 2). Clinical examination dental findings and x-ray findings, as well as the number of agreements Table 2 Summary of the dental findings and x-ray findings, as well as the number of agreements Page 4 of 8 Moll et al. BMC Oral Health 2013, 13:48 http://www.biomedcentral.com/1472-6831/13/48 Radiographic examination resected (18.7%), and 3 root residues (1.8%) were found. In five cases, the maxillary sinus showed shadowing (3%). The results of the radiological findings are shown in Table 2. Of 8,800 maximally possible teeth, 1,011 teeth (11.5%) were missing; of which 553 were wisdom teeth. More than 50% of the teeth were considered to be healthy (n = 4,858). With reference to the x-ray findings carious lesions were found in 212 teeth (2.4%). 2,009 teeth were sufficiently treated with fillings (22.8%) and 429 pros- thetically (4.9%). In contrast, 132 (1.5%) teeth showed in- sufficiently restorative (n = 116) or prosthetical (n = 16) treatment. 133 (1.5%) teeth had fillings or restorations as well as caries. In total, 16 implants were present. (RCF: root canal filling). Comparison of clinical and radiographic findings Further findings: In total, it was possible to establish 171 additional abnormalities/findings: 132 impacted or ectopic wisdom teeth (79.5%), 31 teeth were root-tip Table 3 Number of additional findings (n) in the x-rays (DPR) Type of additional findings Number of additional findings [%] Endodontic (n = 8800) Total 259 (2.9%) sufficient 184 (71%) insufficient 1 insufficiently filled 42 (16%) 2 insufficient homogeneity 2 (1%) 3 over-filled 5 (2%) 4 coronal leakage 11 (4%) combination of 1 - 4 15 (6%) Periapical region (n = 8800) Total 145 (1.7%) periodontal ligament space widened 54 (37%) periapical change 4 (3%) apical raduiolucency teeth with RCF 72 (50%) teeth without RCF 15 (10%) Other (n = 8800) Total 171 (1.9%) impacted or ectopic teeth 132 (77%) root residue 3 (2%) root-tip-resection 31 (18%) shadowing of the maxillary sinus 5 (3%) (RCF: root canal filling). Table 3 Number of additional findings (n) in the x-rays (DPR) Moll et al. Assessment of healthy and carious teeth With an agreement of 93.6%, the results showed no sig- nificant differences between the two investigative methods (p > 0.05). In the assessment of “caries”, as well as insufficient fillings and prosthetic restorations, there were, however, to some extent, substantial differences between clinical and radiological findings. In addition, the x-rays revealed a number of additional findings: end- odontic findings (n = 259), changes in the periapical re- gion (n = 145) and others (n = 171), such as for example impacted or ectopic teeth and shadowing of the maxil- lary sinus. In this study, the parameter “carious teeth” showed a significant difference (p < 0.05) between clinical and x- ray findings. Therefore, the sensitivity for carious teeth is lower then for healthy teeth (healthy: 95.7%, carious: 66.6%). In contrast to the study of Valachovic et al. (1986), we obtained better results with regard to the agreement between clinical and x-ray findings in the as- sessment of healthy and carious teeth. For carious teeth, those authors found with 54%, only a small sensitivity (agreement) of the x-ray findings with the clinical find- ings [10]. Even more unfavorable were the results of Moleander et al. (1993) with 47% [11]. One suspects that the better results in the current study can be traced back to the technical advances in dental radiology. Particu- larly for the detection of interproximal caries lesions, nowadays x-rays, DPRs and specially bitewings, are con- sidered to be the most suitable diagnostic aid. However, Comparison of clinical and radiographic findings BMC Oral Health 2013, 13:48 http://www.biomedcentral.com/1472-6831/13/48 Page 5 of 8 Page 5 of 8 Table 4 Comparison between clinical (C) and radiological findings (R) using the paired-rank-test C 0 1 2 3 4 5 6 7 8 9 Total R 0 996 7 4 0 0 4 0 0 0 0 1011 11.3% 0.08% 0.05% 0.00% 0.00% 0.05% 0.00% 0.00% 0.00% 0.00% 11.5% 1 7 4587 130 18 2 0 0 0 3 111 4858 0.08% 52.1% 1.48% 0.20% 0.02% 0.00% 0.00% 0.00% 0.03% 1.26% 55.2% 2 0 23 176 5 1 0 0 0 0 7 212 0.00% 0.26% 2.0% 0.06% 0.01% 0.00% 0.00% 0.00% 0.00% 0.08% 2.41% 3 1 29 5 1873 51 6 0 0 41 3 2009 0.01% 0.33% 0.06% 21.3% 0.58% 0.07% 0.00% 0.00% 0.47% 0.03% 22.8% 4 0 2 0 43 71 0 0 0 0 0 116 0.00% 0.02% 0.00% 0.49% 0.81% 0.00% 0.00% 0.00% 0.00% 0.00% 1.32% 5 1 0 0 2 0 425 0 0 1 0 429 0.01% 0.00% 0.00% 0.02% 0.00% 4.83% 0.00% 0.00% 0.01% 0.00% 4.88% 6 1 0 0 1 0 11 3 0 0 0 16 0.01% 0.00% 0.00% 0.01% 0.00% 0.13% 0.03% 0.00% 0.00% 0.00% 0.18% 7 0 0 0 0 0 0 0 16 0 0 16 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.18% 0.00% 0.00% 0.18% 8 1 2 3 36 0 0 0 0 91 0 133 0.01% 0.02% 0.03% 0.41% 0.00% 0.00% 0.00% 0.00% 1.03% 0.00% 1.51% Total 1007 4650 318 1978 125 446 3 16 136 121 8800 11.4% 52.8% 3.61% 22.5% 1.42% 5.07% 0.03% 0.18% 1.55% 1.38% 100% (0 = tooth missing, 1 = healthy, 2 = caries, 3 = filling sufficient, 4 = filling insufficient, 5 = prosthetics sufficient, 6 = prosthetics insufficient, 7 = implant, 8 = combination of 2 and 3-6, 9 = suspected caries; number of accordance). Discussion in the context of a comprehensive dental examination. Accordingly, in a large number of cases, it would have been possible to have dispensed with x-rays. Only in the case of 6.4% of teeth there was no agreement between the two methods of investigation. Other studies have also raised the same question [14,15]. The aim of the study was to evaluate the extent to which clinical findings and x-ray findings based on dental panoramic radiography (DPR) correspond with and dif- fer from one another. Interpretation of the results and comparison with the international literature The high level of agreement of both methods of assess- ment according to the criteria for evaluation laid down previously is noteworthy and once more questions the preparation of a DPR as a supplementary diagnostic tool Moll et al. BMC Oral Health 2013, 13:48 http://www.biomedcentral.com/1472-6831/13/48 Moll et al. BMC Oral Health 2013, 13:48 http://www.biomedcentral.com/1472-6831/13/48 Page 6 of 8 the prerequisite for this is that, in the preparation of the x-rays, no errors due to the method or production occur [16] and that the x-rays show no deficiencies in quality [5]. Certainly, bitewings are the radiographic method in- dicated to assess interproximal carious lesions [17]. According to Rushton et al. (2002) bitewing radiographs will reduce the diagnostic yield identified solely by DPR [18]. Therefore, DPRs are unsuitable for carious diagnos- tic. The finding “carious lesion” showed only in 66.6% of cases an agreement between the two investigative methods. Douglass et al. (1986) found comparable re- sults: in 60% of cases certain recognition was achieved with a combination of DPR and bitewing radiographs [19]. Two recent studies in which, in addition, one or more investigators were calibrated, showed a comparably high agreement in the identification of carious lesions [20,21]. The values obtained by Hopcraft and Morgan (2005) using x-rays were, for interproximal caries recog- nition, around 93% to 97% and for occlusal carious le- sions 33% to 42%; however, in addition to DPR, bitewing radiographs were also included in this study [20]. In the study presented here, more carious lesions were found in the clinical examination than in the x-ray findings. This result supports earlier investigations in which, like- wise, the clinical finding was superior to the findings based on DPR [22-24]. In contrast, Heners and Riotte (1978) found more carious lesions using bitewing radio- graphs than in the clinical examination [25]. Therefore, the results of this study can not be compared with the results of our study. Deductive, the results of our study shows that DPR is not a good diagnostic tool to detect carious lesions. For revealing carious lesions Rushton et al. (2002) prefer posterior bitewing radiographs [18]. should be considered that, because of the fuzziness in the DPR, the margins could not always be unambigu- ously evaluated. These points could explain the discrep- ancy between the two investigative methods in relation to the assessment of “insufficient filling”, whereby in 92.8% of teeth an agreement could be found (Table 2). Interpretation of the results and comparison with the international literature In contrast to the assessment of fillings, “insufficient prosthetic” was more frequently made in the x-rays than clinically. A possible explanation for this could be that a clinical, visual-tactile judgement of the frequently sub- gingival located crown margins is possibly more difficult. Accordingly, in routine dental health check of adult pa- tients DPR is not necceassary. This is in accord with other authors [13-15,18]. Consequently, the hypothesis formulated in advance, that clinical and radiological findings show only small differences, was confirmed. At the same time, patho- logical findings (caries, insufficient filling) are more fre- quently uncovered clinically than by using x-rays. Whereas material properties of prosthetic restorations (e.g. precious metals, ceramics) have been well investi- gated, there is a lack of scientific data regarding the quality of provision of fixed artificial dentition from clin- ical practice; compared to other forms of artificial denti- tion, however, it was possible to record high probabilities of survival [27,28]. Taking into account the results presented here, the quality of fixed artificial den- tition and the possibility of their being assessable in x- rays appears to be subject to limitations; it is possible, that because of the two-dimensional presentation in x- rays the number of additional findings could have been overestimated. Assessment of the restorations The assessment of the DPR revealed a substantial num- ber of additional clinically non-conspicuous findings (Table 4). It is here that the strength of the DPR as a supplementary diagnostic aid to the clinical findings lies as, in this way, an additional/substantial gain in informa- tion can be achieved. At the same time, the advantages and disadvantages in the preparation of a DPR in the context of a risk/benefit analysis should be well weighed up. Overall, the differences between the two methods in the assessment of “insufficient filling” and “insufficient pros- thetic restoration” were greater than the agreement. To the best of our knowledge, there have been no data available in the literature, up until now, regarding this question. The extent to which the margin fit can be evaluated in x-rays is considered to be an important criterion for de- ciding whether there is sufficient or insufficient dental provision. The evaluation of the various possibilities of deficient/defective margin fit was carried out in the present study only in terms of “sufficient” or “insuffi- cient”. Material investigations have drawn attention to the fact that an adequate difference in opacity between restoration material (composite) and dental enamel must exist in order to assess quality of the filling [26]. For this reason, it may be possible that in the present study im- precise statements about the margin fit were mostly found in the case of composite restorations. Moreover, it In this study, because DPR provides an overview, it was possible to obtain in total 171 additional findings. Compared with other studies, that is certainly a low num- ber, but the distribution in relation to the corresponding findings (impacted teeth, teeth with root-tip-resection etc.) is, however, very similar when frequencies are com- pared [29,30]. Also, the association between periapical condition and sufficient root filling has already been discussed in some studies [31,32] and is confirmed by the results of this investigation. Accordingly, the second hy- pothesis – that by using DPR a large number of additional Page 7 of 8 Moll et al. BMC Oral Health 2013, 13:48 http://www.biomedcentral.com/1472-6831/13/48 x-ray findings, additional agreement with endodontic and periapical findings could arise. findings can be uncovered – was also confirmed. However, the quantity of additional findings nonetheless makes the DPR an important aid in dental diagnostics. Authors’ contributions MM i d h d MM interpreted the data and wrote the manuscript. MS carried out the radiological examination and performed the determination of the study data. CvS has been involved in revising the manuscript critically for important intellectual content and has given final approval of the version to be published. AZ performed the statistical analysis. EH conceived of the study, and participated in its design, and has been involved in drafting the manuscript. RM has made substantial contributions to conception and design of the study, DZ was the head of the study and has made substantial contributions to conception, design and coordination. All authors read and approved the final manuscript. Acknowledgements h h h k h The authors thanks the dental offices of the military base of the German Federal Armed Forces Cologne, Munster and Wilhelmshaven (Germany) and there teams for supporting the study. Assessment of the restorations Therefore, the preparation of a DPR including meticulous x-ray find- ings in the context of a first dental (check-up) examin- ation can be a helpful measure. In contrast, Rushton and Rushton (2012) in comparing 740 DPR and intraoral ra- diographs showed, that only in 32 DPR additional infor- mation were found [13]. The authors conclude that there is no reason for the use for DPRs in routine dental health examinations [13]. The European Guideline on Radiation in Dental Radiography Issue No. 136 supports this opinion [12]. Furthermore, the assessment of periodontal bone level was dispensed, because of the young age of the study population (25-35 years). In addition, in a first dental (check up) examination the PSR®/PSI is essential for ini- tial periodontal examination [33]. This finding was not available in all cases. Furthermore, Ziebolz et al. (2011) showed, that DPR is of no value in cases of initial peri- odontal problems [33]. Competing interests h h d l h Competing interests The authors declare that they have no financial or other relationships that might lead to competing interests. The authors declare that they have no financial or other relationships that might lead to competing interests. Conclusions There was no difference between clinical and x-ray find- ings. However, with regard to the assessment of carious as well as insufficiently filled or prosthetically treated teeth, there was a clear discrepancy between the two methods of investigation. At the same time, DPR find- ings were inferior to clinical findings in relation to the parameters “carious tooth” and “insufficient filling”. Ac- cordingly, the DPR gives the clinician no additional gain in information in this area. Therefore, it would have been possible to have dispensed with x-rays. Neverthe- less, additional x-ray findings were found. Implication for the practice The results presented in this study indicate that using comprehensive and good clinical dental diagnostics for the assessment of healthy/carious and/or sufficiently/in- sufficiently restored and prosthetically treated teeth, rou- tine DPR can be dispensed with. However, because of the additional and unintended findings, for correct diag- nosis and adequate therapy, the combination of clinical findings and x-ray findings is sensible. Endnote The investigation presented here is a practice-based clin- ical study. Overall, there is a lack of practice-related studies, thus, this study provides a scientific contribution from daily practice. aIn the German Forces, before each deployment abroad, dental findings are documented and x-ray find- ings recorded based on up-to-date dental panoramic radiography (DPR). The clinical findings were obtained under conditions to be found in a dental practice and were undertaken by a number of experienced investigators/skilled dentists with- out prior calibration and knowledge of the participants re- garding the study. In this way, a study-determined effect can be excluded. However, the fact needs to be taken into account that differences may have occurred as a result of individual subjectivity of the examining dentists, different levels of care and clinical experience in the recording of data. Overall, however, a high quality standard appears to predominate in the investigation as, despite the absence of calibration of the clinical investigators, there was a high level of agreement with the x-ray findings. In contrast to this, the x-ray evaluation was carried out with a high stand- ard following prior investigator calibration. This should en- sure that the x-ray findings were recorded completely and comprehensively, with the aim of establishing comparability and in order to assess the additional value of the DPR. Moreover, because of the large number of teeth investigated and strict selection criteria, the results can be viewed as be- ing representative. References 26. 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Cerqueira EM, et al: Genotoxic effects of X-rays on keratinized mucosa cells during panoramic dental radiography. Dentomaxillofac Radiol 2008, 37(7):398–403. 30. Rahn R, Czaja J: Fortuitous findings in panorama layer images. ZWR 1995, 104(2):104–106 [in German]. 5. Peretz B, et al: Common Errors in Digital Panoramic Radiographs of Patients with Mixed Dentition and Patients with Permanent Dentition. Int J Dent 2012:584138. 10.1155/2012/584138. Epub 2012 Feb 8. 31. Hülsmann M, Lorch V, Franz B: An investigation of the frequency and quality of root fillings. Dtsch Zahnarztl Z 1991, 46:296–299 [in German]. 32. Klimek J, Kockapan C, Borchert J: Frequency and quality of root canal fillings in the years 1983 and 1991. Dtsch Zahnarztl Z 1995, 50:154–156 [in German]. 6. Sitzmann F, Benz C: Single image status or orthopantomograph: statement of the DGZMK. Zahnarztl Mitt 1999, 89(1):33 [in German]. 7. Freitag V, Seidel W: On the frequency of secondary findings in panorama layer images. Dtsch Zahnarztl Z 1972, 27:993–996 [in German]. 33. Ziebolz D, Szabadi I, Rinke S, Hornecker E, Mausberg RF: Initial periodontal screen-ing and radiographic findings – A comparison of two methods to evaluate the perio-dontal situation. BMC Oral Health 2011, 11:3. 8. Nitsch JP: The routine use of the orthopantomograph as a diagnostic aid in dental practice. Dtsch Zahnarztl Z 1979, 34:231–232 [in German]. 9. Rushton VE, Horner K: The use of panoramic radiology in dental practice. J Dent 1996, 24(3):185–201. doi:10.1186/1472-6831-13-48 Cite this article as: Moll et al.: Comparison of clinical and dental panoramic findings: a practice-based crossover study. BMC Oral Health 2013 13:48. 10. Moll et al. BMC Oral Health 2013, 13:48 http://www.biomedcentral.com/1472-6831/13/48 Moll et al. BMC Oral Health 2013, 13:48 http://www.biomedcentral.com/1472-6831/13/48 Moll et al. BMC Oral Health 2013, 13:48 http://www.biomedcentral.com/1472-6831/13/48 Page 8 of 8 Page 8 of 8 Author details 1 1Department of Preventive Dentistry, Periodontology and Cariology, University Medical Centre, Robert-Koch Str. 40, Goettingen D-37075, Germany. 2Department of Oral and Maxillofacial Surgery, Hanover Medical School, Hanover, Germany. 3Dental office, German Armed Forces, Munster, Germany. 4Department of Medical Statistics, Hanover Medical School, Hanover, Germany. In the context of the clinical dental examination, only sporadically sensitivity tests were undertaken, so that these could not be included in the study. It is therefore to be assumed that in making the comparison with the Received: 8 June 2013 Accepted: 19 September 2013 Published: 26 September 2013 References Valachovic RW, et al: The use of panoramic radiography in the evaluation of asymptomatic adult dental patients. Oral Surg Oral Med Oral Pathol 1986, 61:289–296. 11. Molander B, et al: Comparison of panoramic and intraoral radiography for the diagnosis of caries and periapical pathology. Dentomaxillofac Radiol 1993, 22:28–32. 12. European Union European Commission: Radiation Protection 136. European guidelines on radiation protection in dental radiology. Luxembourg: Office for Official Publications of the EC; 2004. 13. Rushton MN, Rushton VE: A study to determine the added value of 740 screening panoramic radiographs compared to intraoral radiography in the management of adult (>18 years) dentate patients in a primary care setting. J Dent 2012, 40:661–669. 14. Rushton VE, Horner K, Worthington HV: Routine panoramic radiography of new adult patients in general dental practice: relevance of diagnostic yield to treatment and identification of radiographic selection criteria. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002, 93(4):488–495. 15. Rushton VE, Horner K, Worthington HV: Screening panoramic radiology of adults in general dental practice: radiological findings. Br Dent J 2001, 190(9):495–501. 16. Rushton VE, Horner K, Worthington HV: The quality of panoramic radiographs in a sample of general dental practices. Br Dent J 1999, 186(12):630–633. 17. Pitts NB: Modern concepts of caries measurement. J Dent Res 2004, 83:C43–C47. Spec No C. 18. Rushton VE, Horner K, Worthington HV: Screening panoramic radiography of new adult patients: diagnostic yield when combined with bitewing radiography and identification of selection criteria. Br Dent J 2002, 192:275–279. 19. Douglass CW, et al: Clinical efficacy of dental radiography in the detection of dental caries and periodontal diseases. Oral Surg Oral Med Oral Pathol 1986, 62(3):330–339. 20. Hopcraft MS, Morgan MV: Comparison of radiographic and clinical diagnosis of approximal and occlusal dental caries in a young adult population. Community Dent Oral Epidemiol 2005, 33(3):212–218. References Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit 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: 21. Galcerá Civera V, et al: Clinical and radiographic diagnosis of approximal and occlusal dental caries in a low risk population. Med Oral Patol Oral Cir Bucal 2007, 12(3):E252–E257. 22. Sonnabend E, Ring A: Die Bedeutung der Panoramadarstellung in der Zahnerhaltung und Parodontologie. Dtsch Zahnarztl Z 1972, 27:965–969 [in German]. 23. Hurlburt CE, Wuehrmann AH: Comparison of interproximal carious lesion detection in panoramic and standard intraoral radiography. J Am Dent Assoc 1976, 93:1154–1158. 24. Muhammed AH, Manson-Hing LR: A comparison of panoramic and intraoral radiographic surveys in evaluating a dental clinic population. Oral Surg Oral Med Oral Pathol 1982, 54:108–117. 24. Muhammed AH, Manson-Hing LR: A comparison of panoramic and intraoral radiographic surveys in evaluating a dental clinic population. Oral Surg Oral Med Oral Pathol 1982, 54:108–117. 25. Heners M, Riotte P: Experimental studies on the radiographic presentation of proximal caries in the lateral region. Dtsch Zahnarztl Z 1978, 33:228–230 [in German]. 25. Heners M, Riotte P: Experimental studies on the radiographic presentation of proximal caries in the lateral region. Dtsch Zahnarztl Z 1978, 33:228–230 [in German].
https://openalex.org/W4394976162	https://link.springer.com/content/pdf/10.1007/JHEP04(2024)120.pdf	English	null	Holographic Weyl anomalies for 4d defects in 6d SCFTs	The Journal of high energy physics/The journal of high energy physics	2,024	cc-by	25,743	Published for SISSA by Springer Received: December 7, 2023 Revised: February 20, 2024 Accepted: March 15, 2024 Published: April 19, 2024 Received: December 7, 2023 Revised: February 20, 2024 Accepted: March 15, 2024 Published: April 19, 2024 Keywords: AdS-CFT Correspondence, Anomalies in Field and String Theories, Supergravity Models Published for SISSA by Springer Open Access, © The Authors. Article funded by SCOAP3. 1See [3] for a recent review of defects of various (co-)dimension in QFTs. Holographic Weyl anomalies for 4d defects in 6d SCFTs https://doi.org/10.1007/JHEP04(2024)120 Contents 1 Introduction 1 2 Review 4 2.1 Defect Weyl anomalies 5 2.2 11d SUGRA solutions 7 3 Holographic stress-energy tensor one-point function 12 3.1 Two-charge solutions 14 3.2 Electrostatic solutions 16 4 Defect sphere EE and the defect A-type anomaly 19 4.1 Two-charge solutions 20 4.2 Electrostatic solutions 21 5 On-shell action 25 6 Discussion 29 6.1 Future directions and open questions 32 A Fefferman-Graham coordinates 34 A.1 Two-charge solutions 34 A.2 Electrostatic solutions 36 B Regulating the on-shell action 39 B.1 AdS7 × S4 39 B.2 Renormalized AdS5 volume 40 Contents 1 Introduction 1 2 Review 4 2.1 Defect Weyl anomalies 5 2.2 11d SUGRA solutions 7 3 Holographic stress-energy tensor one-point function 12 3.1 Two-charge solutions 14 3.2 Electrostatic solutions 16 4 Defect sphere EE and the defect A-type anomaly 19 4.1 Two-charge solutions 20 4.2 Electrostatic solutions 21 5 On-shell action 25 6 Discussion 29 6.1 Future directions and open questions 32 A Fefferman-Graham coordinates 34 A.1 Two-charge solutions 34 A.2 Electrostatic solutions 36 B Regulating the on-shell action 39 B.1 AdS7 × S4 39 B.2 Renormalized AdS5 volume 40 2.2 11d SUGRA solutions JHEP04(2024)120 A.2 Electrostatic solutions B Regulating the on-shell action B.2 Renormalized AdS5 volume Holographic Weyl anomalies for 4d defects in 6d SCFTs JHEP04(2024)120 Pietro Capuozzo ,a John Estes ,b Brandon Robinson c and Benjamin Suzzoni a aSTAG Research Centre, University of Southampton, University Road, Southampton, SO17 1BJ, U.K. bDepartment of Chemistry and Physics, SUNY Old Westbury, Old Westbury, NY, United States cINFN, sezione di Milano-Bicocca, Piazza della Scienza 3, I-20126 Milano, Italy E-mail: p.capuozzo@soton.ac.uk, estesj@oldwestbury.edu, brandon.robinson@mib.infn.it, b.suzzoni@soton.ac.uk Pietro Capuozzo ,a John Estes ,b Brandon Robinson c and Benjamin Suzzoni a aSTAG Research Centre, University of Southampton, University Road, Southampton, SO17 1BJ, U.K. bDepartment of Chemistry and Physics, SUNY Old Westbury, Old Westbury, NY, United States cINFN, sezione di Milano-Bicocca, Piazza della Scienza 3, I-20126 Milano, Italy E-mail: p.capuozzo@soton.ac.uk, estesj@oldwestbury.edu, brandon.robinson@mib.infn.it, b.suzzoni@soton.ac.uk Pietro Capuozzo ,a John Estes ,b Brandon Robinson c and Benjamin Suzzoni a aSTAG Research Centre, University of Southampton, University Road, Southampton, SO17 1BJ, U.K. bDepartment of Chemistry and Physics, SUNY Old Westbury, Old Westbury, NY, United States cINFN, sezione di Milano-Bicocca, Piazza della Scienza 3, I-20126 Milano, Italy E-mail: p.capuozzo@soton.ac.uk, estesj@oldwestbury.edu, brandon.robinson@mib.infn.it, b.suzzoni@soton.ac.uk Abstract: In this note, we study 1/4- and 1/2-BPS co-dimension two superconformal defects in the 6d N = (2, 0) AN−1 SCFT at large N using their holographic descriptions as solutions of 11d supergravity. In this regime, we are able to compute the defect contribution to the sphere entanglement entropy and the change in the stress-energy tensor one-point function due to the presence of the defect using holography. From these quantities, we are then able to unambiguously compute the values for two of the twenty-nine total Weyl anomaly coefficients that characterize 4d conformal defects in six and higher dimensions. We are able to demonstrate the consistency of the supergravity description of the defect theories with the average null energy condition on the field theory side. For each class of defects that we consider, we also show that the A-type Weyl anomaly coefficient is non-negative. Lastly, we uncover and resolve a discrepancy between the on-shell action of the 7d 1/4-BPS domain wall solutions and that of their 11d uplift. ArXiv ePrint: 2310.17447 Open Access, © The Authors. Article funded by SCOAP3. 1 Introduction Knowing the spectrum of local operators in a given quantum field theory (QFT) is insufficient to uniquely specify it in field theory space [1], and so operators with non-trivial extension along submanifolds embedded in the background spacetime (‘defects’) play an important role in classifying QFTs [2]. However, the way that the presence of these defects affects, say, correlation functions of local operators depends on the dimension d and geometry of the background manifold Md, the co-dimension d −d and embedding of the d-dimensional defect submanifold Σd, and the couplings between ambient and defect degrees of freedom.1 Thus, it is crucial to characterize allowable defects in a given theory and precisely determine how ambient physical observables change under the deformation by defect operators.f In this effort, some of the most powerful tools that we have come from imposing symmetries on both the ambient and defect theories. The ambient field theories we consider are 6d, supersymmetric, and invariant under 6d flat-space conformal symmetry SO(6, 2); superconformal field theories (SCFTs). The defects that we study in this work are supported on embedded co-dimension 2 submanifolds, Σ4 ,→M6, that will preserve at least 1/4 of the – 1 – total supersymmetries, i.e. N ≥1 4d supersymmetry, as well as an SO(4, 2)×U(1)N ⊂SO(6, 2) global symmetry representing the defect conformal symmetry and U(1)N rotations in M6/Σ4. We will refer to these theories as defect [super]conformal field theories (D[S]CFTs). In the following, we will focus on ambient theories that are maximally superconformal N = (2, 0) SCFTs with gauge algebra AN−1 in the large N limit and the 1/4- and 1/2-BPS co- dimension 2 defects that they support. Despite the highly restrictive symmetries imposed, 6d N = (2, 0) SCFTs and their defect operators pose a challenge to direct study. We know from the worldvolume theory of a stack of coincident M5-branes [4] or M5-branes probing ADE singularities [5] that 6d N ≥(1, 0) SCFTs exist, but generally they have no known Lagrangian description. We also know that 6d SCFTs constructed from M-theory support 4d BPS defect operators engineered at the intersection of orthogonal stacks of M5 branes. Since we often lack a Lagrangian description, our efforts to characterize these d = 4 defect (super)conformal field theories, or D(S)CFTs, are limited to analyzing their global properties using techniques such as anomaly inflow (e.g. [6]) and chiral algebra methods [7]. ( ) [ ] 3For probe branes wrapping AdS5 ⊂AdSd+1, all of 23 of the parity even anomalies can be holographically computed [9] using the work of Graham and Reichert in [12]. 2These 29 terms include 6 terms that break parity on the defect submanifold. The limit case of a co-dimension 1 defect in 5d has 12 (including 3 parity odd) terms in the Weyl anomaly [9, 10]. 2These 29 terms include 6 terms that break parity on the defect submanifold. The limit case of a co-dimension 1 defect in 5d has 12 (including 3 parity odd) terms in the Weyl anomaly [9, 10]. 3For probe branes wrapping AdS5 ⊂AdSd+1, all of 23 of the parity even anomalies can be holographically computed [9] using the work of Graham and Reichert in [12]. 1 Introduction – 2 – aΣ d2 Two-charge N3 24 (1 −y2 +) −N3 6 (q1 + q2) Electrostatic N3 32 −1 96 P a 1+2ka k2a N3 a + nP b=a+1 Nakb N2 a k2a + 3 N2 b k2 b ! −1 24 N3 −P a N3 a k2a Table 1. Holographic defect Weyl anomaly coefficients for the 11d uplift of the 1/4-BPS, two-charge domain wall solution and the 1/2-BPS electrostatic solutions. Table 1. Holographic defect Weyl anomaly coefficients for the 11d uplift of the 1/4-BPS, two-charge domain wall solution and the 1/2-BPS electrostatic solutions. JHEP04(2024)120 The final results of our analysis are collected in table 1. On the first line, the two-charge defect Weyl anomaly coefficients are expressed in terms of the charges q1, q2 and of the location y+, itself expressible in terms of the charges, where the geometry of the SUGRA solution either smoothly caps off or ends on a conical deficit. On the second line, we display the anomalies for the electrostatic solutions, which holographically describe defects in the 6d AN−1 N = (2, 0) SCFT labelled equivalently by a Lie algebra homomorphism ϑ : sl(2) →su(N), the choice of Levi subalgebra l ⊂su(N) associated with the Levi subgroup L = S(U(N1) × . . . U(Nn)), or the Young diagram corresponding to the partition of N = Pn a=1 Na. Additionally, the construction of the electrostatic solutions allows for Zka orbifolds at the location of the ath stack of M5-branes, which in dimensionally reducing to a 4d theory are interpreted as monopole charges for the U(Na) factors. The parameters Na and ka completely determine both aΣ and d2 for the electrostatic solutions. The results that we obtain for these d = 4 defect anomalies have implications and open up questions beyond their roles in 6d SCFTs. Under the (partially) twisted dimensional reduction on genus-g Riemann surface Cg either with Σ4 wrapping two legs along Cg or with Cg orthogonal to Σ4 in M6, the theory containing a defect dual to the electrostatic solution descends to a 4d class−S N = 2 SCFT [19, 20] deformed by a d = 2 surface defect [21, 22] or by (possibly irregular [23–25]) punctures on its UV curve. 1 Introduction That said, there is a tremendous amount that we can learn about the defect theory by studying its conformal anomalies. JHEP04(2024)120 As with any systems preserving an SO(d, 2) global conformal symmetry, putting the ambient theory on a curved Md results in a non-trivial Weyl anomaly. Crucial to our understanding of DCFTs, the theory supported on Σd ,→Md has its own defect-localized contributions to the total Weyl anomaly that are sensitive to both the intrinsic submanifold geometry and its embedding in the ambient space. The resulting defect Weyl anomaly can be far more complicated than that of an ordinary d-dimensional theory. For example, it is common knowledge that the Weyl anomaly in d = 4 is a combination of an ‘A-type’ anomaly ∼aE4, where E4 is the 4d Euler density, and a ‘B-type’ anomaly ∼c\|W\|2 with Wµνρσ denoting the Weyl-tensor [8]. On the other hand, it was recently discovered in [9] that the Weyl anomaly of a d = 4 defect in an ambient theory with d ≥6 has a total of 29 terms.2 The challenge thus far has been finding tractable, non-trivial d = 4 defect systems beyond free theories (e.g. [11]) in which any of the 29 available defect Weyl anomalies can be computed.3 In light of recently discovered 11d supergravity (SUGRA) solutions that holographically describe certain d = 4 BPS defects in 6d SCFTs [13, 14], we have a window on strongly coupled, non-Lagrangian defect systems that can be approached with standard tools in holography to compute quantities known to be controlled by defect anomalies. In this work, we study both the 1/4-BPS ‘two-charge’ solutions in 11d constructed as the uplift of domain wall solutions in 7d gauged SUGRA and the 1/2-BPS ‘electrostatic’ solutions for bubbling geometries [15, 16] built along the lines of those in [17, 18] but with non-compact internal spaces. By holographically computing the one-point function of the stress-energy tensor and the flat defect contribution to the entanglement entropy (EE) of a spherical region co-original with the defect, we will be able to extract two of the 29 possible defect Weyl anomaly coefficients. In doing so, we find two independent pieces of data that characterize these defect systems. 1 Introduction Indeed, due to the non-trivial nature of the fibration via which the 11d SUGRA solution is obtained, the requirement that the 11d spacetime be asymptotically locally in FG parametrization results in a redefinition of the angular coordinates which mixes the U(1) isometry of the 7d spacetime and the U(1) R-symmetry. In the 7d picture, this coordinate redefinition corresponds to a large gauge transformation which maps the theory to a singular gauge. Crucially, the uplift resolves this singularity, thus producing a solution which is perfectly regular from an 11d perspective. When performing the computation of the on-shell action in the original 7d coordinates, which do not manifest the asymptotic local AdS7 × S4 structure of the solution, eq. (5.16) picks up a deformation which recovers precisely the result of [13], up to normalization factors.i At first sight, this result appears to be in tension with the computation of the on-shell action of 7d N = 4 gauged SUGRA performed in [13]. The origin of this discrepancy can be identified in the parametrization of the bulk integrand. Indeed, due to the non-trivial nature of the fibration via which the 11d SUGRA solution is obtained, the requirement that the 11d spacetime be asymptotically locally in FG parametrization results in a redefinition of the angular coordinates which mixes the U(1) isometry of the 7d spacetime and the U(1) R-symmetry. In the 7d picture, this coordinate redefinition corresponds to a large gauge transformation which maps the theory to a singular gauge. Crucially, the uplift resolves this singularity, thus producing a solution which is perfectly regular from an 11d perspective. When performing the computation of the on-shell action in the original 7d coordinates, which do not manifest the asymptotic local AdS7 × S4 structure of the solution, eq. (5.16) picks up a deformation which recovers precisely the result of [13], up to normalization factors.i JHEP04(2024)120 The present work is structured as follows. In section 2, we first review the pertinent aspects of Weyl anomalies for 4d defects and highlight their connection to physical quantities that we will compute in later sections. We will also briefly review the solutions in 11d SUGRA that holographically describe 1/4-BPS and 1/2-BPS co-dimension 2 defects in 6d SCFTs. 1 Introduction For example in the case of Σ4 wrapping Cg = T2, the 4d description is of Gukov-Witten defects in N = 4 SU(N) super-Yang Mills theory [26, 27], whose defect Weyl anomalies are known [28–30]. While the precise map between the 29 defect anomalies and the two independent Weyl anomalies of a surface operator in 4d [31] or the central charges of the 4d SCFT itself is unknown at present, our results provide some insight into how some of the defect data in 6d is reorganized into 4d (defect) Weyl anomalies. For example, from table 1 and the results of [18], we see an interesting relation between the central charge c4d of the dimensionally reduced 4d N = 2 SCFT and the A-type anomaly 1/2-BPS co-dimension 2 defect in the 6d AN−1 N = (2, 0) SCFT dual to the electrostatic solutions: aΣ = N3 32 + c4d. Additionally, we evaluate the action of 11d SUGRA on the uplift of the two-charge solutions. The Hodge dual of the four-form flux is closed, which allows us to recast the on-shell action as an integral over the 10d boundary defined by u = 0, where u is the AdS7 radial direction in Fefferman-Graham (FG) gauge. The volume of the 10d leaves normal to u diverges as u →0. In fact, it is divergent also at non-zero u, due to the infinite volume of the AdS5 subspacetime contained within each leaf. We regulate the first divergence – 3 – by subtracting from the on-shell action the contribution of the AdS7 × S4 vacuum, whose asymptotics match those of the uplift of the two-charge solutions. Finally, we regulate the polynomial divergences in the AdS5 volume, and extract the prefactor of the remaining logarithmic divergence, which is a universal quantity. Our result is in given in eq. (5.16), which we reproduce here for the reader’s convenience: S(ren) OS log = N3(4q1q2 −2(q1 + q2)y+(1 −y+) + 5y+(1 −y2 +)) 1920y+ . (1.1) (1.1) At first sight, this result appears to be in tension with the computation of the on-shell action of 7d N = 4 gauged SUGRA performed in [13]. The origin of this discrepancy can be identified in the parametrization of the bulk integrand. 1 Introduction In section 3, we compute the holographic stress-energy tensor one-point function for both the 1/4-BPS, two-charge solution and a generic 1/2-BPS electrostatic solution, which we use to find the defect B-type Weyl anomaly that we call d2. In section 4, we holographically compute the defect contribution to the EE of a spherical region, which we use to determine defect A-type Weyl anomaly, aΣ. In section 5 we compute the on-shell action for the 11d uplift of the two-charge solutions and highlight a discrepancy with the same computation done in the domain wall description in 7d N = 4 gauged SUGRA. In section 6, we discuss comparisons to field theory results and future directions. In addition, we collect some useful intermediate results in the appendices. In appendix A, we detail the asymptotic maps of the metrics for the solutions we consider into Fefferman- Graham form. Furthermore, in appendix B, we discuss the details of the regulating scheme for the on-shell action including the vacuum solution that we use in background subtraction as well as the renormalized volume of the AdS5 geometry. 2.1 Defect Weyl anomalies Up to a total derivative, the Weyl anomaly of an ordinary 4d CFT has two independent contributions,4 T µµ = 1 4π2 (−a4dE4 + c\|W\|2). (2.1) (2.1) The first term proportional to the Euler density E4 is the so-called “A-type” anomaly in the classification of [8], which exists in all even-dimensional CFTs and is unique in that it transforms as a total derivative under Weyl transformations. The second term given by the square of the Weyl tensor is a “B-type” anomaly. In arbitrary even-dimensional CFTs, there is generally a tower of B-type anomalies each of which is exactly Weyl invariant and built out of non-topological, rank- d 2 monomials in curvatures. The Weyl anomaly coefficients of a 4d CFT control correlation functions of the stress-energy tensor [33], and have strong upper and lower bounds on their ratio [34]; a4d also appears in the EE [35], and obeys an ‘a’-theorem under renormalization group (RG) flows [36, 37]. For 4d SCFTs with an R-symmetry, a4d and c are both related to the cubic and mixed R-anomalies through non-perturbative formulae [38]. JHEP04(2024)120 The Weyl anomaly of a conformal defect supported on Σd ,→Md is much richer due to the additional freedom of building submanifold conformal invariants out of not only the intrinsic curvature but also the normal bundle curvature, the pullback of curvature tensors from the ambient space, and the second fundamental form for the embedding. For conformal defects on Σ4 ,→Md of co-dimension 2 or greater,5 there are a total of 23 anomalies respecting submanifold parity [9].6 The complete form of the 4d defect Weyl anomaly is cumbersome, and so we will only display the parts relevant to the computations in the following sections (see eq. 3.1 of [9] for the full expression): T µµ\|Σ4 ⊃ 1 (4π)2 −aΣE4 + d2J2 + . . . . (2.2) (2.2) The first term is recognizable as the defect A-type anomaly proportional to the intrinsic Euler density, E4, of Σ4. The second term J2 is a B-type anomaly built out of a complicated linear combination of the submanifold pullback of the ambient curvatures, connection on the normal bundle, normal bundle curvature, and the second fundamental form for the embedding (see eq. 3.2 of [9] for the full expression). 4This basis is not unique, and one can exchange either E4 or W 2 for Branson’s Q-curvature [32] and a total derivative, which gives a basis for the 4d Weyl anomaly that is particularly convenient for holography. 5The limit case of co-dimension one is far more restricted and only leads to 9 parity even anomalies [9, 10]. 6There are an additional 6 parity odd defect Weyl anomalies, but as of yet, there are neither any known physical quantities in which they appear nor any no-go theorem to forbid them. 2 Review In this section, we will very briefly review some key background material in order to orient the subsequent computations. In the first subsection, we will introduce the two defect Weyl anomalies and discuss the physical quantities that they control, which will be the focus of – 4 – the computations to follow. In the second subsection, we will give a short overview of the two solutions to 11d SUGRA that will be the focus of our holographic study. the computations to follow. In the second subsection, we will give a short overview of the two solutions to 11d SUGRA that will be the focus of our holographic study. 2.1 Defect Weyl anomalies Importantly, J2 does not contain a term like the pullback of \|W\|2 or the square of the intrinsic Weyl tensor, and so is not analogous to the B-type anomaly of a standalone 4d CFT above. While it is unclear what physics the vast majority of terms in the full expression of the defect Weyl anomaly control, the two anomalies displayed above appear in two physical quantities that will be the primary focus of the following work. – 5 – The first quantity we will analyze is the one-point function of the stress-energy tensor. For a d-dimensional conformal defect embedded in a d-dimensional CFT, conformal symmetry preserved by the defect constrains the form of the one-point function of the stress-energy tensor a distance x⊥away from the defect to be of the form [39, 40] ⟨T ab⟩= −hT (d −d −1)δab \|x⊥\|d , ⟨T ij⟩= hT (d + 1)δij −d xi ⊥xj ⊥ \|x⊥\|2 \|x⊥\|d , (2.3) (2.3) where a, b index directions parallel to the defect and i, j label directions normal to the defect. By starting from the defect geometry Σ4 = R4 ,→Rd and then finding the totally transverse log divergent parts of the effective action in the presence of a linear ambient metric perturbation [9, 41], it can be shown that the normalization of the stress-energy tensor one-point function is determined by JHEP04(2024)120 hT = − Γ d 2 −1 π d 2 (d −1) d2 . (2.4) (2.4) In the case that we are particularly interested in for the following work, i.e. d = 6, hT = −1 5π3 d2 . (2.5) (2.5) There is a constraint on the sign of d2 that follows from the assumption that the average null energy condition (ANEC) holds in the presence of a defect. That is, the statement of the ANEC is that for any state \|Ψ⟩of a QFT, the expectation value of the stress-energy tensor projected along a null direction vµ in that state satisfies Z ∞ −∞ dλ ⟨Ψ\|Tµν\|Ψ⟩vµvν ≥0 , (2.6) (2.6) where λ parametrizes the null geodesic. From eq. 7In fact, it has recently been argued that the quantum null energy condition (QNEC), which is a stronger energy condition valid in any ambient QFT and reduces to ANEC in a certain limit (see e.g. [42]), holds in the presence of a defect [43]; putting d2 ≤0 and any other sign constraint derived from such energy conditions on even firmer ground. 2.1 Defect Weyl anomalies (2.4), we see that by taking the ambient theory to be a CFT and \|Ψ⟩to be the vacuum state of the theory deformed by a defect and orienting the null ray vµ to be parallel to the defect and separated by a distance x⊥in the normal direction, hT ≥0, which implies d2 ≤0 [9, 30].7 The other physical quantity controlled by defect Weyl anomalies that we will study below is the contribution to the EE of a spherical region of size R centered on Σ4 = R1,3 ,→R1,d−1. Following the same logic that formed the basis of the proof for 2d defects [30, 44], it was shown in [9] that for a 4d defect of co-dimension d −4, the coefficient of the universal, i.e. the log divergent, part of the defect EE is SEE[Σ] log = −4 aΣ + 1 4 (d −4)(d −5) d −1 d2 log R ϵ , (2.7) (2.7) where ϵ ≪R is a UV cutoff scale and log denotes dropping the leading non-universal divergences as well as the trailing scheme-dependent terms. 7In fact, it has recently been argued that the quantum null energy condition (QNEC), which is a stronger energy condition valid in any ambient QFT and reduces to ANEC in a certain limit (see e.g. [42]), holds in the presence of a defect [43]; putting d2 ≤0 and any other sign constraint derived from such energy conditions on even firmer ground. – 6 – For a conformal defect on Σ4, we will use a background subtraction scheme to isolate the defect contribution to the EE. That is, our computations below will use For a conformal defect on Σ4, we will use a background subtraction scheme to isolate the defect contribution to the EE. That is, our computations below will use 4aΣ + 2 5d2 = −R∂R (SEE[Σ] −SEE[∅]) \|R→0 , (2.8) (2.8) where SEE[∅] is the EE computed without the defect, i.e. the EE of the vacuum of the 6d ambient theory. Thus, combining the computation of d2 from ∆⟨Tij⟩with the result of eq. (2.8), we can compute the defect A-type anomaly unambiguously. Unlike d2, however, there is no constraint on the sign of aΣ. Indeed, in the simple case of a free scalar on a 5d manifold with a boundary, aΣ > 0 for Neumann (Robin) boundary conditions, while aΣ < 0 for Dirichlet [10].8 JHEP04(2024)120 8Note we are using the conventions for the definition of the 4d defect A-type anomaly aΣ as in [9], which differs from the defect A-type anomaly, a, in [10] by aΣ ↔−a/5760. 2.2 11d SUGRA solutions Two-charge solutions. We now briefly review the domain wall solutions in 7d N = 4 gauged SUGRA found in [13] and uplifted to 11d in [14]. The bosonic 7d gauged SUGRA action built from the metric g, two scalars Φ1,2 and two U(1) gauge fields A1,2 takes the following form: S = − 1 16πG(7) N Z d7x q \|g\| R −1 2\|∂µΦI\|2 −ˆg2V (Φ) −1 4 2 X I=1 e⃗aI ⃗ΦF 2 I ! . (2.9) (2.9) Using ⃗a1 = ( √ 2, p 2/5), ⃗a2 = (− √ 2, p 2/5), the potential is given by = ( √ 2, p 2/5), ⃗a2 = (− √ 2, p 2/5), the potential is given by V = −4e−1 2 (⃗a1+⃗a2)⃗Φ −2 e 1 2 (⃗a1+2⃗α2)⃗Φ + e 1 2 (2⃗a1+⃗α2)⃗Φ + 1 2e2(⃗a1+⃗a2)⃗Φ . (2.10) (2.10) The domain wall solution to eq. (2.9) describing the double analytic continuation of a charged black hole is given by ds2 7 = (yP(y)) 1 5 ds2 AdS5 + y(yP(y)) 1 5 4Q(y) dy2 + yQ(y) (yP(y)) 4 5 dz2, (2.11) (2.11) where the polynomials P, Q are given by where the polynomials P, Q are given by P(y) = H1(y)H2(y), (2.12a) Q(y) = −y3 + µy2 + ˆg2 4 P(y), (2.12b) (2.12a) (2.12b) where HI(y) = y2 + qI, I ∈{1, 2}. The gauge fields in this solution9 are given by AI = r 1 −µ qI qI HI(y) + aI dz . (2.13) (2.13) 8Note we are using the conventions for the definition of the 4d defect A-type anomaly aΣ as in [9], which differs from the defect A-type anomaly, a, in [10] by aΣ ↔−a/5760. f 9Note that, in general, the action in eq. (2.9) does not qualify as a consistent truncation of 11d supergravity. The 7d solutions considered here, however, are characterized by F1 ∧F2 = 0; this guarantees that their uplift produces consistent solutions of the 11d theory [45]. – 7 – In order to find BPS solutions, SUSY forces µ = 0. For both qI ̸= 0, the solutions are 1/4-BPS, while setting one charge, say q2, to zero allows for 1/2-BPS solutions. In the following, we will refer to the former 1/4-BPS cases as ‘two-charge solutions’ and the latter 1/2-BPS cases as ‘one-charge solutions’. The coordinate y ranges from y+, the largest root of Q(y), to infinity. 2.2 11d SUGRA solutions To have a smooth geometry one can choose the gauge so that AI(y+) = 0 by appropriate choice of the aI. Setting ˆg = 2, the AdS5 × S1 geometry does not have a conical deficit provided z ∈[0, 2π) (this will be assumed in the uplift to 11d). At y = y+, the geometry either has a smooth cap or a conical deficit 2π ˆn−1 ˆn with ˆn related to y+ by the constraint ˆn Q′(y+) = y2 +. The conditions Q(y+) = 0 and ˆn Q′(y+) = y2 + can be solved to determine q1 and q2 in terms of ˆn and y+ as follows JHEP04(2024)120 qI = y+  3ˆn + 1 ˆnˆg2 −y+ ± 2 ˆg s (1 + 3ˆn)2 4ˆg2ˆn2 −y+  , (2.14) (2.14) where q1 and q2 are chosen with opposite signs for the square root. This has real solutions provided 0 ≤y+ ≤y+,max with y+,max = (1 + 3ˆn)2/4ˆg2ˆn2. It will be useful later to notice that the sum q1 + q2 is always non-negative as is evident from q1 + q2 2y+ = 3ˆn + 1 ˆnˆg2 −y+ ≥ 3ˆn + 1 ˆnˆg2 −y+,max = (ˆn −1)(3ˆn + 1) 4ˆg2ˆn2 ≥0. (2.15) q1 + q2 2y+ = 3ˆn + 1 ˆnˆg2 −y+ ≥ 3ˆn + 1 ˆnˆg2 −y+,max = (ˆn −1)(3ˆn + 1) 4ˆg2ˆn2 ≥0. 2.2 11d SUGRA solutions (A.1) is a function of the y, ψ, and ζ coordinates and also depends on the qI’s and aI’s. Note that in eq. (A.1), we have introduced the slightly abusive shorthand where each of the ˆf’s displayed in eq. (A.1) is a function of the y, ψ, and ζ coordinates and also depends on the qI’s and aI’s. Note that in eq. (A.1), we have introduced the slightly abusive shorthand sin x ≡sx , cos x ≡cx , (2.20) (2.20) in order to compactly express some of the more cumbersome expressions, and we will adopt this notation throughout the following sections. Continuing on, the uplifted four-form field strength can be inferred from JHEP04(2024)120 ⋆11F4 κ2 = −2( ˆH(X0 + 2(X1 + X2)) −2X2 0 + 2(X2 0 −X2 1)s2 ζ + 2(X2 0 −X2 2)c2 ψc2 ζ)Υ7 (2.21) + c2 ζcψsψ 2X0X2 (X2 ⋆7 dX0 −X0 ⋆7 dX2) ∧dψ + cζsζ 2X1 ⋆7 dX1 ∧dζ − cζsζ 2X0X2 (X2s2 ψ ⋆7 dX0 + X0c2 ψ ⋆7 dX2) ∧dζ + cζsζ 4X2 1 dζ ∧(dϕ1 + 2A1) ∧⋆7dA1 −cζcψ 4X2 2 (cζsψdψ + sζcψdζ) ∧(dϕ2 + 2A2) ∧⋆7dA2 (2.21) where we have set ˆg = 2. We also defined where we have set ˆg = 2. We also defined ˆH = X2(H2 −q2c2 ψ)c2 ζ y2 + X1s2 ζ . (2.22) (2.22) Electrostatic solutions. In this subsection, we review the construction of an infinite class of ‘bubbling’ solutions to 11d SUGRA with AdS5 × S1 boundary geometries that holographically describe 1/2-BPS co-dimension 2 defects in 6d SCFTs [14]. There is a long history of AdS5 compactifications in 11d SUGRA and M-theory holographically dual to 4d N = 2 SCFTs, e.g. [15, 16, 46, 47]. The class into which the solutions of [13, 14] are embedded are a particular type of Lin-Lunin-Maldacena (LLM) ‘bubbling’ geometries [15, 16]. Recall that the general LLM solution consists of an 11d geometry with a warped product AdS5 × S2 over M4 realized as a U(1)χ-fibration over a 3d base space B3 supported by four-form flux. The data that specifies the solution is encoded in a function that satisfies a non-linear Toda equation on B3, which is generically difficult to solve. However, by imposing that B3 has an additional U(1)β isometry, the Toda equation can be cast in an axi-symmetric form that can be solved more easily. 2.2 11d SUGRA solutions (2.15) (2.15) The 7d solutions above were uplifted in [14] using the standard ansatz for the Kaluza- Klein (KK) reduction of 11d SUGRA on S4 [45]; namely, the 11d metric and four-form flux are given by the 7d data as follows, The 7d solutions above were uplifted in [14] using the standard ansatz for the Kaluza- Klein (KK) reduction of 11d SUGRA on S4 [45]; namely, the 11d metric and four-form flux are given by the 7d data as follows, ds2 11 = ˜∆1/3ds2 7 + ˆg−2 ˜∆−2/3 " X−1 0 dµ2 0 + 2 X I=1 X−1 I dµ2 I + µ2 I(dϕI + ˆgAI)2# (2.16a) ⋆11F4 = 2ˆg 2 X i=0 X2 i µ2 i −˜∆Xi Υ7 + ˆg ˜∆X0Υ7 + 1 2ˆg 2 X i=0 ⋆7d ln Xi ∧d(µ2 i ) (2.16b) + 1 2ˆg2 2 X I=1 X−2 I d(µI)2 ∧(dϕI + ˆgAI) ∧⋆7FI (2.16a) (2.16b) here Υ7 is the 7d volume form and we defined where Υ7 is the 7d volume form and we defined X1 = (yH2(y)) 2 5 H1(y) 3 5 , X2 = (yH1(y)) 2 5 H2(y) 3 5 , X0 = (X1X2)−2 , ˜∆= 2 X i=0 Xiµ2 i , (2.17) (2.17) as well as as well as µ1 = sin ζ , µ2 = cos ψ cos ζ . (2.18) µ0 = sin ψ cos ζ , µ1 = sin ζ , µ2 = cos ψ cos ζ . (2.18) µ0 = sin ψ cos ζ , µ1 = sin ζ , µ2 = cos ψ cos ζ (2.18) µ0 = sin ψ cos ζ , µ0 = sin ψ cos ζ , Explicitly, the 11d metric above for the two-charge 1/4-BPS solutions can be brought into the form Explicitly, the 11d metric above for the two-charge 1/4-BPS solutions can be brought into the form ds2 11 = ˆf2 AdSds2 AdS5 + ˆf2 y dy2 + ˆf2 z dz2 + ˆf2 ϕidϕ2 i + ˆf2 zϕidzdϕi + ˆf2 ψdψ2 + ˆf2 ζ dζ2 + ˆfψζdψdζ (2 ds2 11 = ˆf2 AdSds2 AdS5 + ˆf2 y dy2 + ˆf2 z dz2 + ˆf2 ϕidϕ2 i + ˆf2 zϕidzdϕi + ˆf2 ψdψ2 + ˆf2 ζ dζ2 + ˆfψζdψdζ , (2.19) (2.19) – 8 – where each of the ˆf’s displayed in eq. 2.2 11d SUGRA solutions . ∧dxd is the volume form on a d-dimensional manifold M. In this notation, the coordinates {r, η, β} span B3, κ11 = πℓ3 P /2, and where we have adopted the notation where ΥM := p \|gM\|dx1 ∧. . . ∧dxd is the volume form on a d-dimensional manifold M. In this notation, the coordinates {r, η, β} span B3, κ11 = πℓ3 P /2, and V ′ ≡∂ηV, ˙V ≡r∂r(V ), σ ≡V ′′(2 ˙V −¨V ) + ( ˙V ′)2. (2.24) (2.24) In this background, away from sources, the electrostatic potential V (r, η) satisfies ¨V (r, η) + r2V ′′(r, η) = 0, (2.25) (2.25) subject to the boundary condition ∂rV \|η=0 = 0. Exploiting the U(1)β isometry imposed on B3, the line charge distribution ϖ(η) specifying the solution is related to the Laplace potential V by subject to the boundary condition ∂rV \|η=0 = 0. Exploiting the U(1)β isometry imposed on B3, the line charge distribution ϖ(η) specifying the solution is related to the Laplace potential V by ϖ(η) = lim r→0+ ˙V (r, η). (2.26) (2.26) Given an appropriate ϖ(η), the solution to eq. (2.25) can be expressed in terms of a Green’s function, G(r, η, η′), as Given an appropriate ϖ(η), the solution to eq. (2.25) can be expressed in terms of a Green’s function, G(r, η, η′), as V (r, η) = −1 2 Z dη′G(r, η, η′)ϖ(η′). (2.27) (2.27) By the symmetry of the problem, the Green’s function can be written simply using the method of images as [14, 18] By the symmetry of the problem, the Green’s function can be written simply using the method of images as [14, 18] G(r, η, η′) = 1 p r2 + (η −η′)2 − 1 p r2 + (η + η′)2 . (2.28) (2.28) The complete description of the solution to the 11d SUGRA field equations is thus given by finding a ϖ(η) that obeys a set of necessary conditions. For a generic ϖ(η), the constraints that follow from charge conservation and regularity (modulo Ak singularities on M4) of the full 11d geometry were given in [47]. Satisfying these constraints determines the profile of ϖ(η) to be a continuous, convex piecewise linear function of η with integer slope, whose slope decreases by integer values at discrete ηa. In general, the boundary conditions and symmetry imposed on V in solving eq. 2.2 11d SUGRA solutions Further facilitating finding general solutions to the axi-symmetric Toda equation, one can perform a Bäcklund transformation to map to a Laplace-type equation on R3, and so the problem is turned into an ‘electrostatic’ one [6, 14, 18, 48–50]. Hence, the class of bubbling geometries reviewed below will be referred to as ‘electrostatic solutions’ in the following sections.i In the formulation as a Laplace-type equation, finding a solution to the SUGRA equations of motion amounts to specifying a linear charge density ϖ which determines the electrostatic potential V . Exploiting the axial symmetry of the problem on B3, we take ϖ = ϖ(η) to be aligned along the η-axis, i.e. the fixed point of the U(1)β rotations. The bosonic sector – 9 – of these solutions takes the form of these solutions takes the form of these solutions takes the form ds2 11 = κ 2 3 11 ˙V σ 2V ′′ ! 1 3 4ds2 AdS5 + 2V ′′ ˙V σ dΩ2 2 + 2(2 ˙V −¨V ) ˙V σ dβ + 2 ˙V ˙V ′ 2 ˙V −¨V dχ !2 (2.23a) + 2V ′′ ˙V dr2 + 2 ˙V 2 ˙V −¨V r2dχ2 + dη2 ! ! ≡f2 AdSds2 AdS5 + fS2dΩ2 2 + f2 βdβ2 + f2 χdχ2 + f2 βχdβdχ + f2 3 (dr2 + dη2) , C3 = 2κ11 σ ˙V ˙V ′ −ση dβ −2 ˙V 2V ′′dχ ∧ΥS2 , (2.23b) ds2 11 = κ 2 3 11 ˙V σ 2V ′′ ! 1 3 4ds2 AdS5 + 2V ′′ ˙V σ dΩ2 2 + 2(2 ˙V −¨V ) ˙V σ dβ + 2 ˙V ˙V ′ 2 ˙V −¨V dχ !2 (2.23a) + 2V ′′ ˙V dr2 + 2 ˙V 2 ˙V −¨V r2dχ2 + dη2 ! ! (2.23a) V V V ! ! ≡f2 AdSds2 AdS5 + fS2dΩ2 2 + f2 βdβ2 + f2 χdχ2 + f2 βχdβdχ + f2 3 (dr2 + dη2) , C3 = 2κ11 σ ˙V ˙V ′ −ση dβ −2 ˙V 2V ′′dχ ∧ΥS2 , (2 ≡f2 AdSds2 AdS5 + fS2dΩ2 2 + f2 βdβ2 + f2 χdχ2 + f2 βχdβdχ + f2 3 (dr2 + dη2) , C3 = 2κ11 σ ˙V ˙V ′ −ση dβ −2 ˙V 2V ′′dχ ∧ΥS2 , (2.23b) (2.23b) JHEP04(2024)120 where we have adopted the notation where ΥM := p \|gM\|dx1 ∧. . 2.2 11d SUGRA solutions (2.25) require ϖ(0) = 0. However, there are generally two cases for the behavior of ϖ as η increases. – 10 – In the first case, apart from the zero at the origin, ϖ has a zero at some value η = ηc > 0 where the internal space closes off. The geometry of the 11d SUGRA solution is then a warped product of AdS5 over the compact internal space M6 = Cg ×M4, and holographically describes a 4d theory that descends from the compactification of a 6d SCFT on a Riemann surface Cg. The generic charge distribution is decomposed into n + 1 ‘regular’ intervals with positive slope and an ‘irregular’ interval [ηn, ηc] with negative slope fixed by ratios of four-form flux. The data associated with the kinks between the regular parts of the charge distribution, namely a partition of N, label a regular puncture on Cg, while the data specifying the slope of the irregular interval is mapped to an irregular puncture [18]. This construction — reminiscent of other spindle compactifications engineering 4d SCFTs [17, 51–58] — was argued in [18] to be the SUGRA dual to class-S constructions [19] of certain classes of Argyres-Douglas theories [59] by analyzing anomalies and counting of Coulomb and Higgs branch operators in the field theory. While we will not study these types of solutions further here, we will mention some of their properties as they pertain to the results of holographic calculations of defect anomalies. JHEP04(2024)120 The second case, relevant for our study, is where ϖ(η) has non-trivial support over the whole range η ∈[0, ∞) [14]. Since ϖ(η) never turns around to hit the η-axis, the geometry M6 in the 11d SUGRA solution is non-compact, and the 11d geometry can be engineered to be asymptotically locally AdS7 × S4 where the geometry of the conformal boundary of the AdS7 factor is AdS5 × S1. These solutions are, thus, interpreted as holographically describing co-dimension 2 defect operators in 6d SCFTs, where the defect operator ‘lives’ at the conformal boundary of AdS5. 2.2 11d SUGRA solutions As a simple example of a line charge density that gives rise to a non-compact geometry, it was shown in [14] that the one-charge solution reviewed in the previous subsection can be recast in the language of the electrostatic solutions as a ϖ(η) with two segments: ϖ(η) =    1 + 1 √1−4q1 η, η ∈ 0, N 2 √1 −4q1 η + N/2, η ∈ N 2 √1 −4q1, ∞ . (2.29) (2.29) Due to ϖ being continuous and piecewise linear, we will refer to the solution engineered by eq. (2.29) as a ‘single kink solution’. This relation between the q2 →0 limit of the two-charge solutions and the simple single kink line charge distribution for the electrostatic solutions will be useful in later sections as a consistency check for our computations. We should also note that the constraint that the change in slope of ϖ(η) is integral forces q1 = j2−1 4j2 for j ∈N. Due to ϖ being continuous and piecewise linear, we will refer to the solution engineered by eq. (2.29) as a ‘single kink solution’. This relation between the q2 →0 limit of the two-charge solutions and the simple single kink line charge distribution for the electrostatic solutions will be useful in later sections as a consistency check for our computations. We should also note that the constraint that the change in slope of ϖ(η) is integral forces q1 = j2−1 4j2 for j ∈N. j Generalizing beyond the single kink solutions, the constraints on ϖ(η) realizing a defect solution allow for a generic n-kink charge profile. Since ϖ(η) is piecewise linear, its behavior on the ath interval, where η ∈[ηa, ηa+1] and a ∈{0, 1, . . . , n}, can be written as [6, 14] ϖa(η) =  1 + n X b=a+1 kb  η + a X b=1 ηbkb (2.30) ≡pa+1η + δa+1, (2.30) ≡pa+1η + δa+1, where in the second line we have introduced a convenient short hand for the slope pa+1 and intercept δa+1 of the line continued from the ath segment. From the boundary condition ϖ(0) = 0 it is understood that η0 = 0, and due to the semi-infinite domain of support we take ηn+1 →∞. – 11 – η η1 η2 ηn ϖ(η) . . . N/2 . . . 2.2 11d SUGRA solutions kn k2 ηn η2 k1 η1 η η1 η2 ηn ϖ(η) . . . N/2 . . . kn k2 ηn η2 k1 η1 Figure 1 Figure 1. (Left) A generic line charge distribution ϖ(η), with n kinks at positions ηa along the axis of cylindrical symmetry, specifying a solution to the axially symmetric Laplace equation in R3. The AdS7 × S4 vacuum corresponds to the single-kink (n = 1) charge distribution with k1 = 1; the location of the kink is then given by η1 = N/2. (Right) The Young Tableau corresponding to the partition N = 2 Pn a=1 kaηa = Pn a=1 Na. The height and width of the a-th block are given by the location ηa ∈Z and slope change ka ∈Z of the a-th kink in ϖ(η), respectively. The AdS7 × S4 vacuum is associated to the 1 of su(N) determined by n = k1 = 1 and η1 = N/2. . . . kn k2 ηn η2 k1 η1 JHEP04(2024)120 Figure 1 Figure 1. (Left) A generic line charge distribution ϖ(η), with n kinks at positions ηa along the axis of cylindrical symmetry, specifying a solution to the axially symmetric Laplace equation in R3. The AdS7 × S4 vacuum corresponds to the single-kink (n = 1) charge distribution with k1 = 1; the location of the kink is then given by η1 = N/2. (Right) The Young Tableau corresponding to the partition N = 2 Pn a=1 kaηa = Pn a=1 Na. The height and width of the a-th block are given by the location ηa ∈Z and slope change ka ∈Z of the a-th kink in ϖ(η), respectively. The AdS7 × S4 vacuum is associated to the 1 of su(N) determined by n = k1 = 1 and η1 = N/2. As a simple visualization of an arbitrary distribution, see the left side of figure 1. Note that from the constraint following from the quantization of four-form flux N = 2 Pn a=1 ηaka along with the quantization of the ηa and their ordering along the η-axis (0 < . . . < ηa < ηa+1 < . . . 2.2 11d SUGRA solutions < ηn), there is a natural interpretation of the data (ηa, ka) specifying the charge distribution as a Young diagram, which is displayed on the right side of figure 1.i 1 In the language of the field theory description, the Young diagram corresponding to the specific ϖ(η) is in correspondence to both the Lie algebra homomorphism ϑ : sl(2) →g and to the choice of Levi subalgebra l of the AN−1 gauge algebra. Furthermore, the slope change ka ∈Z between the (a −1)th and ath intervals corresponds to the monopole charge at the R4/Zka orbifold point located at (r, η) = (0, ηa) in the internal manifold. These points are the holographic realization of the non-Abelian summands su(ka) of the global symmetry algebra [47].i Lastly, for use in future computations, it will be convenient to define the ‘moments’ of the potential as in [14] mj = n X a=1 (pa −pa+1)ηj a = n X a=1 kaηj a. (2.31) (2.31) For most of the following, we will only need the first and third moments For most of the following, we will only need the first and third moments m1 = N 2 and m3 = X a N3 a 8k2a (2.32) (2.32) respectively. 11Note that the differences in sign are due the fact that we are using the convention that, in units of L2d, the scalar curvature R < 0 for a space of constant “negative curvature”; whereas the authors of [60] use the opposite convention, R > 0. 10Our index conventions in this section are that µ, ν, . . . are AdS7 indices, a, b, . . . are S4 indices, and i, j, . . . are 6d indices on the conformal boundary of AdS7.f 3 Holographic stress-energy tensor one-point function In this section, we will compute the contribution of a co-dimension 2 defect to the one-point function of the stress-energy tensor of the ambient 6d SCFT. To do so, we will reduce the – 12 – 11d SUGRA backgrounds described in the previous section on the internal S4 and employ the holographic renormalization methods of [60]. In their original formulation, these methods are meant to apply to asymptotically AdS solutions of pure Einstein-Hilbert gravity; therefore, we must ensure that the presence of the four-form flux in the dimensionally reduced M-theory solutions does not necessitate a modification of those methods. In both two-charge and electrostatic solutions, we will show that the field strength decays sufficiently fast as the conformal boundary of AdS7 is approached so that it produces a vanishing contribution to the field equations on the boundary. Following the general procedure in [60], we begin by recasting the 11d metric as a perturbation h11 about the AdS7 × S4 vacuum: JHEP04(2024)120 ds2 11 = gAdS7×S4 + h11. (3.1) (3.1) Dimensionally reducing on the internal S4 then leads to the 7d line element Dimensionally reducing on the internal S4 then leads to the 7d line element Dimensionally reducing on the internal S4 then leads to the 7d line element ds2 7 = 1 + ¯ς 5 gAdS7 + ¯h7, (3.2)il (3.2) where gAdS7 is the metric on AdS7, the 7d field h7 captures the fluctuations about the AdS7 geometry, and ς is the trace of the fluctuations in the internal manifold. Bars indicate zero modes on the internal space; for instance,10 where gAdS7 is the metric on AdS7, the 7d field h7 captures the fluctuations about the AdS7 geometry, and ς is the trace of the fluctuations in the internal manifold. Bars indicate zero modes on the internal space; for instance,10 ¯ς = 3 4 Z S4 √gS4 habg(0) ab . (3.3)f (3.3) Mapping the 7d line element into Fefferman-Graham (FG) gauge, ds2 7 = L2 u2 du2 + g , (3.4) (3.4) metric g admits the power series expansion where the 6d metric g admits the power series expansion where the 6d metric g admits the power series expansion 12 g = g(0) + g(2)u2 + g(4)u4 + g(6)u6 + h(6)u6 log u2 + . . . 3.1 Two-charge solutions In this subsection, we will focus on the 11d uplift of the two-charge solutions described in section 2.2 and compute ⟨Tij⟩with the methods described above. In order to isolate the contributions from the holographic dual to the defect, we will employ a background subtraction scheme where we remove the contributions from vacuum AdS7 × S4. Before jumping in to the computation of ⟨Tij⟩, we need to carefully check that we can properly utilize our chosen holographic renormalization scheme. One of the crucial assumptions in the construction of eq. (3.6a) is that Einstein’s equations near the boundary of the dimensionally reduced AdS7 geometry are not modified by contributions coming from non-trivial fluxes, such as the four-form curvature F4. So, we must be careful to make sure that in the asymptotic small u region, the components of the variation of the FMNPQF MNPQ part of the 11d SUGRA action involving AdS7 directions fall off sufficiently fast so as to not modify the boundary equations of motion.fif JHEP04(2024)120 For the solutions in eqs. (2.19) and (2.21), it suffices to show the fall-off conditions for the single charge case. Setting q2 →0 and a2 →0, transforming ϕI →φI −2aIz, and mapping to FG gauge as in appendix A.1, a quick computation shows the small u behavior to be (up to overall numerical prefactors) F MNP a FbMNP ∼c2 θgab + . . . , F MNP φ1 Fφ1MNP ∼s2 θ + . . . , F MNP θ FθMNP ∼1 + . . . , F MNP z FzMNP ∼q2 1(13 −5c2θ)u8 + . . . , F MNP z Fφ1MNP ∼q1s2 θu4 + . . . , F MNP y FyMNP ∼q2 1s2 2θu12 + . . . , where gab are components along the S2 ⊂S4 and the AdS5 components of the variation vanish. From the zz- and zφ1-components of the variation of F 2 4 , we can see that the contributions to the boundary equations of motion dies at worst as u4 as u →0. The analysis of the two-charge solution follows similarly, and so we can proceed using eq. (3.6a) without modification. 3 Holographic stress-energy tensor one-point function , (3.5) (3.5) the 6d stress-energy tensor one-point function can be computed the 6d stress-energy tensor one-point function can be computed ⟨Tij⟩dxidxj = 3L5 8πG(7) N g(6) −A(6) + S 24 (3.6a) = N3 4π3 g(6) −A(6) + S 24 , (3.6b) (3.6a) (3.6b) where A(6) and S are rank-2 tensors built out of g(0), and in the second line we have used the holographic map to field theory quantities 1 G(7) N = vol(S4) G(11) N , G(11) N = 24π7ℓ9 P , L3 = πNℓ3 P , vol(S4) = L4π2 6 . (3.7) (3.7) Note that in our conventions the internal S4 has curvature scale L2/4. Explicit expressions for A(6) and S are provided in [60].11 Once the appropriate vacuum subtraction is performed, the defect contribution to hT , and therefore to d2, can be extracted via eq. (2.3) and eq. (2.5). 11Note that the differences in sign are due the fact that we are using the convention that, in units of L2d, the scalar curvature R < 0 for a space of constant “negative curvature”; whereas the authors of [60] use the opposite convention, R > 0. – 13 – 3.1 Two-charge solutions Allowing for q2 ̸= 0 modifies the variation of FMNPQF MNPQ but crucially does not introduce any leading terms in the small u expansion.fi Now that we have established that the variation of F 2 4 decays sufficiently fast near the AdS7 boundary, we can proceed using the logic of [60] recapped above to compute ⟨Tij⟩. To do so, we first map eq. (2.19) to FG gauge as in eq. (A.4), which we reproduce here for clarity ds2 FG = L2 u2 (du2 + ˆαAdSds2 AdS5 + ˆαzdz2) + L2s2 θ ˆαzφ1dzdφ1 + L2c2 ℵc2 θ ˆαzφ2dzdφ2 + L2 4 (ˆαθdθ2 + s2 θ ˆαφ1dφ2 1 + c2 θ(ˆαℵdℵ2 + c2 ℵˆαφ2dφ2 2) + ˆαθℵdθdℵ). The ˆα metric functions are given in eq. (A.6). In order to put the dimensionally reduced metric in the form of eq. (3.2), we then write ds2 FG as a fluctuation around AdS7 × S4 The ˆα metric functions are given in eq. (A.6). In order to put the dimensionally reduced metric in the form of eq. (3.2), we then write ds2 FG as a fluctuation around AdS7 × S4 ds2 = (g(0) µν + hµν)dxµdxν (3.8) ds2 = (g(0) µν + hµν)dxµdxν (3.8) – 14 – where g(0) µν dxµdxν = L2du2 u2 + L2 u2 1 + u2 2 + u4 16 ! ds2 AdS5 + 1 −u2 2 + u4 16 ! dz2 ! + L2 4 dΩ2 4. (3 (3.9) Using the expressions in eq. (A.6), we can compute the zero modes of the fluctuations around the AdS7 directions Using the expressions in eq. (A.6), we can compute the zero modes of the fluctuations around the AdS7 directions ¯h7 = −2L2(q1 + q2) 15 u4(ds2 AdS5 −5dz2). (3.10) (3.10) JHEP04(2024)120 Similarly, the trace fluctuations on the S4 are found to be Similarly, the trace fluctuations on the S4 are found to be ς = 10q2c2ℵc2 θ + 5(q2 −2q1)c2ℵ+ 2q1 −3q2 8 u4 + . . . . (3.11) (3.11) Integrating the internal space fluctuations over the S4 gives ¯ς = 0. The vanishing of the zero modes of the trace fluctuations means that the dimensionally reduced metric is already in FG form. The resulting stress-energy tensor one-point function is ⟨Tij⟩dxidxj = N3 192π3 1 −32 5 (q1 + q2) ds2 AdS5 −5dz2 . (3.12) (3.12) As it stands, eq. 12This subtlety can be seen in familiar cases like in choosing Neumann/Robin or Dirichlet conditions for scalar fields in the presence of a boundary [61] or magnetic (‘t Hooft) line operators in gauge theories, see e.g. [2], as well as in more exotic contexts such as surface operators in ambient theories with unusual deformations [62]. 3.1 Two-charge solutions (3.12) contains information associated with the holographic description of the physics of both the ambient and defect theories, and hence, the universal divergences that it contains are ambiguous. A commonly method used to isolate quantities associated with the defect degrees of freedom is the so-called vacuum (or background) subtraction scheme. In short, this scheme involves using a SUGRA solution that is holographically dual to the theory without a defect — or, in some sense, containing a “trivial” defect. A crucial part of this construction is that the vacuum used to regulate the theory and remove the contributions sourced by ambient degrees of freedom must have the same asymptotic behavior in the UV as the original, unregulated SUGRA solution. For our purposes in both the immediate case of the two-charge solution and in the electrostatic solutions below, the holographic description of the large N limit of ambient theory — the 6d AN−1 N = (2, 0) SCFT with no defect insertions — is well known to be simply Ads7 × S4, which can be obtained from the two-charge solution by taking the qI →0 limit. We will use this limit to compute ⟨T (vac) ij ⟩, which will be subtracted from eq. (3.12) to obtain the change in the stress-energy tensor one-point function due to the presence of the defect. Before proceeding, we note that there are limitations to using vacuum subtraction that, while not affecting any of the computations below, are nonetheless important to keep in mind. As is alluded to above, in general it may not always be obvious how to correctly choose the background to be removed, as the notion of a “defect” is not always well-defined.12 Furthermore, it is not clear that background subtraction is a justifiable scheme for computing 12This subtlety can be seen in familiar cases like in choosing Neumann/Robin or Dirichlet conditions for scalar fields in the presence of a boundary [61] or magnetic (‘t Hooft) line operators in gauge theories, see e.g. [2], as well as in more exotic contexts such as surface operators in ambient theories with unusual deformations [62]. – 15 – non-universal quantities due to a lack of a systematic way to account for finite counterterms. However, for the log-divergent quantities that we compute in this work, such subtleties can be ignored. 3.1 Two-charge solutions A more precise, alternative method for holographically computing defect quantities would involve generalizing the standard tools in holographic renormalization, but as of yet, correctly identifying a set of suitable covariant counterterms for holography with AdS submanifolds remains an open problem. With an understanding of the utility of the background subtraction scheme in hand, we can now compute ⟨T (vac) ij ⟩computed using vacuum AdS7×S4, which we obtain by taking qI →0 in eq. (3.10). This limit kills the fluctuations and gives the exact AdS7 metric upon dimensional reduction, as expected. So, taking qI →0 in eq. (3.12) yields the vacuum 1-pt function JHEP04(2024)120 ⟨T (vac) ij ⟩dxidxj = N3 192π3 ds2 AdS5 −5dz2 . (3.13) (3.13) Subtracting this vacuum contribution from eq. (3.12) computes the change in the stress- energy tensor one-point function due to the introduction of the holographic dual to the field theory defect: ∆⟨Tij⟩dxidxj = −N3(q1 + q2) 30π3 (ds2 AdS5 −5dz2), (3.14) (3.14) which recovers the results in [13] up to subtraction of the contribution from the AdS7 × S4 vacuum. Using eq. (2.3) we arrive at hT = N3(q1 + q2) 30π3 (3.15) (3.15) Thus, one of the B-type anomaly coefficients for 1/4-BPS co-dimension 2 operators in a 6d N = (2, 0) AN−1 SCFT holographically described by the two-charge solutions is found to be d2 = −1 6N3(q1 + q2). (3.16) (3.16) Recall that in eq. (2.15), we found that the linear combination q1 + q2 ≥0 for all ˆn. Further, we know that eq. (2.6) implies d2 ≤0, and so all of the two-charge solutions studied in [13, 14] are consistent with the defect ANEC. In figure 2, we reproduce the curves for solutions obeying eq. (2.15) as appear in [13, 14] together with the region excluded by consistency with defect ANEC. We see that, indeed, all of the ˆn = 1, 2, 3 solutions lie above the line q1 + q2 ≥0 with only ˆn = 1 saturating the bound at q1 = q2 = 0. 3.2 Electrostatic solutions So, – 17 – in the u →0 limit, there are no surviving contributions to the equations of motion in the dimensionally reduced geometry coming from the variation of the F 2 4 term.l in the u →0 limit, there are no surviving contributions to the equations of motion in the dimensionally reduced geometry coming from the variation of the F 2 4 term.l We can now proceed with [60]. First, we rewrite the metric in eq. (A.16) as fluctuations around AdS7 × S4. The perturbation away from AdS7 × S4 takes the form h11 = L2 u2 αAdS −1 −u2 2 −u4 16 ! ds2 AdS5 + L2 u2 αz −1 + u2 2 −u4 16 ! dz2 + L2 4 (αθ −1)dθ2 + L2s2 θ 4 (αφ −1)dφ2 + L2c2 θ 4 (αS2 −1)dΩ2 2 + L2s2 θαzφdzdφ. (3.18) (3.18) Fixing χ = −z −φ and β = 2z + φ, we can compute the zero modes for the AdS7 part of the fluctuations, JHEP04(2024)120 JHEP04(2024)120 ¯h7 = L2 m3 −m3 1 30m3 1 u4ds2 AdS5 + L2 m3 1 −m3 6m3 1 u4dz2 + . . . . (3.19) (3.19) The trace S4 fluctuations are found to be The trace S4 fluctuations are found to be The trace S4 fluctuations are found to be ς = (1 −5c2θ)m3 1 −m3 16m3 1 u4 −11(1 −5c2θ)m3 1 −m3 216m3 1 u6 + . . . . (3.20) (3.20) Integrating ς over the S4, we find the zero modes ¯ς = 0. The reduced geometry g7 = 1 + ¯ς 5 g(0) + ¯h7 (3.21) (3.21) is thus already in FG form. So, the dimensionally reduced metric is ds2 7 =L2 u2 " du2 + 1 + u2 2 + u4 16 + (m3 −m3 1)u6 30m3 1 ! ds2 AdS5 + 1 −u2 2 + u4 16 + (m3 1 −m3)u6 6m3 1 ! dz2 # , (3.22) (3.22) where we have suppressed higher powers of u. From this expression for ds2 7, we can easily read off g(0), g(2), g(4), and g(6). Note if we take n = 1 and k1 = 1, then m3 = m3 1 = N3/8, and so in this limit, eq. (3.22) reduces to the exact AdS7 metric, which is expected from eqs. (2.23a), (2.25), and (2.30).i where we have suppressed higher powers of u. 3.2 Electrostatic solutions Prior to approaching the holographic computation of ∆⟨Tij⟩for the electrostatic solutions using the methods outlined above, we again must verify that the boundary equations of motion in the dimensionally reduced geometry are unmodified by the four-form flux. From eq. (2.23b), we can compute F4. For brevity, we will immediately define r = ϱcω and η = ϱsω to map eq. (2.23b) into (ϱ, ω) coordinates on the internal space and adopt (z, φ) using eq. (A.14) as – 16 – Figure 2. The solutions to the constraint in eq. (2.15) for ˆn = 1 (red), ˆn = 2 (blue), and ˆn = 3 (green) on the (q1, q2) plane, reproduced from [13, 14]. The shaded regions correspond to the two-charge configurations for which Q(y) = 0 admits no real solutions (region I) or which violate the defect ANEC (region II). JHEP04(2024)120 Figure 2. The solutions to the constraint in eq. (2.15) for ˆn = 1 (red), ˆn = 2 (blue), and ˆn = 3 (green) on the (q1, q2) plane, reproduced from [13, 14]. The shaded regions correspond to the two-charge configurations for which Q(y) = 0 admits no real solutions (region I) or which violate the defect ANEC (region II). our angular coordinates and compute the large ϱ expansion to leading order in each component F4 2κ11 = " c2 ωs3 ω 5m3 −2m3 1 ϱ3 dϱ ∧dz + 3cωs2 ωm1dω ∧dz (3.17) + s3 ω 4(m3 −m3 1) ϱ3 dϱ ∧dφ + cωs2 ω 6(m3 1 −m3) ϱ2 dω ∧dφ # ∧vol(S2) + . . . (3.17) where we have fixed Cz = −2 following the discussion in appendix A.2.f Now, we can check the fall off of the contribution of the variation of F 2 4 to the equations of motion. Keeping Cz = −2 fixed and transforming into FG gauge, we find the leading behavior in the small-u expansion (up to numerical factors) FuMNP FuMNP ∼s2 2θ(m3 1 −m3)2u6 + . . . , FzMNP FzMNP ∼(13 −5c2θ)(m3 1 −m3)2u8 + . . . , FφMNP FzMNP ∼(m3 1 −m3)s2 θu4 + . . . , FaMNP FbMNP ∼gS4 + . . . , where a, b are indices for S4 coordinates {θ, φ, S2}, gS4 is the metric on the unit S4 in S1 × S2 coordinatization. Note that the variations in the AdS5 directions vanish identically. 3.2 Electrostatic solutions From this expression for ds2 7, we can easily read off g(0), g(2), g(4), and g(6). Note if we take n = 1 and k1 = 1, then m3 = m3 1 = N3/8, and so in this limit, eq. (3.22) reduces to the exact AdS7 metric, which is expected from eqs. (2.23a), (2.25), and (2.30).i Proceeding with the computation in the same way as the previous subsection, we find that the holographic stress-energy tensor one-point-function takes the form ⟨Tij⟩dxidxj = −N3(3m3 1 −8m3) 960π3m3 1 ds2 AdS5 −5dz2 . (3.23) (3.23) Regulating this result by subtracting the AdS7 × S4 vacuum contribution ⟨T (vac) ij ⟩in eq. (3.13) produces ∆⟨Tij⟩dxidxj = −N3(m3 1 −m3) 120π3m3 1 ds2 AdS5 −5dz2 . (3.24) (3.24) – 18 – – 18 – As a quick check, computing the trace of eq. (3.24) gives ∆ T ii = 0 as expected due to defect conformal symmetry. Comparing eq. (3.24) to eq. (2.3), we find As a quick check, computing the trace of eq. (3.24) gives ∆ T ii = 0 as expected due to defect conformal symmetry. Comparing eq. (3.24) to eq. (2.3), we find hT = m3 1 −m3 15π3 . (3.25) (3.25) We can thus read off the defect Weyl anomaly coefficient d2 from eq. (2.5): We can thus read off the defect Weyl anomaly coefficient d2 from eq. (2.5): d2 = −m3 1 −m3 3 (3.26a) = −1 24 N3 − X a N3 a k2a ! , (3.26b) (3.26a) (3.26b) JHEP04(2024)120 where in the second line we have rewritten d2 in terms of the parameters Na and ka which are more suitable for comparison to field theory. For any partition of N = P a Na, it is clear that d2 ≤0. The upper bound d2 = 0 is only saturated in the vacuum case n = 1, k1 = 1 where there is no defect. There is a non-trivial consistency check on the value of d2 in the n = 1 case. As mentioned above, the 11d uplift of the 1/2-BPS one-charge solutions is related to the single- kink electrostatic solutions by setting n = 1 and k1 = 1/√1 −4q1. Plugging these values into eq. (3.26b) results in d2 = −N3q1/6. Checking this against the one-charge solutions found by taking q2 →0 in eq. (3.16), we also find d2 = −N3q1/6. 3.2 Electrostatic solutions Thus, the values of d2 computed in the two-charge and n-kink electrostatic solutions are consistent in this limit. 4 Defect sphere EE and the defect A-type anomaly In the following subsections we will use the techniques developed in [63, 64] to holographically compute the defect contribution to the EE of a spherical region in the dual 6d AN−1 N = (2, 0) SCFT at large N for both the 1/4-BPS two-charge and 1/2-BPS electrostatic co-dimension 2 defects. Leveraging the results of the previous section and eqs. (2.7) and (2.8), we will be able to compute the defect A-type anomaly aΣ.l To facilitate the discussion below, let us briefly review some of the relevant background concepts for defect EE. We will restrict our discussion here to the holographic duals to 6d (D)SCFTs. To start, we will need the Ryu-Takayanagi (RT) formula for holographic EE [65–67], which we write agnostic to the presence of a defect as SEE = Amin 4GN . (4.1) (4.1) The quantity Amin is the area of the extremal surface that minimizes the bulk area functional subject to the condition that the surface anchored at the conformal boundary of AdS7 is homologous to the entangling region in the dual theory. For our computations below, we take the entangling region in the 6d SCFT at a fixed time slice to be a Euclidean 5-ball B = B5 ,→R5 of radius R. When we consider the theory deformed by a flat embedding of a Lorentzian defect on Σ = R1,3, we will take the defect to be co-original with the entangling surface such that ∂B ∩Σ = S2 sitting along the equator of ∂B. – 19 – By including a defect in the field theory, there are subtleties that arise in directly applying eq. (2.7). On the field theory side, SEE will now have short-distance divergences near ∂B due to highly entangled UV modes in both ambient and defect localized theories. In the holographic description, one needs to adopt a suitable regularization scheme that isolates the defect contribution to SEE; we will use a background subtraction scheme (SEE[Σ] −SEE[∅]) akin to the one used in computing the holographic stress-energy tensor one-point function. One further complication in the holographic computation is the fact that the FG expansion is generally not globally defined, and so one must be careful to find the asymptotic form of the map to FG gauge in order to define the UV cutoff slice at fixed AdS7 radius Λ ≫L. 4 Defect sphere EE and the defect A-type anomaly A general formula for finding the asymptotic form of the FG transformation and cutoff slice was found in [64], which we will use in the computations below. JHEP04(2024)120 Since we are considering a spherical entangling region, the solution for Amin takes a particularly simple form; even in the presence of a defect. It was shown in [63] that for a bulk geometry realizing the defect symmetry group SO(2, d) × SO(d −d), the relative warp factors of the AdSd+1 and Sd−d−1 spaces are largely immaterial, and the logic of [68] can be generalized to prove eq. (2.7) for these backgrounds. In the process, the authors of [63] proved that for the holographic defect spherical EE the surface Amin is simply a hemispherical region extending into the bulk anchored at B. For the 11d backgrounds corresponding to both the two-charge and electrostatic solutions that we consider, if we write the line element on the AdS5 in the form ds2 AdS5 = 1 w2 (dw2 −dt2 + dr2 ∥+ r2 ∥dΩ2 2) , (4.2) (4.2) then Amin is the surface w2 + r2 ∥= R2. We will exploit the simplicity of the minimal surface to great effect in the subsequent computations. 4.1 Two-charge solutions To begin computing the defect spherical EE for the two-charge solutions, we need to express the area functional A in terms of the metric functions, ˆf in eq. (2.19) with the AdS5 factor written as in eq. (4.2). Evaluating on the extremal surface r2 ∥+ w2 = R2, we regularize the w integration by introducing a UV cutoff ϵw ≪1 and performing the integral over the angular coordinates ϕ1, ϕ2 and z to obtain Amin[Σ] = 8π4L9R Z ∞ ϵw dw √ R2 −w2 w3 I = 4π4 R2 ϵ2w −log 2R ϵw + . . . ! I , (4.3) (4.3) where we have defined the remaining integral I ≡ Z dψdζ Z Λy(ϵu,ψ,ζ) y+ dy ˆf3 AdSfy q (4 ˆf2 ψ ˆf2 ζ −ˆf4 ψζ)( ˆf2 ϕ1 ˆf4 zϕ2+ ˆf2 ϕ2 ˆf4 zϕ1−4 ˆf2 ϕ1 ˆf2 ϕ2 ˆf2z ). (4 ζ) dy ˆf3 AdSfy q (4 ˆf2 ψ ˆf2 ζ −ˆf4 ψζ)( ˆf2 ϕ1 ˆf4 zϕ2+ ˆf2 ϕ2 ˆf4 zϕ1−4 ˆf2 ϕ1 ˆf2 ϕ2 ˆf2z ). (4.4) (4.4) Despite the initially complicated appearance of the integrand upon substituting the form of the metric functions in eq. (A.1), we find after a bit of algebra that the remaining integral drastically simplifies to I = 1 8 Z dψ dζ cψc2 ζsζ Z Λy(ϵu,ψ,ζ) y+ dy y. (4.5) (4.5) – 20 – – 20 – Using the double-cutoff prescription to compute I as in [64, 69], we first map the radial coordinate y to the FG coordinate u leaving the remaining angular coordinates ψ and ζ in their original frame. We then impose a cutoff ϵu ≪1, which induces a cutoff in large y, Λy(ϵu, ψ, ζ). Recalling the asymptotic FG map in appendix A.1 used in the previous section and recasting the FG angular coordinates ℵ, θ in terms of ψ, ζ, we find that Λy(ϵu, ψ, ζ) = 1 ϵ2u + 1 2 + 3 −10q1 −9q2 −2q2c2ψc2 ζ + (2q1 −q2)c2ζ 48 ϵ2 u + . . . . (4.6) (4.6) Evaluating the integral I with this cutoff is straightforward, yielding JHEP04(2024)120 I = 1 24ϵ4u + 1 24ϵ2u + 1 960(15 −16(q1 + q2) −40y2 +) + . . . (4.7) (4.7) In order to find the contributions coming from the defect, we must regulate the ϵu divergences present in Amin. 4.1 Two-charge solutions In order to do so, we employ the same vacuum subtraction scheme as was used in computing ∆⟨Tij⟩above. For the two-charge solution, the vacuum is obtained by setting q1 = q2 = 0 and a1 = a2 = 0, which sets y(vac) + = 1. Recomputing Amin[∅] for the vacuum solution and subtracting it from Amin[Σ], the regulated area functional gives Amin[Σ] −Amin[∅] = −π4L9 30 (2q1 + 2q2 + 5(y2 + −1)) R2 ϵ2w −log 2R ϵw + . . . ! , (4.8) (4.8) free from ϵu divergences. In order to compute aΣ for the defect theory, we insert eq. (4.8) in eq. (4.1). Mapping to field theory quantities by L3 = 4πNℓ3 P and GN = 24π7ℓ9 P , we can read off the coefficient of the universal part of the defect sphere EE from eq. (4.1) −R∂R(SEE[Σ] −SEE[∅])\|R→0 = −N3 30 (2(q1 + q2) + 5(y2 + −1)). (4.9) (4.9) Hence, using d2 = −N3 6 (q1 + q2) derived above in eq. (2.8) we find g d2 = −N3 6 (q1 + q2) derived above in eq. (2.8) we find aΣ = N3 24 (1 −y2 +). (4.10) (4.10) One interesting consequence of this computation is that one can show that A-type anomaly of the general two-charge solution must satisfy aΣ ≥0. To see this more clearly, recall from eq. (2.15) that y+ ≤3ˆn + 1 4ˆn ≤1. (4.11) (4.11) The second inequality follows from ˆn ∈N, and so the upper bound is saturated only for ˆn = 1. Thus, for all consistent two-charge solutions, aΣ ≥0. 4.2 Electrostatic solutions Continuing with the logic used in the previous subsection, we now turn our attention to the electrostatic solutions. Our starting point for the computation is in transforming the metric in eq. (2.23a) using eq. (A.14) and reading off the metric functions. Since only Cz = −2 – 21 – gives an asymptotic form for the metric suitable for mapping into FG gauge, we fix the transformation χ = −z −φ and β = 2z + φ and arrive at gives an asymptotic form for the metric suitable for mapping into FG gauge, we fix the transformation χ = −z −φ and β = 2z + φ and arrive at ds2 11 = f2 AdSds2 AdS5 + fS2dΩ2 2 + f2 z dz2 + f2 φdφ2 + f2 zφdzdφ + f2 ϱdϱ2 + f2 ωdω2. (4.12) (4.12) We will also write the AdS5 line element as in eq. (4.2). We will also write the AdS5 line element as in eq. (4.2). Plugging in the expression for the minimal surface, r2 ∥+w2 = R2, into the area functional, we first integrate over the two S2 factors as well as the angular coordinates z ∈[0, 2π] and φ ∈[0, 2π], which yields Amin[Σ] = 32π4R Z dw √ R2 −w2 w3 I[Σ] , (4.13) JHEP04(2024)120 JHEP04(2024)120 (4.13) where I[Σ] ≡ Z π/2 0 dω Z Λϱ(ϵu,ω) 0 f3 AdSf2 S2fωfϱ q 4f2z f2φ −f4zφ . (4.14) (4.14) Note that we have introduced the large ϱ cutoff, Λϱ, that was induced by the small u cutoff in FG gauge ϵu: Λϱ(ϵu, ω) = 2m1 ϵ2u + 2m3 1s2 ω −(1 + 5c2ω)m3 48m2 1 ϵ2 u + s2 ω m3 −m3 1 36m2 1 ϵ4 u + . . . . (4.15) (4.15) Since the metric functions f are independent of w, the w integral can be performed over [ϵw, ∞), where ϵw ≪1, Amin[Σ] = 16π4 R2 ϵ2w −log 2R ϵw + O(ϵ0 w) ! I[Σ] . (4.16) (4.16) Using the expressions for the metric functions in eq. (2.23a) in terms of the potential, we find that I can be expressed as a total derivative. To see this more clearly, we note that in (ϱ, ω) coordinates I[Σ] = 64κ3 11 Z π/2 0 dω Z Λϱ(ϵu,ω) 0 dϱ ϱ2cω ˙V V ′′ . 4.2 Electrostatic solutions (4.17) (4.17) Switching to (r, η) coordinates and using the Laplace equation ¨V = −r2V ′′, we arrive a I[Σ] = −32κ3 11 Z Λη 0 dη Z Λr 0 dr ∂r ˙V 2 , (4.18) (4.18) where we have mapped the asymptotic cutoff in ϱ back to the (r, η) frame, Λr = Λϱ(ϵu, ω)cω , Λη = Λϱ(ϵu, ω)sω . (4.19) (4.19) The remaining integral in I is identical to the one found in computing the central charge for the compact electrostatic solutions in [18] and again in [14]. For clarity, let us analyze I in detail here. We can integrate the total derivative in eq. (4.18) and find that the surviving contributions come from the boundary of the region in the ϱ−ω quarter-plane spanned by the η-axis at ω = π/2 and the contour at fixed ϱ = Λϱ between ω = 0 and ω = π/2. The integral – 22 – along the η-axis can be decomposed into the regions of η ∈[0, ηn] and η ∈[ηn, Λϱ(ϵu, π/2)]; in the latter region, the line charge density takes the form λ(η) = η + m1. In all, along the η-axis can be decomposed into the regions of η ∈[0, ηn] and η ∈[ηn, Λϱ(ϵu, π/2)]; in the latter region, the line charge density takes the form λ(η) = η + m1. In all, I[Σ] 32κ3 11 = Z ηn 0 dηϖ(η)2 \| {z } I1 + Z Λϱ(ϵu,π/2) ηn dη(η + m1)2 \| {z } I2 − Z ω=π/2 ω=0 ˙V 2 Λrd(Λρ(ϵu, ω)) \| {z } I3 , (4.20) (4.20) where ˙V 2 Λ in I3 is held at fixed r = Λr in the integration over ω. where ˙V 2 Λ in I3 is held at fixed r = Λr in the integration over ω. Λr Let’s take each of the I’s individually, starting with I2. Performing the integral is trivial and leads to the small ϵu expansion JHEP04(2024)120 I2 = 8m2 1 3ϵ6u + 4m3 1 ϵ4u + 13m3 1+2m3 6ϵ2u + 8m3+m3 1−18m2 1ηn−18m1η2 n−6η3 n 18 +... . (4.21) (4.21) The integral I3 can also be easily taken. First, we expand the integrand using the large ϱ expansions of the potential in eq. (A.10). Then after computing dΛr(ϵu, ω), we expand in small ϵu and integrate term-by-term in ω ∈[0, π/2], which gives The integral I3 can also be easily taken. 4.2 Electrostatic solutions (4.26) (4.26) – 23 – – 23 – We then arrive at the expression for the regulated I: We then arrive at the expression for the regulated I: I[Σ] −I[∅] 32κ3 11 = 2m3 + 13m3 1 15 + 1 3 n X a=0 p2 a+1(η3 a+1 −η3 a) + n X a=0 δa+1pa+1(η2 a+1 −η2 a) + n X a=0 δ2 a+1(ηa+1 −ηa)), (4.27) I[Σ] −I[∅] 32κ3 11 = 2m3 + 13m3 1 15 + 1 3 n X a=0 p2 a+1(η3 a+1 −η3 a) + n X a=0 δa+1pa+1(η2 a+1 −η2 a) n (4.27) + n X a=0 δ2 a+1(ηa+1 −ηa)), (4.27) which recovers the result of the integral for the non-compact electrostatic solutions in [14]. Thus, the regulated minimal area is given by Amin[Σ] −Amin[∅] = 29π4κ3 11 R2 ϵ2w −log 2R ϵw + O(1) ! (I[Σ] −I[∅]). (4.28) (4.28) JHEP04(2024)120 Proceeding with the computation of aΣ, we feed eq. (4.28) in eq. (4.1) to get SEE. Computing the log derivative with respect to R of the regularized minimal area functional at R = 0 gives the universal part of defect entanglement entropy Proceeding with the computation of aΣ, we feed eq. (4.28) in eq. (4.1) to get SEE. Computing the log derivative with respect to R of the regularized minimal area functional at R = 0 gives the universal part of defect entanglement entropy R∂R(SEE[Σ] −SEE[∅]) = −(I[Σ] −I[∅]), (4.29) (4.29) where we mapped to the field theory variables using G(11) N = 213π4κ3 11 and κ11 = L3/8N. Using eq. (2.8) we can read off the A-type anomaly coefficient using d2 = −1 3(m3 1 −m3) where we mapped to the field theory variables using G(11) N = 213π4κ3 11 and κ11 = L3/8N. Using eq. (2.8) we can read off the A-type anomaly coefficient using d2 = −1 3(m3 1 −m3) aΣ = (Pn a=1 kaηa)3 4 + 1 12 n X a=0 (p2 a+1(η3 a+1 −η3 a) + 3δa+1pa+1(η2 a+1 −η2 a) + 3δ2 a+1(ηa+1 −ηa)). (4.30) Recall that the ηa are ordered by 0 = η0 < η1 < . . . < ηn, and so (ηj a+1 −ηj a) > 0 for any j ∈N and for all a. Further, the orbifold parameters are non-negative ka ∈N, and so by definition are the pa, and in addition 2δa ∈N. Hence, we see that aΣ ≥0. 4.2 Electrostatic solutions First, we expand the integrand using the large ϱ expansions of the potential in eq. (A.10). Then after computing dΛr(ϵu, ω), we expand in small ϵu and integrate term-by-term in ω ∈[0, π/2], which gives I3 = 8m3 1 3ϵ6u + 8m3 1 3ϵ4u + 5m3 1 + 2m3 6ϵ2u + m3 1 + 14m3 45 + . . . . (4.22) (4.22) Combining I2 and I3, we see Combining I2 and I3, we see I2 −I3 = 4m3 1 3ϵ4u + 4m3 1 3ϵ2u + 4m3 + m3 1 −10ηn(η2 n + 3ηnm1 + 3m2 1) 30 + . . . . (4.23) (4.23) Lastly, we need to take care of the integral I1. To do so, we break up the integral over η ∈[0, ηn] into a sum over the intervals [ηa, ηa+1] for a = 0, . . . , n −1 with η0 = 0. Then, using ϖa = pa+1η + δa+1 over each interval we find = 1 3 n−1 X a=0 p2 a+1(η3 a+1 −η3 a) + 3δa+1pa+1(η2 a+1 −η2 a) + 3δ2 a+1(ηa+1 −ηa) . (4.24) (4.24) Combining everything we get Combining everything we get I[Σ] 32κ3 11 = 4m3 1 3ϵ4u + 4m3 1 3ϵ2u + 4m3 + m3 1 30 + 1 3 n X a=0 (p2 a+1η3 a+1 −η3 a) + n X a=0 δa+1pa+1(η2 a+1 −η2 a) + n X a=0 δ2 a+1(ηa+1 −ηa), (4.25) (4.25) where we slightly abuse the notation by setting ηn+1 = 0 in this sum to make the expressions a bit more compact. The ϵu divergences in I[Σ] need to be regulated. We again adopt the background subtraction scheme as before, where the background vacuum AdS7 × S4 solution is obtained by taking n = 1 and k1 = 1. Taking this limit in eq. (4.25) yields I[∅] 32κ3 11 = 4m3 1 3ϵ4u + 4m3 1 3ϵ2u −5m3 1 6 + . . . . 4.2 Electrostatic solutions Note that the inequality is saturated at n = k1 = 1 i.e. aΣ = 0, which is expected since this line charge density configuration corresponds to having no defect. For completeness, we can rewrite aΣ in terms of the ranks, Na, of the factors in the Levi subalgebra l ⊂AN−1 and their associated monopole charges, ka, aΣ = N3 32 −1 96 n X a=1  1 + 2ka k2a N3 a + n X b=a+1 Nakb N2 a k2a + 3N2 b k2 b ! . (4.31) (4.31) While the definite sign of aΣ is a bit less clear in terms of the gauge algebra data, it is nonetheless non-negative following from eq. (4.30). As we mentioned toward the end of section 3.2, there is a non-trivial consistency check of our results in eq. (4.30) from the comparison to the one-charge (q2 →0) solutions. Setting n →1 and k1 →1/√1 −4q1 in eq. (4.30) results in aΣ n=1 = N3 48 1 + 2q1 − p 1 −4q1 . (4.32) (4.32) Looking back to the computation of aΣ for the two-charge solutions, we need the largest root of Q(y) with q2 →0, which is simply y+(q1) = 1 2(1 + √1 −4q1). Plugging y+(q1) into eq. (4.10) exactly matches eq. (4.32). Looking back to the computation of aΣ for the two-charge solutions, we need the largest root of Q(y) with q2 →0, which is simply y+(q1) = 1 2(1 + √1 −4q1). Plugging y+(q1) into eq. (4.10) exactly matches eq. (4.32). – 24 – – 24 – We now compare aΣ to the computations of the ‘defect central charge’ for these solutions. The ‘defect central charge’ was computed in [14] using the standard formula for the central charge c4d of standalone 4d N = 2 SCFTs at large N holographically dual to AdS5 solutions in M-theory [70] c4d = 25π3κ3 11 (2πℓP )9 Z M6 ˙V σ 2V ′′ ! 3 2 , (4.33) (4.33) which applies to 11d metrics of the form ds2 11 = κ2 11 ˙V σ 2V ′′ ! 1 3 (ds2 AdS5 + ds2 M6). (4.34) JHEP04(2024)120 (4.34) This formula had been used to find the holographic central charge dual to electrostatic solutions with compact internal space engineering irregular punctures [18, 71]. Despite the integrals in eqs. 4.2 Electrostatic solutions (4.27) and (4.33) having the same form, the crucial difference is in the interpretation of the result: the relative difference between aΣ and c4d is a factor of −2d2/5. Lastly, while monotonicity of the universal part of the defect sphere EE has yet to be tested for 4d DCFTs, in the case of a co-dimension 4 Wilson surface in 6d SCFTs the universal defect contribution to the sphere EE does not behave monotonically under defect RG flows (see e.g. [72]). Due to the relative sign in ∆SEE and the fact that only aΣ is known to obey a weak defect a-theorem,13 it is expected that eq. (4.29) is not a monotone along defect RG flows. 13The recent entropic proof in [43] of the irreversibility of defect RG flows in addition to the dilaton effective action methods (á la [37]) in [73] have firmly established the existence of at least a weak defect a-theorem. 5 On-shell action Given a solution to the SUGRA equations of motion, one of the most basic quantities that one can compute is the on-shell action. Holographically, the on-shell action is mapped to the free energy of the theory, and so with an even dimensional spherical boundary, it has universal divergences related to anomalies. In this section, we evaluate the on-shell action for the 11d uplift of the two-charge solutions. This will facilitate a comparison to the same quantity computed in the realization as a domain wall in 7d N = 4 gauged SUGRA, which was previously found in [13]. Crucially, by employing the equations of motion, the 11d action can be recast as a boundary integral. Then by fixing a gauge where C6 has vanishing components along cycles, e.g. the dz ∧ΥAdS5 cycle, that vanish at y = y+, the regulated on-shell action computed in the background subtraction scheme is then symmetric in the qI, as expected from the uplift of the 7d theory. Interestingly, the 11d result does not match the on-shell action computed in [13] for the 7d domain wall. We argue the discrepancy is related to the change of variables defined in eq. (A.5). On the 11d side, the change of variables is induced by the transformation to FG gauge. In the 7d theory, this corresponds to making a large gauge transformation, which can be seen in the uplift given in eq. (2.16). Interestingly, the choice of FG gauge seems to correspond to a singular gauge in the 7d theory. We note that the change of coordinates mixes spatial rotations around the defect with the R-symmetry, which is specific to co-dimension two defects. 13The recent entropic proof in [43] of the irreversibility of defect RG flows in addition to the dilaton effective action methods (á la [37]) in [73] have firmly established the existence of at least a weak defect a-theorem. – 25 – Moving on, the starting point for computing the on-shell action for the two-charge solutions in eq. (2.16) is the bosonic part of the 11d SUGRA action Moving on, the starting point for computing the on-shell action for the two-charge solutions in eq. 5 On-shell action (2.16) is the bosonic part of the 11d SUGRA action S = 1 16πG(11) N Z M d11x √−g11 R −1 48FMNPQF MNPQ + 1 8πG(11) N Z ∂M KΥ∂M + SCS, (5 1) (5.1) where Υ∂M is the natural volume form associated to the metric induced on the boundary ∂M, while K is the trace of the boundary extrinsic curvature KMN = −1 2(∇MνN + ∇NνM) with νM denoting the components of the outward-pointing normal vector to ∂M and where capital Latin indices M, N ∈{0, . . . , 10}. Using the equations of motion for the 11d metric we can write the bulk term as JHEP04(2024)120 √−g11 R −1 48FMNPQF MNPQ d11x = −1 3F4 ∧⋆F4. (5.2) (5.2) Note that for this particular solution, the four-form flux obeys the equation (5.3) d ⋆F4 = 0, (5.3) d ⋆F4 = 0, and consequently the Chern-Simons term SCS vanishes. As a further consequence of the equations of motion for the four-form flux, we can freely exchange ⋆F4 for dC6, which due to C6 being better behaved will make the following computation a bit easier. Using this fact and the bulk equations of motion, the bulk integrand can be expressed as a total derivative. Thus, the on-shell action can be written as a boundary integral SOS = 1 16πG(11) N Z ∂M 2KΥ∂M −1 3F4 ∧C6 =: SOS,GHY + SOS,bulk. (5.4) (5.4) The particular solutions we are interested in are asymptotically locally AdS7 × S4. So, in order to regularize the boundary integral, we first map the metric into FG form as in eq. (A.4) using the explicit asymptotic coordinate transformation derived in eq. (A.3). That is, we will define a regulating hypersurface at u = ϵu that will become ∂M as we take ϵu →0. Note that due to the presence of an AdS5 factor, an additional regularization procedure will have to be applied, which we will address later. The particular solutions we are interested in are asymptotically locally AdS7 × S4. So, in order to regularize the boundary integral, we first map the metric into FG form as in eq. (A.4) using the explicit asymptotic coordinate transformation derived in eq. (A.3). That is, we will define a regulating hypersurface at u = ϵu that will become ∂M as we take ϵu →0. 5 On-shell action (5.8) (5.8) JHEP04(2024)120 Using this gauge transformation we find the asymptotic expansion of ˜C6 to be Using this gauge transformation we find the asymptotic expansion of ˜C6 to be Using this gauge transformation we find the asymptotic expansion of ˜C6 to be ˜C6 = L6 1 u6 + 3 2u4 − 1 16u2 (2q1−3(5+q2)+10q2c2ℵc2 θ+5(q2−2q1)c2θ) dz ∧ΥAdS5+... ( ˜C6 = L6 1 u6 + 3 2u4 − 1 16u2 (2q1−3(5+q2)+10q2c2ℵc2 θ+5(q2−2q1)c2θ) dz ∧ΥAdS5+... . (5.9) (5.9) It is useful to note that the ΥAdS5 ∧dz component of ˜C6 does not change under the coordinate transformation ϕI →φI, once the shift in the gauge transformation is taken into account.i It is useful to note that the ΥAdS5 ∧dz component of ˜C6 does not change under the coordinate transformation ϕI →φI, once the shift in the gauge transformation is taken into account. Next, we need to find F4, which we can easily compute from eq. (2.21). We then map into FG coordinates, fix ˆg = 2, and expand in small u. Keeping the most relevant Next, we need to find F4, which we can easily compute from eq. (2.21). We then map into FG coordinates, fix ˆg = 2, and expand in small u. Keeping the most relevant singular terms, we find F4 = L3 8 (" 3c2 θsθdφ1 ∧dθ + c3 θ 2 5s2 θ (2q1 −q2c2ℵ−q2) du ∧dφ1 + 16q1du ∧dz u3 # ∧ΥS2 + sθ\|cℵ\| 2 du ∧dφ1 ∧ 5q2c2 θs2ℵdθ ∧dφ2 + 8q2dz ∧ 2sℵ cℵ dθ −s2θdℵ u3 ) + . . . . (5.10) (5.10) Now that we have the asymptotics of the metric, ˜C6, and F4, we are in position to compute the on-shell action for the two-charge solutions. To begin, we first examine the Gibbons-Hawking- York (GHY) term. We note that after mapping to FG coordinates as in eq. (A.4), the volume form on the regulating cutoff slice at u = ϵu can be easily seen to have small ϵu expansion = L10 16 1 ϵ6u + 1 ϵ4u + 5 16ϵ2u + 5 5c2θ(q2−2q1)+2q1+q2(10c2 θc2ℵ−3) 432 ! ΥAdS5∧dz∧ΥS4+... , (5.11) where we denote ΥS4 := \|cℵ\|c2 θsθdϕ1 ∧dϕ2 ∧dθ ∧dℵ. 5 On-shell action Note that due to the presence of an AdS5 factor, an additional regularization procedure will have to be applied, which we will address later. Before beginning the computation in earnest, we will need the asymptotic u ≪1 expansions of F4 and C6. First, we compute C6 from eq. (2.21), which yields C6 = L6 ( 1 2q2c2 ζc2 ψdϕ2 + 1 2q1s2 ζdϕ1 + " y(y2 + q2) − c2 ζ 2y (q2c2ψ (y (y −a2 −1) + q1) C6 = L6 ( 1 2q2c2 ζc2 ψdϕ2 + 1 2q1s2 ζdϕ1 + " y(y2 + q2) − c2 ζ 2y (q2c2ψ (y (y −a2 −1) + q1) +2q1y (a1 −y + 1) + q2y (y −a2 −1) −q2q1) # dz ) ∧ΥAdS5. (5.5) +2q1y (a1 −y + 1) + q2y (y −a2 −1) −q2q1) # dz ) ∧ΥAdS5. (5.5) (5.5) We can then use the residual gauge freedom to shift C6 7→C6 + dΛ5 =: ˜C6 such that ˜C6 has no surviving dz ∧ΥAdS5 components at y = y+. At y = y+, we can use the values for aI determined from AI(y+) = 0 to show C6(y+) = L6 1 2q2c2 ζc2 ψdϕ2 + 1 2q1s2 ζdϕ1 + y+H2(y+)dz ∧ΥAdS5, (5.6) (5.6) – 26 – – 26 – where the terms in ΥAdS5 ∧dz depending on the angular coordinates vanish due to a common factor of Q(y+) appearing in their coefficients. By demanding that the ΥAdS5 ∧dz part of C6 vanishes at y = y+, we find the appropriate gauge transformation to be where the terms in ΥAdS5 ∧dz depending on the angular coordinates vanish due to a common factor of Q(y+) appearing in their coefficients. By demanding that the ΥAdS5 ∧dz part of C6 vanishes at y = y+, we find the appropriate gauge transformation to be Λ5 = −zL6y+H2(y+)ΥAdS5. (5.7) (5.7) To implement the FG cutoff, we change coordinates to ϕI →φI as defined in eq. (A.5). This modifies the vanishing condition for C6 at y = y+, resulting in a shifted gauge transformation with the shift given by To implement the FG cutoff, we change coordinates to ϕI →φI as defined in eq. (A.5). This modifies the vanishing condition for C6 at y = y+, resulting in a shifted gauge transformation with the shift given by Λ5 →Λ5 + zL6c2 ζc2 ψa2q2 + zL6s2 ζa1q1. 5 On-shell action A quick calculation also shows the trace of the extrinsic curvature on the cutoff slice to be given by K = −6 L + 2ϵ2 u L −3ϵ4 u 4L + 25c2θ(q2−2q1)+10q1+50q2c2 θc2ℵ−15q2+9 ϵ6 u 72L +..., (5.12) K = −6 L + 2ϵ2 u L −3ϵ4 u 4L + 25c2θ(q2−2q1)+10q1+50q2c2 θc2ℵ−15q2+9 ϵ6 u 72L +..., (5.12) (5.12) where we have dropped terms at O(ϵ8 u) that depend on the charges but do not contribute to the final result as ϵu →0. Thus, we find SOS,GHY = −vol(AdS5) π2L9 8G(11) N 2 ϵ6u + 4 3ϵ4u + 5 24ϵ2u + . . . . (5.13) (5.13) – 27 – – 27 – Note that despite K and Υ∂M containing non-trivial dependence on the charges, the end result in eq. (5.13) is independent of the charges to O(ϵ0 u), and the ϵ0 u part of the GHY term explicitly vanishes. Moving on to find SOS,bulk, using eqs. (5.9) and (5.10), pulling back on to the u = ϵu hypersurface, and combining with eq. (5.13), we arrive at SOS,bulk = −vol(AdS5) π2L9 16G(11) N 2 3ϵ6u + 1 ϵ4u + 5 8ϵ2u −2q1(q2 + y+(2 + 3y+)) 15y+ (5.14) −32q2 + 48q2y+ + 80y3 + −25 120 ! + . . . . (5.14) JHEP04(2024)120 Thus, combining eqs. (5.13) and (5.14) and subtracting of the on-shell action for the AdS7 × S4 vacuum in eq. (B.7), which is recovered by setting qI = aI = 0 and y+ = 1, the full regulated on-shell action is SOS −S(vac) OS = vol(AdS5) π2L9 120y+G(11) N q1q2 + (q1 + q2)y+(2 + 3y+) + 5y+(y3 + −1) = −vol(AdS5) π2L9 24G(11) N 1 −y2 + −1 5 2q2 1 q1 + y2 + + 2q2 2 q2 + y2 + !! = −vol(AdS5) π2L9 24G(11) N 1 −y2 + −1 5(2a1q1 + 2a2q2) . (5.15) (5.15) Note that choosing a different Λ5 while maintaining regularity at y = y+ does not change the final result. Further, using the form of the regulated AdS5 volume in appendix B.2, the log divergent part of the on-shell action for the two-charge solutions is given by Note that choosing a different Λ5 while maintaining regularity at y = y+ does not change the final result. 6 Discussion JHEP04(2024)120 In this work, we have analyzed solutions in 11d SUGRA that holographically describe 1/4− and 1/2−BPS co-dimension 2 defects in the 6d AN−1 N = (2, 0) SCFT at large N. In this work, we have analyzed solutions in 11d SUGRA that holographically describe 1/4− and 1/2−BPS co-dimension 2 defects in the 6d AN−1 N = (2, 0) SCFT at large N. Our holographic computations of the defect contribution to the one-point function of the stress energy tensor have revealed simple expressions for the defect Weyl anomaly coefficient d2 in section 3. For the 1/4-BPS two-charge solutions specified by charges q1, q2, we have found that d2 ∝N3(q1 + q2). For the 1/2-BPS electrostatic solutions determined by a potential solving a Laplace-type equation with moments mj, d2 ∝(m3 1 −m3) ∝N3 −P a N3 a where N = P a Na. Using the 4d form of the defect ANEC, which states d2 ≤0, we have demonstrated that all of the allowed two-charge solutions found in [13] and the electrostatic solutions in [14] obey the bound and are thus consistent with this known defect energy condition [43]. We were also able to compare against a similar computation for the two-charge solutions done in 7d N = 4 gauged SUGRA, and found an agreement with ⟨Tij⟩in [13]. ⟨ j⟩ [ ] In section 4, we used the tools developed in [63, 64] to holographically compute the contribution of flat, co-dimension 2 defects to the EE of a spherical region in the dual field theory. By isolating the universal, log-divergent part of the defect sphere EE, we were able to find closed form expressions for the A-type anomaly aΣ for both defect systems considered. Since we know that the universal part of the defect sphere EE, is a linear combination of aΣ and d2 as in eq. (2.7), by combining ∆⟨Tij⟩and ∆SEE, we have a direct computation of aΣ: for the two-charge solutions we found aΣ ∝N3(1 −y2 +) where y+ is the largest root of the quartic polynomial in eq. (2.12b), while aΣ for the electrostatic solutions in eq. (4.30) is a complicated function of the data of the line charge distribution that specifies the solution. For the electrostatic solutions, we have shown that the computation of the holographic ‘central charge’ in [14] is proportional to the universal part of the defect sphere EE. 14To be clear, there is a discrepancy in the normalization between the expression in eq. (5.19) and that found in eq. (4.7) of [13]. Our expression normalizes the sign convention in the computation of the on-shell action between uplifted 11d solution and the 7d domain-wall description and fixes missing factors of 2π, coming from the integral over z, and L5. 5 On-shell action Further, using the form of the regulated AdS5 volume in appendix B.2, the log divergent part of the on-shell action for the two-charge solutions is given by S(ren) OS log = − N3 1920y+ q1q2 + (q1 + q2)y+(2 + 3y+) + 5y+(y3 + −1) = N3(4q1q2 −2(q1 + q2)y+(1 −y+) + 5y+(1 −y2 +)) 1920y+ . (5.16) (5.16) Before comparing to the 7d result, we first consider what would happen if we were to perform the computation without making the coordinate transformation in eq. (A.5). In particular, we consider φI = ˜φI + 2aInIz. (5.17) (5.17) The bulk integrand F4 ∧C6 picks up new cross terms which are absent when n1 = n2 = 0. The remaining terms are unmodified due to the regularity condition imposed on C6 at y = y+. The new terms are The bulk integrand F4 ∧C6 picks up new cross terms which are absent when n1 = n2 = 0. The remaining terms are unmodified due to the regularity condition imposed on C6 at y = y+. The new terms are 3L9 8 n2q2a2c2 ζc2 ψ + n1a1q1s2 ζ ΥS4 ∧ΥAdS5 ∧dz + . . . ⊂F4 ∧C6 . (5.18) (5.18) Integrating these along the boundary changes the on-shell action as follows, SOS 7−→SOS −vol(AdS5) π2L9 24G(11) N 1 5(2n1a1q1 + 2n2a2q2) . (5.19) (5.19) – 28 – – 28 – In particular, choosing n1 = n2 = 1 reproduces the 7d result given in [13]14 and corresponds to using the original coordinates ϕI. Examining the uplift defined in eq. (2.16), we can see that the coordinate transformations given by eq. (5.17) and eq. (A.5) correspond to large gauge transformations in the 7d description. Thus from the 7d point of view, the choice of FG gauge, with n1 = n2 = 0 corresponds to a singular gauge choice. Uplifts of lower dimensional solutions to higher dimensional ones are not necessarily unique, and it is possible that there are other 11d geometries which correspond to other gauge choices, such as one with n1 = n2 = 1. 6 Discussion Further, we were able to show that the complicated sum over line charge density data that appears in aΣ is the same sum that determines the large N ‘central charge’ c4d(= a4d) for the compact electrostatic solutions describing 4d N = 2 SCFTs; the important difference is that the defect aΣ has an additional contribution of N3/32. In both classes of defects, we have also shown that aΣ ≥0, where the inequality is only saturated for a trivial defect. Curiously, in section 5, we showed that the holographically renormalized on-shell action for the 11d uplift of the two-charge solutions using the full form of the radial cutoff in FG – 29 – gauge and found that the log divergent part of the action cannot be written in terms of either aΣ, as was expected from the same computation done in 7d gauged SUGRA description of the two-charge defects [13]. We ultimately identified the source of this discrepancy in the parametrization of angular variables, ϕI versus φI, in the bulk integrand. The map to FG gauge results in a redefinition of ϕI →φI which mixes the U(1) normal bundle rotations around the defect and the U(1) R-symmetry. While the uplifted 11d SUGRA description is perfectly regular after the map to φI, the 7d gauged SUGRA picture sees this redefinition as a large gauge transformation resulting in a singular gauge. At the level of the on-shell action in the original ϕI coordinates, eq. (5.16) picks up extra terms which, accounting for the normalization discussed in footnote 14, recovers the 7d results in [13]. JHEP04(2024)120 With the holographic predictions for aΣ and d2 in hand, let us compare to results in the field theory at large N. We will focus entirely on the 1/2-BPS electrostatic solutions in the following comparisons. Defect supersymmetric Casimir energy. In ordinary 4d SCFTs with R-symmetry placed on S1 β × S3, the supersymmetric localized partition function can be decomposed as a product of an exponential prefactor multiplying the superconformal index ZS1 β×S3 = e−βECI. (6.1) (6.1) The supersymmetric Casimir energy (SCE), EC, can be expressed in terms of the conformal anomalies a and c [74, 75] of the theory, the equivariant integral of the anomaly polynomial [76], or ‘t Hooft anomalies [77]. 15Evidence for a version of this conjecture for d = 2 defects gathered from studying various examples appeared to support the claim, and in [80], a relation between the SCE and hT was established using the chiral algebra description of the defect insertion, which gives a much stronger argument for EC being controlled by d2. We thank Maxime Trépanier for pointing out the chiral algebra proof in [80] to us. 6 Discussion Given the results in [78] for the localized partition functions a 1/2-BPS co-dimension 2 defect in a 6d N = (2, 0) AN−1 SCFT labelled by ϑ wrapping Σ = S1 β × S3 ⊂S1 β × S5, it was conjectured in [79] that the change in the exponential prefactor due to the introduction of the defect was in fact the defect SCE and could be related to defect conformal anomalies.15 Now that we have holographic predictions for two defect anomalies, we can look for a superficial match to this field theory quantity. As a very brief overview, we start the comparison by putting the ambient theory on the squashed S1 β × S5 b and reducing along the S1 factor. The localized partition function of the 6d N = (2, 0) AN−1 SCFT in the unrefined limit becomes the partition function of 5d N = 2 U(N) super-Yang-Mills theory on S5 b, which determines the ambient SCE EC[∅] ≡c 24 , where c = N(N2 −1)(b + b−1)2 + N −1. (6.2) (6.2) The quantity c in this picture is the central charge of the 2d WN-algebra on the plane orthogonal to the directions that defect will eventually wrap [7, 78]. The introduction of a co-dimension 2 defect breaks the gauge algebra to the Levi subalgebra l = s [Ln a=1 u(Na)]. The most general 1/2-BPS defect configuration allows for monodromy parameters ⃗w = (w1, . . . , wn) for the Levi factors. The change in the SCE due to introducing the defect along – 30 – Σ labelled by ϑ : sl(2) →g with monodromy parameters ⃗w was found to be given by [78, 79] EC[Σ]ϑ,⃗w −EC[∅] = 1 2(b + b−1)2[(ˆϱl, ˆϱl) −(ˆϱg, ˆϱg)] + 1 2(⃗w, ⃗w), (6.3) = −1 6 N3 − n X a=1 N3 a −3(⃗w, ⃗w) ! . (6.3) In the second line we took the limit b →1, and replaced the scalar product of the Weyl vectors — denoted ˆϱl and ˆϱg for l and g = su(N), respectively — with (ˆϱl, ˆϱl) = 1 12 n X a=1 (N3 a −Na), (ˆϱg, ˆϱg) = 1 12(N3 −N). (6.4) (6.4) JHEP04(2024)120 Turning off the monodromy parameters16 (wa = 0) in eq. (6.3) we see the superficial relation EC[Σ]ϑ,⃗0 −EC[∅] = 4d2\|ka→1 , (6.5) (6.5) where on the right hand side we take all orbifold parameters ka →1 in eq. (3.26b). 6 Discussion Since the expression for the defect SCE in terms of explicit defect Weyl anomalies is still unknown and 4d DCFTs have 23 possible parity even anomalies, we cannot definitively state that the defect SCE is determined solely by d2. We note, though, that a similar relation was found for co-dimension 4 Wilson surface defects: the defect SCE in that case was also related to the 2d DCFT equivalent of d2. Since 2d DSCFT preserving at least N = (2, 0) supersymmetry have only two independent Weyl anomalies,17 which for the Wilson surface defect can be clearly distinguished from one another [30], it was conjectured that d2 alone fixed the defect SCE [79]. So, while it is not inconceivable that d2 could appear in the defect SCE for co-dimension 2 defects, we leave establishing the precise relation for future work. R-anomalies. Ordinarily in 4d SCFTs, there are non-perturbative formulae that relate the A-type and B-type Weyl anomalies to ‘t Hooft anomalies for the superconformal R symmetry [38]. In [73], it was conjectured that aΣ obeys the same relation to defect R- anomalies as a standalone theory:18 aΣ = 9krrr −3kr 32 , (6.6) (6.6) 16In light of the compact LLM-type solutions found recently in [81] where the additional internal U(1) symmetry is broken by the presence of scalar fields, which are interpreted as monodromy parameters, it may be possible to pin down a more precise relation between EC and defect anomalies by computing ⟨Tµν⟩if similar non-compact solutions allowing for wa ̸= 0 can be constructed.i 17This was first proven for superconformal surface defects in 4d N = 2 SCFTs in [31], and later, it w proven for 2d defects in the 6d N = (2, 0) theory in [82]. 18 18It was also conjectured that a B-type defect anomaly built out of the square of intrinsic Weyl tensor (cΣ\| ¯ W\|2) obeys the usual relation [38] cΣ = 9krrr −5kr 32 . However, the basis used in [9] did not include \| ¯ W\|2. From the Gauss-Codazzi and Ricci relations, \| ¯ W\|2 is related to several anomalies in the original basis (none of which include d2). So it is unclear at the this time, what observables can be used to compute cΣ. Though it is reasonable to expect that the defect limit of ⟨TµνTρσ⟩may be the appropriate correlator to compute cΣ, proving this is the subject of future work. 6 Discussion – 31 – where krrr and kr are the cubic and mixed U(1)r R-anomalies. Importantly for the defect theory written in 4d N = 1 language, the superconformal rΣ symmetry is a linear combination of the Cartan generator of the ambient SU(2)R R-symmetry and the generator of normal bundle rotation Mφ [73] rΣ = 2 3(2r6d −Mφ). (6.7) (6.7) It was further stated in [73] that precisely for the types of defects holographically described by the electrostatic solutions considered above, in order to determine the R and mixed anomaly we should use the counting formulae [6] krrr = 2 27(nv −nh) + 8 9nv, kr = 2 3(nv −nh), (6.8) JHEP04(2024)120 (6.8) where nv is the number of 4d vector multiplets and nh is the number hypermultiplets. In turn, both nh and nv are determined by the Young diagram data. As we have pointed out around eq. (4.30), the defect A-type anomaly contains a con- tribution that is precisely of the form of the central charge c4d of 4d SCFTs engineered from irregularly punctured Riemann surface compactifications of 6d N = (2, 0) AN−1 series SCFTs dual to electrostatic solutions of the type studied above. Further, in [6, 18], a match was found between the holographic computation of c4d of the dual 4d SCFT and the large N behavior of the central charge computed in the field theory using the R-anomalies and eq. (6.8). However since we have found aΣ = c4d +N3/32, it is clear that the naïve application of eq. (6.6) and eq. (6.8) do not directly match. 6.1 Future directions and open questions As we have seen in the computation of the A-type anomaly for co-dimension 2 defects in the 6d N = (2, 0) AN−1 series SCFTs, there is a connection to the central charge of a 4d SCFT engineered on a Riemann surface with regular punctures, at least in the large N limit. It is natural, then, to wonder how the rest of the data contained in the other 22 parity even defect Weyl anomalies can be used to characterize the lower dimensional theory, or whether the remaining unknown defect Weyl anomalies are vanishing or fixed by aΣ and d2. For BPS Wilson surfaces in 6d preserving at least 2d N = (2, 0) supersymmetry, the defect supersymmetry imposes non-trivial relations among the B-type defect Weyl anomalies [82], but as of yet, there is no known relation imposed by 4d N = 2 defect supersymmetry. JHEP04(2024)120 A special case of dimensional reduction of the 6d N = (2, 0) AN−1 theory is taking the Riemann surface to be T2, which reduces to 4d N = 4 SU(N) super Yang-Mills theory. The co-dimension 2 defects labelled by ϑ : sl(2) →su(N) in the parent theory that we have holographically studied above wrapped on T2 reduce to Gukov-Witten type defects. In the absence of complex structure deformations on T2, all of the defect Weyl anomalies are equal to one another and are ∝N2 −P a N2 a [28–30, 95], which is closer in appearance to d2 in eq. (3.26b) than aΣ in eq. (4.30). However, an exact relation to determine the anomalies of the Gukov-Witten defect from the higher dimensional defect anomalies is as of yet unknown. Defect Weyl anomalies and ‘t Hooft anomalies. As we saw in the attempt to match the any of the holographic results for aΣ or d2 to large N field theory computations, there are points of tension that should be resolved. One of the biggest issues, though, is that the putative relation between defect ‘t Hooft anomalies and defect Weyl anomalies seemed to disagree with the holographic results. While it remains a possibility that the issue stems from the holographic side of the story, there is an open question on the field theory side that must be addressed as well. Namely, the formulae conjectured in [73] only relate two of the twenty-three parity even defect Weyl anomalies to the defect R-anomalies for co-dimension ≥2 4d defects. 6.1 Future directions and open questions The work that we have presented in this paper is only scratching the surface of 4d defects. While a full accounting of all of the defect Weyl anomalies of these systems through computing entropies, correlation functions, or other physical quantities is not currently possible, there are a number of questions opened up by our analysis that we will leave for future work. Probe branes. Even though we have access to the full 11d SUGRA bubbling geometry solution, it is useful to consider limit cases where we can instead appeal to a probe brane construction. By finding κ-symmetric embeddings of probe M5-branes in an AdS7 × S4 background wrapping AdS5 ⊂AdS7 and an S1 living either in the internal S4 or in the AdS7, we expect to be able to holographically study defects engineered by Young diagrams associated to totally symmetric or totally antisymmetric representations of su(N) similar to the co-dimension 4 Wilson surface defects from M2 and M5 probe branes [72, 83, 84]. One advantage of studying these defect systems using probe brane holography is that we will have clearer access to the study of defect RG flows, which will provide holographic tests of the defect aΣ-theorem in a strongly coupled theory, a means to study defect phase transitions, and a setting to test the monotonicity of the defect sphere EE along an RG flow [72]. Further taking inspiration from AdS5 holography [85–87], if one was able to construct a κ-symmetric probe M5 brane embedding in global AdS7, say with an S1 × S5 boundary, one could try to compare to recent results in type IIB probe brane holography and supersymmetric localization in 3d/5d systems on a sphere [88, 89]. These questions are currently being investigated in work currently in progress. – 32 – Dimensional reduction. By (partial) topologically twisted dimensional reduction on a Riemann surface or a 3-manifold, 6d SCFTs can be used to engineer large classes of 4d [22, 90] and 3d [91, 92] theories. Further, we can enrich the algorithm to determine the lower dimensional theory by starting from a 6d theory deformed by their natural co-dimension 2 and 4 defects to end up with a dimensionally reduced theory possibly with defects [21, 93, 94]. 6.1 Future directions and open questions That is, only the E4 and \|W\|2 structures in the defect anomaly have been supersymmetrized. A similar supersymmetrization of the defect Weyl anomaly for 2d defects limited to be sensitive only to the intrinsic geometry of the defect submanifold was carried out in [96]. This naturally leads one to wonder if it is possible to supersymmetrize the full defect Weyl anomaly including the anomalies containing the second fundamental form and normal bundle curvature in order to arrive at a complete set of non-perturbative formulae for defect Weyl anomalies. Acknowledgments The authors would like to thank Pieter Bomans, Michael Gutperle, and Andy O’Bannon for useful discussions throughout this work. We would also like to thank Pieter Bomans and Michael Gutperle for comments on the draft. JE would also like to thank the EIC – 33 – Theory Institute at BNL for partial financial support and warm hospitality while this work was being completed. The work of PC is supported by a Mayflower studentship from the University of Southampton. The work of BR is supported by the INFN. The work of BS is supported in part by the STFC consolidated grant ST/T000775/1. This material is based upon work supported by the U.S. Department of Energy, Office of Science, Office of High Energy Physics under Award Number DE-SC0024557. Disclaimer. This report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof. JHEP04(2024)120 A Fefferman-Graham coordinates The starting point for computing holographic quantities associated with the two-charge solutions and electrostatic solutions is finding the asymptotic transformation which maps their respective metrics into FG gauge. In this appendix, we will first derive the transformations of eq. (2.19) and find the asymptotic expressions for the metric functions in FG gauge. We will also derive the transformation of eq. (2.23a) into FG gauge. In this process, we will find necessary conditions on the mixing of two of the angular coordinates that allow for the metrics to be put into FG form. The interpretation of this mixing of angular coordinates is interpreted in the field theory language as an identification of the defect superconformal R-symmetry. A.1 Two-charge solutions In this subsection, we will focus on putting the two-charge solutions in FG gauge. The explicit forms of the metric functions in eq. (2.19) are as follows: ˆf2 AdS = κ2/3 "c2 ζ q1+y2q2−q2c2ψ+2y2 2y +y q2+y2 s2 ζ #1/3 , (A.1a) ˆf2 y = κ2/3 ˆf2 AdSy 4(q1+y2)(q2+y2)−4y3 , (A.1b) (A.1a) (A.1b) 2/3 "c2 ζ c2ψ (a2+1) 2q2y+a2 2y3−q2 q1+y2+(a2+1) 2q2y+ a2 2−2 y3 2y ˆf4 AdS ˆf2 z = κ2/3 "c2 ζ c2ψ (a2+1) 2q2y+a2 2y3−q2 q1+y2+(a2+1) 2q2y+ a2 2−2 y3 2y ˆf4 AdS " 2yfAdS + s2 ζ y a2 1−1 y+ q2+y2+(a1+1) 2q1 ˆf4 AdS + c2 ζ q1+y2q2+2y2 2 ˆf4 AdSy # , (A.1c) ˆf2 ϕ1 = κ2/3 q1+y2s2 ζ 4 ˆf4 AdS , (A.1d) AdS + s2 ζ y a2 1−1 y+ q2+y2+(a1+1) 2q1 ˆf4 AdS + c2 ζ q1+y2q2+2y2 2 ˆf4 AdSy # , (A.1c) 2 2 ˆf2 ϕ1 = κ2/3 q1+y2s2 ζ 4 ˆf4 AdS , ˆf2 ϕ1 = κ2/3 q1+y2s2 ζ 4 ˆf4 AdS , (A.1d) – 34 – ˆf2 ϕ2 = κ2/3 c2 ψc2 ζ q2+y2 4 ˆf4 AdS , (A.1e) ˆf2 zϕ1 = κ2/3 s2 ζ a1q1+a1y2+q1 ˆf4 AdS , (A.1f) ˆf2 zϕ2 = κ2/3 c2 ψc2 ζ a2q2+a2y2+q2 ˆf4 AdS , (A.1g) ˆf2 ψ = κ2/3 c2 ζ q2−q2c2ψ+2y2 8 ˆf4 AdS , (A.1h) ˆf2 ζ = κ2/3 q1c2ζ +2q2c2 ψs2 ζ +q1+2y2 8 ˆf4 AdS , (A.1i) ˆf2 ψζ = κ2/3 q2cψcζsψsζ 2 ˆf4 AdS , (A.1j) ˆf2 ϕ2 = κ2/3 c2 ψc2 ζ q2+y2 4 ˆf4 AdS , ˆf2 zϕ1 = κ2/3 s2 ζ a1q1+a1y2+q1 ˆf4 AdS , ˆf2 zϕ2 = κ2/3 c2 ψc2 ζ a2q2+a2y2+q2 ˆf4 AdS , ˆf2 ψ = κ2/3 c2 ζ q2−q2c2ψ+2y2 8 ˆf4 AdS , ˆf2 ζ = κ2/3 q1c2ζ +2q2c2 ψs2 ζ +q1+2y2 8 ˆf4 AdS , ˆf2 ψζ = κ2/3 q2cψcζsψsζ 2 ˆf4 AdS , JHEP04(2024)120 (A.1i) (A.1j) where we denote κ = ˆg3Nℓ3 P/2. where we denote κ = ˆg3Nℓ3 P/2. where we denote κ = ˆg3Nℓ3 P/2. We seek an asymptotic map from {y, ψ, ζ} to the FG coordinates {u, ℵ, θ} in the large-y/small-u regime. A.1 Two-charge solutions , (A.6d) ˆαzφ1 = q1u4 −q1u6 + . . . , (A.6e) ˆαzφ2 = q2u4 −q2u6 + . . . , (A.6f) ˆαθ = 1 + 5q2c2ℵ+ 2q1 −3q2 12 u4 + . . . , (A.6g) ˆαℵ= 1 + 5(q2 −2q1)c2θ −10q2c2ℵs2 θ −6q1 −q2 24 u4 + . . . , (A.6h) ˆαθℵ= 5q2s2θs2ℵ 12 u4 + . . . . (A.6i) ˆαφ1 = 1 + 10q2c2ℵc2 θ + 5(q2 −2q1)c2θ + 14q1 −11q2 24 u4 + . . . , (A.6c) ˆαφ2 = 1 + 10q2c2ℵc2 θ + 5(q2 −2q1)c2θ −6q1 + 9q2 24 u4 + . . . , (A.6d) ˆαzφ1 = q1u4 −q1u6 + . . . , (A.6e) ˆαzφ2 = q2u4 −q2u6 + . . . , (A.6f) ˆαθ = 1 + 5q2c2ℵ+ 2q1 −3q2 12 u4 + . . . , (A.6g) ˆαℵ= 1 + 5(q2 −2q1)c2θ −10q2c2ℵs2 θ −6q1 −q2 24 u4 + . . . , (A.6h) ˆαθℵ= 5q2s2θs2ℵ 12 u4 + . . . . (A.6i) JHEP04(2024)120 (A.6i) If we had not transformed to φI, we would not have been able to put the metric in FG form. We can see this in the original ϕI coordinates, where ˆαzϕI has an O(1) term which is proportional to aI. FG gauge requires ˆαzϕI ∼u4, which would mean setting aI = 0. However, the values of the aI’s are set by regularity, i.e. aI = − qI qI + y2 + , (A.7) (A.7) and so, we cannot simply tune them to zero without also setting the corresponding qI = 0, which lands us on the pure AdS7 × S4 solution. and so, we cannot simply tune them to zero without also setting the corresponding qI = 0, which lands us on the pure AdS7 × S4 solution. A.1 Two-charge solutions By solving ˆf2 y dy2 + ˆf2 ψdψ2 + ˆf2 ζ dζ2 + ˆf2 ψζdψdζ = L2 u2 du2 + L2 4 c2 θ ˆαℵdℵ2 + ˆαθdθ2 + ˆαθℵdθdℵ (A.2)i (A.2) order by order in u, we find that the appropriate asymptotic map is order by order in u, we find that the appropriate asymptotic map is order by order in u, we find that the appropriate asymptotic map is y = 1 u2 + 1 2 + (2q1 −q2) c2θ −2q2c2ℵc2 θ −10q1 −9q2 + 3 48 u2 + . . . , ψ = ℵ+ q2s2ℵ 24 u4 + . . . , ζ = θ −s2θ q1 −q2c2 ℵ 24 u4 + . . . , (A.3) (A.3) 24 where we have suppressed higher orders in u due to their cumbersome expressions. To complete this map, we need to identify κ = L3, where L denotes the radius of the asymptotic AdS7 spacetime.i where we have suppressed higher orders in u due to their cumbersome expressions. To complete this map, we need to identify κ = L3, where L denotes the radius of the asymptotic AdS7 spacetime.i Mapping all of the other metric functions in eq. (2.19), we find the FG form of the metric to be ds2 FG = L2 u2 (du2 + ˆαAdSds2 AdS5 + ˆαzdz2) + L2s2 θ ˆαzφ1dzdφ1 + L2c2 ℵc2 θ ˆαzφ2dzdφ2 + L2 4 (ˆαθdθ2 + s2 θ ˆαφ1dφ2 1 + c2 θ(ˆαℵdℵ2 + c2 ℵˆαφ2dφ2 2) + ˆαθℵdθdℵ), (A.4) (A.4) where we have transformed the angular coordinates using where we have transformed the angular coordinates using (A.5) ϕI = φI −2aIz. (A.5) Note that since ϕI and z are all 2π-periodic and aI ∈Z/2, the new angular coordinates φI are also 2π-periodic. The metric functions have the asymptotic behavior ˆαAdS = 1 + u2 2 + 3 −2q1 + 3q2 −10q2c2ℵc2 θ + 5(2q1 −q2)c2θ 48 u4 + . . . , (A.6a) ˆαz = 1 −u2 2 + 3 −2q1 + 3q2 −10q2c2ℵc2 θ + 5(2q1 −q2)c2θ 48 u4 + . . . , (A.6b) (A.6a) – 35 – – 35 – – 35 – ˆαφ1 = 1 + 10q2c2ℵc2 θ + 5(q2 −2q1)c2θ + 14q1 −11q2 24 u4 + . . . , (A.6c) ˆαφ2 = 1 + 10q2c2ℵc2 θ + 5(q2 −2q1)c2θ −6q1 + 9q2 24 u4 + . . . A.2 Electrostatic solutions We now turn to deriving the FG form of the metric for the electrostatic solutions. Finding the asymptotic expansions of the metric factors in eq. (2.23a) requires explicit expressions for ˙V , ¨V , ˙V ′, V ′′, and σ. We can compute the indefinite integral in V for a trial line charge distribution ϖa(η) = p1+aη + δ1+a, −1 2 Z dη′G(r,η,η′)ϖa(η′) = p1+a 2 q r2+(η+η′)2− q r2+(η−η′)2 (A.8) −ηtanh−1 η+η′ p r2+(η+η′)2 ! +ηtanh−1 η−η′ p r2+(η−η′)2 ! + δ1+a 2 tanh−1 η+η′ p r2+(η+η′)2 ! +tanh−1 η−η′ p r2+(η−η′)2 !! , (A.8) and then build up the full potential by summing over the intervals. Clearly, evaluating the result above in the η′ →∞region leads to linear and logarithmic divergences. However, when evaluating derivatives of the right-hand side above, these divergences are eliminated, and only derivatives of V appear in all of the computations carried out below and in the main body of the text. The asymptotically AdS7 × S4 region corresponds to the limits r, η →∞. In order to facilitate the expansion of the derivatives of the electrostatic potential in this region, we redefine r = ϱcω and η = ϱsω, with ω ∈[0, π/2], so that f2 3 (dr2 + dη2) →f2 ϱdϱ2 + f2 ωdω2, (A.9) f2 3 (dr2 + dη2) →f2 ϱdϱ2 + f2 ωdω2, (A.9) – 36 – with f2 ϱ = f2 3 and f2 ω = f2 3 ϱ2. The AdS7 × S4 region now lies in the ϱ →∞limit. We can compute the asymptotic expansions of the derivatives of the electrostatic potential in this region in terms of its moments as follows, ˙V = ϱsω+m1sω−m3c2 ωsω 2ϱ2 + m5 (7c2ω−1)c2 ωsω 16ϱ4 +... , (A.10a) ¨V = −m1c2 ωsω+ m3 (5c2ω+1)c2 ωsω 4ϱ2 −m5 (28c2ω+63c4ω+29)c2 ωsω 64ϱ4 +... , (A.10b) ˙V ′ = 1+ m1c2 ω ϱ + m3 (3−5c2ω)c2 ω 4ϱ3 + 3m5 (21c4ω−28c2ω+15)c2 ω 64ϱ5 +... , (A.10c) V ′′ = m1sω ϱ2 −m3 (5c2ω+1)sω 4ϱ4 + m5 (28c2ω+63c4ω+29)sω 64ϱ6 +... . (A.10d)i (A.10a) JHEP04(2024)120 (A.10d) From these expressions, we can also find the asymptotic behavior of σ in terms of the moments of the electrostatic potential to be σ = 1+ 2m1 ϱ −m2 1 (c2ω−3) 2ϱ2 + m3 (1−3c2ω) 2ϱ3 + m3m1 (1−12c2ω+3c4ω) 8ϱ4 +... . (A.10e)i (A.10e) Together, these expansions can be inserted into the definitions of the metric functions in eq. A.2 Electrostatic solutions Note that this transformation parallels the one taken in [18], where Cz = 1/C is fixed by the ratio of four-form flux through two 4-cycles, which in turn fixes the mixing parameter between the U(1) symmetries leading to U(1)r symmetry ∂χ = ∂z + 1 C∂φ in the field theory. Here we are following the conventions of [14] where the corresponding C is negative. We then find that the metric functions for the transformed coordinates display the following asymptotic behavior, JHEP04(2024)120 f2 φ L2 = (aφ + bφ)2 u2 −1 8 (2aφ + bφ)2c2θ + bφ(4aφ + 3bφ) + . . . , (A.15a) f2 zφ L2 = 2(aφ + bφ) u2 + 1 4(2aφCz + bφ(Cz −2) −(2aφ + bφ)(Cz + 2)c2θ) + . . . , (A.15b) f2 z L2 = 1 u2 + 1 8(Cz(Cz + 4) −(Cz + 2)2c2θ) + . . . , (A.15c) (A.15a) (A.15c) where we introduced the AdS7 radius L = (16m1κ11)1/3. Setting aφ = −bφ = −1 removes the 1/u2 divergences in the asymptotic expansions of f2 φ and f2 zφ. In particular, f2 φ = L2s2 θ/4+. . .. This identifies the φ-circle as the internal S1 ⊂S4. Furthermore, f2 z = L2/u2 +. . ., as required for the external S1 ⊂AdS7. The final requirement to achieve an FG parametrization is that f2 zφ ∼O(u2). Eliminating the u0 behavior of f2 zφ fixes Cz ≡−2. Recalling the role of Cz, we see that the defect superconformal R-symmetry is ∂χ = ∂z −2∂φ.i Having identified the correct combination of angular variables, we can at once express the metric in FG gauge as ds2 FG = L2 u2 (du2 + αAdSds2 AdS5 + αzdz2) + L2s2 θαzφdzdφ + L2 4 s2 θαφdφ2 + c2 θαS2dΩ2 2 + αθdθ2 , (A.16) (A.16) where the metric functions have asymptotic behavior αAdS = 1+ u2 2 + 1 96 10c2 θ+ m3 (1−5c2θ) m3 1 u4+ m3−m3 1 c2 θ 18m3 1 u6 ... , (A.17a) αz = 1−u2 2 + 1 96 10c2 θ+ m3 (1−5c2θ) m3 1 u4+ m3−m3 1 (5c2θ−13) 72m3 1 u6+... , (A.17b) αφ = 1+ m3−m3 1 (5c2θ−7) 48m3 1 u4+ m3 1−m3 (10c2θ−17) 108m3 1 u6+... , (A.17c) αS2 = 1+ m3−m3 1 (5c2θ+3) 48m3 1 u4+ m3 1−m3 (5c2θ+4) 54m3 1 u6+... A.2 Electrostatic solutions (2.23a) to give (2m1)1/3 κ2/3 11 f2 AdS = 4ϱ+4m1+ 5m3c2ω+4m3 1s2 ω+m3 3m1ϱ + 4 m3−m3 1 s2 ω 3ϱ2 +... , (A.11a) (2m1)1/3 s2ωκ2/3 11 f2 S2 = 2m1−(1+5c2ω)m3+4s2 ωm3 1 3ϱ2 + 8m1 m3 1−m3 s2 ω 3ϱ3 +... , (A.11b) (2m1)1/3 κ2/3 11 f2 ϱ = 2m1 ϱ2 −(1+5c2ω)m3−2s2 ωm3 1 3ϱ4 + 4m1 m3−m3 1 s2 ω 3ϱ5 +... , (A.11c) (2m1)1/3 κ2/3 11 f2 β = 4ϱ+m1 (c2ω−3)+ (1+5c2ω)m3+4m3 1s2 ω 3m1ϱ +... , (A.11d) (2m1)1/3 κ2/3 11 f2 χ = 4ϱ+4m1c2ω+ 5m3c2ω+4m3 1s2 ω+m3 3m1ϱ +... , (A.11e) (2m1)1/3 κ2/3 11 f2 βχ = 8ϱ−8m1s2 ω+ 2 5m3c2ω+4m3 1s2 ω+m3 3m1ϱ +... . (A.11f) (A.11a) (A.11f) We again look for an asymptotic map to a set of coordinates {u, θ} in terms of which the metric is in FG form. By taking ϱ = ϱ(u, θ) and ω = ω(u, θ), and expanding in small u to solve We again look for an asymptotic map to a set of coordinates {u, θ} in terms of which the metric is in FG form. By taking ϱ = ϱ(u, θ) and ω = ω(u, θ), and expanding in small u to solve f2 ϱdϱ2 + f2 ωdω2 = L2 u2 du2 + L2 4 αθdθ2 (A.12) (A.12) order by order, we find order by order, we find ρ = 2m1 u2 + 2m3 1c2 θ + m3 (5c2θ −1) 48m2 1 u2 + m3 −m3 1 c2 θ 36m2 1 u4 + . . . , (A.13a) ω = θ + π 2 − m3 1 + 5m3 s2θ 96m3 1 u4 + m3 1 −m3 s2θ 216m3 1 u6 + . . . . (A.13b) – 37 – – 37 – The asymptotic expansions of f2 χ, f2 β, and f2 βχ under the above transformation reveal an ambiguity as to which of the angular coordinates should be identified as parametrizing the external S1 ⊂AdS7 and which as parametrizing the internal S1 ⊂S4 upon mapping to FG gauge. That is, both are characterized by 1/u2 divergences at small-u, so that the resulting asymptotic metric is not in FG gauge. To resolve this issue, we introduce χ = (1 + Cz)z + aφφ, β = −Czz + bφφ, (A.14) (A.14) where Cz ∈Z and aφ and bφ are arbitrary constants. A.2 Electrostatic solutions , (A.17d) αzφ = m3 1−m3 4m3 1 u4−m3 1−m3 4m3 1 u6+... , (A.17e) αθ = 1+ m3 1−m3 24m3 1 u4+ m3−m3 1 (5c2θ+9) 216m3 1 u6+... . (A.17f) – 38 – Note that, upon being evaluated on the single kink electrostatic profile in eq. (2.29), the asymptotic metric above recovers the q2 = 0 instance of eq. (A.4); in particular, the coordinate φ maps over to φ1, while ℵand φ2 correspond to, respectively, the polar and azimuthal angles on the asymptotic internal S2 ⊂S4 in the electrostatic description. B Regulating the on-shell action In this appendix, we collect some of the details of the regulating scheme for the computation of the on-shell 11d supergravity action evaluated on the two-charge solutions. Below we compute the vacuum AdS7 × S4 on-shell action, which we use in the background subtraction scheme. This value also provides a good diagnostic for the known limiting case, qI = aI = 0 for the two charge solutions, that recovers the vacuum geometry. We also briefly discuss computing the renormalized volume of the AdS5 submanifold of the 11d spacetime. JHEP04(2024)120 B.1 AdS7 × S4 B.1 AdS7 × S4 . . (B.7) (B.7) JHEP04(2024)120 Finally, we note that since d ⋆F4 = 0 we can introduce a gauge potential C6 so that dC6 = ⋆11F4. We can then perform the bulk integral of F4 ∧C6 over the radial cutoff slice at u = ϵu with the pullback of the six-form potential being given by C6 u=ϵu = 3L6 1 3ϵ6u + 1 2ϵ4u + 5 16ϵ2u −11 48 + 5ϵ2 u 256 + ϵ4 u 512 + ϵ6 u 12288 ! dz ∧ΥAdS5. (B.8) (B.8) Crucially, we have used the residual gauge freedom to fix the six-form potential to be regular at the origin of AdS7, i.e. we pick a gauge such that C6 u=2 = 0. In this gauge, the on-shell action computed using C6 gives the same result as above. B.1 AdS7 × S4 In this subsection, we compute the on-shell action for the vacuum AdS7 × S4 geometry that we use in our background subtraction scheme. The data which specifies this solution to the bosonic theory in eq. (5.1) is the metric ds2 11 = L2 dx2 + cosh2(x)ds2 AdS5 + sinh2(x)dz2 + L2 4 dΩ2 4 , (B.1) (B.1) which is an AdS5 slicing of AdS7 with x ∈[0, ∞), and the four-form flux and its Hodge dual F4 = −3L3 8 ΥS4 , (B.2a) ⋆11F4 = 6L6 cosh5(x) sinh(x)dx ∧dz ∧ΥAdS5 . (B.2b) F4 = −3L3 8 ΥS4 , (B.2a) 6 5 F4 = −3L3 8 ΥS4 , (B.2a) ⋆11F4 = 6L6 cosh5(x) sinh(x)dx ∧dz ∧ΥAdS5 . (B.2b) (B.2b) Since we are working with the AdS7×S4 vacuum, the transformation to FG gauge is simply x = −ln(u/2) , (B.3) (B.3) where the FG radial coordinate is valued u ∈[0, 2]. In FG gauge, the metric takes the form ds2 11 = L2 u2 du2 + 1 + u2 2 + u4 16 ! ds2 AdS5 + 1 −u2 2 + u4 16 ! dz2 ! + L2 4 dΩ2 4 . (B.4) (B.4) The four-form flux has no functional dependence on x and is unchanged in transforming to FG gauge, while the seven-form flux becomes ⋆11F4 = 6L6 1 u7 + 1 u5 + 5 16u3 −5u 256 −u3 256 − u5 4096 ! du ∧dz ∧ΥAdS5 + . . . . (B.5) (B.5) With the asymptotics of the metric and fluxes in hand, we can easily compute the on-shell action. Note that the GHY term for the vacuum AdS7 ×S4 solution is trivially identical to the expression found in eq. (5.13), and so we will not reproduce it here. The bulk action is then – 39 – computed from the F4 ∧⋆F4 term, which after inserting eqs. (B.2a) and (B.5), introducing a radial cutoff at u = ϵu ≪1, and integrating over the AdS7 × S4 geometry gives S(vac) OS,bulk = −L9π2 8G(11) N vol(AdS5) 1 3ϵ6u + 1 2ϵ4u + 5 16ϵ2u −11 48 + . . . . (B.6) (B.6) Combining with the GHY term, we find Combining with the GHY term, we find S(vac) OS = −π2L9 8G(11) N vol(AdS5) 1 3ϵ6u + 5 3ϵ5u + 1 2ϵ4u + 1 ϵ3u + 5 16ϵ2u + 5 48ϵu −11 48 + . . ds2 AdS5 = dx2 + sinh2(x) dΩ2 4. (B.9) B.2 Renormalized AdS5 volume Even after accounting for the divergences coming from the asymptotically AdS7 part of the geometry via background subtraction, we are still left to deal with the volume of the AdS5 factor in the on-shell action. In order to regularize the remaining polynomial divergences and read off the universal log-divergent part of the on-shell action, we will simply treat the intrinsic parts of the AdS5 geometry using standard counterterms in holographic renormalization and neglecting any divergences associated with the embedding. This renormalization scheme is admittedly simplistic as it only treats the set of counterterms associated with the intrinsic geometry of the AdS5 submanifold. However, since the background subtraction scheme leaves behind only divergences from the volume of the AdS5 and we choose the boundary geometry to be S4 ,→R6, only defect Weyl anomalies constructed purely from the intrinsic geometry should contribute, which will be accounted for in the scheme we have chosen. The caveat is that there may be structures for which we have not accounted in the full set of 11d counterterms, which is difficult to construct, whose pullback to the AdS5 submanifold contains terms that contribute to the log divergence in a similar way. Absent a full holographic renormalization scheme for solutions to SUGRA dual to defects, which would replace background subtraction scheme as well, this scheme choice constructing counterterms only for the intrinsic geometry of the AdS5 submanifold is the best tool available. Moving on, the volume of AdS5 has well known divergences. In order to systematically remove them and reveal any universal log-divergent terms, we consider AdS5 in global coordinates with an S4 boundary: ds2 AdS5 = dx2 + sinh2(x) dΩ2 4. (B.9) ds2 AdS5 = dx2 + sinh2(x) dΩ2 4. (B.9) – 40 – For simplicity, we consider the round metric on S4. Computing the AdS5 volume requires regulating the large x behavior, and so we introduce a radial cutoff Λx ≡−log ϵx 2 for ϵx ≪1. Then, expanding in small ϵx For simplicity, we consider the round metric on S4. Computing the AdS5 volume requires regulating the large x behavior, and so we introduce a radial cutoff Λx ≡−log ϵx 2 for ϵx ≪1. Then, expanding in small ϵx vol(AdS5) = 8π2 3 Z Λx 0 dx sinh4(x) = 2π2 3ϵ4x −4π2 3ϵ2x −π2 log ϵx 2 + . . . . B.2 Renormalized AdS5 volume (B.10) (B.10) We regulate the volume using covariant counterterms19 added on the radial cutoff slice that are standard in AdS5 holographic renormalization [60, 97] SCT,1 = −1 4 Z dΩ4 q \|gϵx\| = −2π2 3ϵ4x + 2π2 3ϵ2x −π2 4 + . . . , (B.11a) SCT,2 = 1 48 Z dΩ4 q \|gϵx\|Rϵx = 2π2 3ϵ2x −π2 3 + . . . , (B.11b) (B.11a) JHEP04(2024)120 (B.11b) where p \|gϵx\| = (1 −ϵx)4p \|gS4\|/16ϵ2 x and Rϵx = 12 csch2(ϵx) are the volume form and the intrinsic Ricci scalar on the cutoff slice, respectively, built from the induced AdS5 metric. Adding these counterterms to the bulk action, we see that the holographically renormalized volume of the unit AdS5 takes the well-known form vol(AdS5) = −π2 log ϵx 2 + . . . . (B.12) (B.12) To complete the regularization of the on-shell actions for the vacuum AdS7 × S4 and two- charge solutions and extract the universal contributions to the defect free energy, we replace vol(AdS5) = −π2 log(ϵx/2) wherever it appears. Open Access. 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